Heat shock protein 83: Biological Overview | References
Gene name - Heat shock protein 83
Synonyms - Hsp90
Cytological map position - 63B11-63B11
Function - heat shock protein
Keywords - suppresses of phenotypic variability, optimizes PolII pausing, promotes anaphase-promoting complex function during cell cycle exit, regulates asymmetric cell division, regulates Piwi-interacting RNA pathway, required for mRNA localization
Symbol - Hsp83
FlyBase ID: FBgn0001233
Genetic map position - chr3L:3193216-3197059
Classification - HSP90 family protein, Histidine kinase-like ATPase
Cellular location - cytoplasmic and nuclear
|Recent literature||Choutka, C., DeVorkin, L., Go, N. E., Hou, Y. C., Moradian, A., Morin, G. B. and Gorski, S. M. (2017). Hsp83 loss suppresses proteasomal activity resulting in an upregulation of caspase-dependent compensatory autophagy. Autophagy: 1-17. PubMed ID: 28806103
The two main degradative pathways that contribute to proteostasis are the ubiquitin-proteasome system and autophagy but how they are molecularly coordinated is not well understood. This study demonstrates an essential role for an effector caspase in the activation of compensatory autophagy when proteasomal activity is compromised. Functional loss of Hsp83, the Drosophila ortholog of human HSP90 (heat shock protein 90), resulted in reduced proteasomal activity and elevated levels of the effector caspase Dcp-1. Surprisingly, genetic analyses showed that the caspase was not required for cell death in this context, but instead was essential for the ensuing compensatory autophagy, female fertility, and organism viability. The zymogen pro-Dcp-1 was found to interact with Hsp83 and undergo proteasomal regulation in an Hsp83-dependent manner. This work not only reveals unappreciated roles for Hsp83 in proteasomal activity and regulation of Dcp-1, but identifies an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo.
|Karney-Grobe, S., Russo, A., Frey, E., Milbrandt, J. and DiAntonio, A. (2018). HSP90 is a chaperone for DLK and is required for axon injury signaling. Proc Natl Acad Sci U S A. PubMed ID: 30275300
Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration. A loss-of-function pharmacological screen was carried out in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth. The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes. These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90-client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve. This phenomenon is evolutionarily conserved in Drosophila. Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. These findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.
The molecular chaperone Heat shock protein 90 (Hsp90) promotes the maturation of several important proteins and plays a key role in development, cancer progression, and evolutionary diversification. By mapping chromatin-binding sites of Hsp90 at high resolution across the Drosophila genome, an unexpected mechanism was uncovered by which Hsp90 orchestrates cellular physiology. It localizes near promoters of many coding and noncoding genes including microRNAs. Using computational and biochemical analyses, it was found that Hsp90 maintains and optimizes RNA polymerase II pausing via stabilization of the negative elongation factor complex (NELF). Inhibition of Hsp90 leads to upregulation of target genes, and Hsp90 is required for maximal activation of paused genes in Drosophila and mammalian cells in response to environmental stimuli. These findings add a molecular dimension to the chaperone's functionality with wide ramifications into its roles in health, disease, and evolution (Sawarkar, 2012).
Since its discovery, most of the cellular functions of Hsp90 have been attributed to stabilization of client proteins involved in signaling (Rutherford, 1998; Workman, 2007). The results outlined in this study argue for an additional, and hitherto ignored, role of this chaperone at a different level of the information-processing pathway - gene transcription. Although this activity may be a result of the function of Hsp90 to stabilize a protein-forming paused pol II complex, it ultimately results in a much wider control of cellular behavior (Sawarkar, 2012).
These studies linking Hsp90 with pol II pausing were suggested solely by genomic comparisons of several digital data sets arising from a single homogeneous cell line. Use of inhibitors, rather than slow-acting RNAi, with rapid and genome-wide follow-up of downstream gene activity led to a mechanism with minimal complications arising from secondary effects of Hsp90 inhibition, such as cell-cycle arrest and cell death. The upregulation of Hsp90 targets within minutes of radicicol treatment seen on a global scale confirms the conjecture that the immediate transcriptional effect of Hsp90 inhibition is via its chromatin function and not as an effect on cytosolic function (Sawarkar, 2012).
The list of Hsp90 targets includes several genes important for growth homeostasis. c-myc, a potent protooncogene, exhibits pol II pausing in mammalian cells and Hsp90 binding in Drosophila cells. Given the conservation of the process described in this study, it is highly possible that human Hsp90 also targets the paused c-myc promoter. Hsp90 depletion, thus, may also alter expression of c-Myc, p53, and other signaling components in human cells via pol II pausing, a possibility that is significant in light of anticancer activities of Hsp90 inhibitors (Sawarkar, 2012).
In addition to c-myc and p53, other Hsp90 targets such as bantam and mir-278 are implicated in apoptosis and growth control. Their direct regulation by the chaperone caused by release of paused pol II in the gene body adds yet another aspect to Hsp90 biology. In addition to the chaperone's role in Argonaute function, its involvement in miRNA-gene expression may represent a major pathway by which Hsp90 orchestrates cellular physiology and pathology (Sawarkar, 2012).
Recently, Hsp90 was shown to bind HIV promoter and regulate its expression and eventually viral infectivity (Vozzolo, 2010). The viral genome exhibits pol II pausing at its promoter. Moreover NELF-E depletion results in increased virion production. Taken together with the current results, it is most likely that Hsp90 targets the paused pol II located on HIV promoter via NELF-E and, similar to several genes reported in this study, affects release of paused pol II from the viral locus. Thus, the fundamental observation with Drosophila cells may help explain several findings of biomedical importance, paving a way for a more rational drug design (Sawarkar, 2012).
Most heat shock loci are targeted by heat shock factor (HSF) at high temperature, leading to their activation. A comparison between binding sites of HSF mapped in heat-shocked S2 cells (Guertin, 2010) and those of Hsp90 in normal cells reported in this study showed that three-quarters of a total 437 HSF targets were occupied by the chaperone before heat shock, implying a collaboration between HSF and Hsp90 at chromatin. Given that HSF itself is a client of Hsp90 (Zou, 1998), the promoter-bound Hsp90 may assist HSF binding to nearby heat shock elements or its activity thereafter. Notably, not all loci bound by HSF show elongation factor recruitment on polytene chromosomes. It is possible that HSF binds to many sites but activates genes only in the context of bound Hsp90 and paused polymerase. It should be noted that HSF binds very strongly to only one locus prior to heat shock, Hsp83 locus (Guertin, 2010), the promoter of which is also targeted by Hsp90. The control of this locus by promoter-bound Hsp90 may be assisted by HSF and shows an important motif in the chaperone circuitry (Sawarkar, 2012).
What targets Hsp90 to specific sites across chromatin? Given the association of Hsp90 with pausing factors, it's likely that paused complex acts as a docking site for Hsp90. NELF complex may be only one of the several pausing factors required by Hsp90 to get recruited to chromatin as Hsp90 occupancy only mildly changes upon NELF knockdown. No pausing of pol II is reported in yeast S. cerevisiae, and it lacks NELF complex, suggesting that TSS association of Hsp90 may be metazoan specific. It will be highly interesting to see whether Hsp90 is associated with distinct chromatin regions even in the yeast (Sawarkar, 2012).
This study provides several lines of evidence for a functional association between Hsp90 and pol II pausing. From genome-wide comparison of Hsp90 ChIP and pol pausing data sets and biochemical interaction between Hsp90 and pol pause factors to functional links in culture cells and flies, the results buttress the model of Hsp90's action through NELF-mediated regulation of pol II pausing. Hsp90 inhibition does not cause upregulation of all target paused genes, for example CecC and CecA1. This is visible in the nonuniform changes in gene expression of radicicol-treated cells. Knockdown of the best characterized pol pause factor, NELF complex, has previously been shown to upregulate only a small fraction of paused genes but downregulate many more genes. This may be attributed to a competition between nucleosomal occupancy and pol II binding or another pause-associated regulatory factor like Polycomb. The results with Hsp90 inhibition thus reiterate that there are different layers of regulation, and a final picture is an integrated outcome of a variety of these processes in a gene-specific way (Sawarkar, 2012).
The molecular details of how Hsp90 is involved in pol II pausing may share similarities with how Hsp90 poises steroid receptors for activation. The chaperone may keep the paused pol II components such as NELF and pol II itself in a conformation receptive for signal-mediated phosphorylation by P-TEFb. Radicicol causes paused pol II to be instantaneously but inefficiently released in elongation mode owing to NELF malfunction, causing a transient increase in expression of many genes. On the other hand, in absence of Hsp90, P-TEFb may not be able to phosphorylate and activate pol II efficiently following an extracellular signal such as LPS. It should be noted that P-TEFb is thought to be a client of Hsp90 (O'Keeffe, 2000). Additionally, activators of transcription such as Trx are also degraded after a few hours of radicicol treatment (Tariq, 2009), suggesting a possible feedback regulation of increased transcription upon Hsp90 inhibition (Sawarkar, 2012).
Past studies have linked Hsp90 depletion with cellular effects via its cytosolic function of protein stabilization. In light of the current results, earlier observations need to be reinterpreted to accommodate the direct effects of Hsp90 inhibition on gene expression. Hsp90 is thought to dampen phenotypic manifestation of genetic variants (Rutherford, 1998) in protein-coding as well as cis-regulatory regions (Jarosz, 2010). The relative contribution of cytosolic and chromatin-bound Hsp90 to different categories of genetic variants needs to be assessed. The recent demonstration of Hsp90's function in signal-mediated transactivation of the inducible nitric oxide synthase (iNOS) gene in addition to stabilization of iNOS protein (Luo, 2011) underlines the need for reanalysis. In this regard, it is important to devise methods to distinguish cytosolic and nuclear functions of Hsp90. Either a nucleus-specific cochaperone network or nuclear entry of Hsp90 could be targeted by small-molecule regulators. Given the biomedical importance of Hsp90 in cancer and infectious diseases, development of such tools would be the next significant milestone. Thus the work presented here unites two rather disparate branches of biology -- molecular chaperones and gene regulation -- and it should open a new avenue of therapeutic importance for integrating vast amounts of data in both fields (Sawarkar, 2012).
Neural stem cells (NSCs) have the ability to exit quiescence and reactivate in response to physiological stimuli. In the Drosophila brain, insulin receptor (InR)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway triggers NSC reactivation. However, intrinsic mechanisms that control the InR/PI3K/Akt pathway during reactivation remain unknown. This study identified heat shock protein 83 (Hsp83/Hsp90), a molecular chaperone, as an intrinsic regulator of NSC reactivation. Hsp83 is both necessary and sufficient for NSC reactivation by promoting the activation of InR pathway in larval brains in the presence of dietary amino acids. Both Hsp83 and its co-chaperone Cdc37 physically associate with InR. Finally, reactivation defects observed in brains depleted of hsp83 were rescued by over-activation of the InR/PI3K/Akt pathway, suggesting that Hsp83 functions upstream of the InR/PI3K/Akt pathway during NSC reactivation. Given the conservation of Hsp83 and the InR pathway, this finding may provide insights into the molecular mechanisms underlying mammalian NSC reactivation (Huang, 2018).
How the InR/PI3K/Akt pathway is regulated during NSC reactivation is poorly understood. This study shows that molecular chaperone Hsp83/Hsp90, together with its co-chaperone Cdc37, play a role in the reactivation of Drosophila NSCs. Mechanistically, Hsp83 and Cdc37 physically associate with InR and are important for the activation of the InR/PI3K/Akt pathway in NSCs. Therefore, this study demonstrates that Hsp83 serves as an intrinsic factor within NSCs that is necessary for the activation of the InR/PI3K/Akt pathway and, in turn, reactivation of NSCs. This evidence suggests that Hsp83 and Cdc37 regulate the protein folding and activation of InR in the nervous system (Huang, 2018).
The role of Hsp83 in NSC reactivation at early larval stages is distinct from its known role in centrosomes or NSC polarity. Drosophila Hsp83 is a core centrosomal component required for proper mitotic spindle formation and chromosome segregation. In Drosophila larval CNS, Hsp83 and co-chaperone Sgt1 are required for the stabilization of Polo and centrosome organization in NSCs. Hsp83 and Sgt1 are also required for the establishment of NSC polarity via the LKB1/AMPK pathway in third-instar larvae. However, sgt1 RNAi in NSCs did not display any phenotypes during NSC reactivation, suggesting that Hsp83 interacts with different co-chaperones to control NSC reactivation and cortical polarity at different developmental stages. Consistent with this notion, Cdc37, but not other co-chaperones of Hsp83, was found to be required for NSC reactivation. The proliferation of MB NSCs were unaffected by hsp83 knockdown, Therefore, Hsp83 promotes NSC reactivation rather than general cell proliferation. Consistent with these observations, there is no significant difference in proliferation between hsp83 mutant and wild-type eye imaginal discs in the proliferating zone. Interestingly, in pupal eyes that undergo terminal differentiation, Hsp83 is required for cell-cycle exit by activating the anaphase-promoting complex/cyclosome. This study found cytokinesis defects in cdc37-depleted NSCs, but not in NSCs depleted of Hsp83. This observation is consistent with a known role of Cdc37 in cell division and cytokinesis in Drosophila (Huang, 2018).
Hsp90 plays a key role in signal transduction and appears to bind to its substrates in a near native state poised for activation by binding of ligand or other factors. Since Hsp83 overexpression is sufficient to drive the activation of InR/PI3K/Akt pathway and trigger premature NSC reactivation, Hsp83 likely plays an active role in promoting NSC reactivation by binding to InR at a late stage of folding poised for activation by dILP binding. Furthermore, in the absence of dietary amino acids, the expression of hsp83 is downregulated, likely partially contributing to the inactivation of the InR pathway. It is proposed that InR is a target of Hsp83 and Cdc37 during NSC reactivation. The physical association among Hsp83, Cdc37, and InR was strongly supported by PLA assays, and both in vitro and in vivo BiFC. Although tandem affinity purification-mass spectrometry in Drosophila S2 cells implied an interaction between Hsp83 and InR, this study failed to detect a consistent interaction between Hsp83 and InRintra in S2 cells in co-immunoprecipitation experiments, probably due to the transient nature of this interaction. In addition, genetic interaction experiments indicate that Hsp83 activates the InR/PI3K/Akt pathway to promote NSC reactivation. Taken together, InR is likely a client of Hsp83 in Drosophila NSCs. Consistent with these findings, in human fibroblasts, Hsp90 co-immunoprecipitated with intracellular InR β. Furthermore, Hsp90 facilitates the maturation of the InR precursor in the ER and, in turn, is required for cell surface expression of InR in both bovine adrenal medullary chromaffin cells and human kidney HEK293 cells. Therefore, the interaction between the Hsp90 chaperone family and InR may be conserved from Drosophila to humans. In mammals, the expression level of Hsp90 in the brain is the highest among all tissues. Although mammalian Hsp90 proteins are heavily implicated in neurodegenerative diseases, their function in brain development is not well understood. Hsp90/Cdc37 stabilize the intracellular domain of Ryk, a Wnt receptor required for neurogenesis. Furthermore, Hsp90 stabilizes hypoxia-inducible factor-1, which promotes NSC proliferation under hypoxia. It remains to be determined whether the interaction between mammalian Hsp90 and InR is conserved during mammalian NSC development (Huang, 2018).
The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. A genetic screen in Drosophila was designed to identify genes required for cell cycle exit. This screen utilized a reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, it is proposed that Cdh1/Fzr is a client protein of Hsp83. The results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis (Bandura, 2013).
Analysis of Hsp83 mutant clones indicates that the cell cycle exit delay is primarily due to increased Cyclin/Cdk activity. In addition, the levels of several APC/C target proteins, including Cyclin A, are increased in cells lacking Hsp83. Removing one copy of an inhibitor of the APC/CCdh1 suppresses the Hsp83 cell cycle exit phenotype. These data are consistent with a model in which Cdh1/Fzr is a client protein of Hsp83, and Hsp83 restrains Cyclin/Cdk activity after cell cycle exit by ensuring maximal activity of the APC/CCdh1. In this way, Hsp83 could provide yet another layer of control over the core cell cycle machinery to ensure that cells stop dividing on schedule during development (Bandura, 2013).
Several mechanisms have been implicated in controlling permanent cell cycle exit upon terminal differentiation. E2F and Rb family members influence cell proliferation by transcriptional regulation, Cyclin/Cdks and CKIs regulate cell proliferation via control of cell cycle protein phosphorylation, and the APC/C targets pro-proliferative proteins for proteasome-dependent degradation. However, it has been clear that additional factors must exist that modulate the activity of known cell cycle regulators to ensure timely cell cycle exit. This study has provide evidence of an additional layer of control imposed on the cell cycle machinery by the molecular chaperone Hsp90 (Bandura, 2013).
Cells mutant for the Drosophila Hsp90 homologue, Hsp83, or expressing an RNAi against Hsp83 experienced a delay in cell cycle exit. Hsp83 mutant cells also experienced ectopic increases in both E2F and Cyclin/Cdk activity. The data indicate that the increased Cyclin/Cdk activity was the primary defect, which secondarily caused an increase in E2F activity only at time points shortly after wild-type cells have exited the cell cycle. It was further found that the Hsp83 mutant cells contained increased levels of Cyclin A, Cyclin B and Geminin proteins, consistent with reduced APC/C function. This finding has led to a hypothesis that Cdh1/Fzr may be a client protein of Hsp83. The cell cycle exit effect resulting from Hsp83 knock-down was suppressed by reducing the dosage of Rca1, which provided support to this hypothesis (Bandura, 2013).
Although the number of cells undergoing mitosis in the mutant and RNAi clones was significantly increased compared to control clones (1-1.24% versus 0%), Hsp83 loss-of-function resulted in a rather small number of cells delaying cell cycle exit. Why was this? One reason is that the cell cycle exit mechanism is quite robust. To provide a comparison, rbf1 mutant clones in pupal tissues exhibit a mitotic index of ~9% only from 24-28 APF and dap mutant cells do not experience any cell cycle exit delay. Although the Hsp83 mutant cells have a mitotic index of only ~1%, ectopic cell divisions continue until approximately 40 APF, timing consistent with one extra cell division. In addition, direct overexpression of Rca1 to inhibit Fzr delays cell cycle exit with similar timing as the Hsp83 mutation, and also results in a modest increase in mitotic index in pupal tissues. It is not surprising that Hsp836-55, a hypomorphic mutation in something that is thought to optimize the function of Cdh1/Fzr, would produce a much more subtle effect (Bandura, 2013).
This study is the first providing evidence that Cdh1/Fzr could be an Hsp90 client. In budding yeast, it has been demonstrated that another chaperone, the CCT chaperonin, is required for the folding of both Cdc20 and Cdh1 and therefore is necessary for all APC/C activity. While the CCT chaperonin functions in the bulk folding of nascent proteins, the Hsp90 family of chaperones generally promotes more subtle structural changes to potentiate the function of its clientele. It is proposed that Hsp90 is specifically required to optimize the function of APC/CCdh1 and not APC/CCdc20. Indeed, the fact that it was possible to observe Hsp90 mutant cells undergoing mitosis indicates that the function of the APC/CCdc20 is intact, as cells lacking functional Cdc20/Fzy arrest in mitosis (Bandura, 2013).
Several studies have demonstrated that mutation or inhibition of fzr can delay cell cycle exit, but it is not clear which target protein(s) of the APC/CCdh1 are responsible for this delay. One likely candidate for the crucial target protein that causes the delay in cell cycle exit is Cyclin A. In Drosophila, Cyclin A normally functions during mitosis, but when ectopically expressed it can also induce entry into S phase. Cyclin A overexpression can drive the G1/S transition even in the absence of Cyclin E, suggesting that Cyclin A/Cdk complexes can directly phosphorylate Cyclin E/Cdk2 targets important for S phase. Further, in mammals it has been demonstrated that Cdk1 is the only essential Cdk and that it is sufficient to drive the entire cell cycle in the absence of interphase Cdks. Indeed, direct overexpression of Cyclin A can cause one complete ectopic cell cycle in differentiating pupal eyes and wings. However, the extra cell cycle induced in the Drosophila embryonic epidermis by mutation of fzr cannot be rescued by also mutating cyclin A, suggesting there may be other crucial target proteins of the APC/CCdh1 in addition to Cyclin A that must be restrained in order to initiate cell cycle exit. APC/C targets that could potentially play a role in cell cycle exit include Cyclins B and B3, Cdc25/Stg phosphatase and the DNA replication factor Orc1. Further studies are needed to address whether restraining these target proteins comprises an important part of the cell cycle exit requirement for APC/CCdh1 (Bandura, 2013).
The current model is that Hsp90 facilitates the function of the Cdh1/Fzr. What could be the purpose for this regulation of the APC/CCdh1? Unlike other ubiquitin ligases that recognize their targets only when they have been post-translationally modified, for example by phosphorylation or hydroxylation, the APC/C recognizes different unmodified substrates at distinct points in the cell cycle. Although it is not entirely understood how this change in substrate specificity occurs, it is clear that it is partially dependent on which co-activator, Cdc20/Fzy or Cdh1/Fzr, is associated with the complex. While Cdc20/Fzy is expressed periodically and this may partially control activation of the APC/CCdc20, Cdh1/Fzr is not periodically transcribed. Cdh1/Fzr is known to be negatively regulated by both Cyclin/Cdk phosphorylation and binding of inhibitors. In addition, activation of Cdh1/Fzr by interaction with Hsp90 in a cell cycle-dependent manner could potentially also be responsible for the timely activation of the APC/CCdh1 (Bandura, 2013).
How could a protein such as Hsp90 that is expressed ubiquitously and at high levels regulate another protein so that it only acts at a specific place and time? Hsp90 function is modulated both by association with co-chaperones and via post-translational modifications, including phosphorylation, acetylation and S-nitrosylation, all of which can direct Hsp90 to particular client proteins (Trepel, 2010). A recent study has even indicated that a portion of cellular Hsp90 is phosphorylated by Wee1, causing it to selectively associate with only some of its clients in a cell cycle-dependent manner (Mollapour, 2010). Although it has not been demonstrated that this specific phosphorylation event plays a role during exit from the cell cycle, these data combined with data indicating that Hsp90 is subject to many post-translational modification raise the possibility that a particular combination of modifications on Hsp90 could create a G1-specific Hsp90 activity that optimizes the function of clients important for cell cycle exit in G1 (Bandura, 2013).
Hsp90 is needed to protect a number of mutated and overexpressed oncoproteins, such as ErbB2/HER2, v-Src, Akt and Bcr-Abl, from misfolding and degradation (Trepel, 2010; Pearl, 2008). Therefore, inhibition of Hsp90 is an efficient way to silence multiple oncogenic signaling pathways simultaneously. As a result, several Hsp90 inhibitors are currently being developed and tested as anti-cancer therapeutics. However, the pleiotropic effects of Hsp90 inhibitors may complicate their clinical efficacy. For example, Hsp90 inhibition results in the release and activation of the heat shock transcription factor 1 (HSF1), and HSF1 has a clear role in supporting the proliferation and survival of transformed cells. In addition, in Drosophila, is had been demonstrated that mutation or inhibition of Hsp90 relieves the suppression of transposon activity, resulting in de novo mutations and revealing that Hsp90 inhibition can be mutagenic (Specchia, 2010). The current findings, which suggest that APC/CCdh1 function is reduced in the absence of Hsp90, identify an additional mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. Mouse and human cells lacking Cdh1/Fzr exhibit multiple markers of genomic instability, including chromosome breaks, anaphase bridges and aneuploidy. Further, Cdh1/Fzr heterozygous mice displayed an increased propensity to develop epithelial tumors, suggesting a tumor suppression function for Cdh1/Fzr. Overall, these data highlight a need to target Hsp90 inhibitors not only to tumor cells, but specifically to Hsp90-oncoprotein client interactions within tumor cells. Current efforts by researchers to design Hsp90 inhibitors that disrupt specific Hsp90-co-chaperone interactions, or to develop Hsp90 isoform- and cell location-specific inhibitors are underway (Trepel, 2010). This approach of designing more precisely targeted Hsp90 inhibitors will be important to reduce undesirable pro-proliferative and mutagenic effects of Hsp90 inhibition (Bandura, 2013).
Juvenile hormone (JH) receptors, Met and Gce, transduce JH signals to induce Kr-h1 expression in Drosophila. Dual luciferase assay identified a 120-bp JH response region (JHRR) in the Kr-h1α promoter. Both in vitro and in vivo experiments revealed that Met and Gce transduce JH signals to induce Kr-h1 expression through the JHRR. DNA affinity purification identified the chaperone protein Hsp83 as one of the proteins bound to the JHRR in the presence of JH. Interestingly, Hsp83 physically interacts with the PAS-B and bHLH domains of Met, and JH induces Met-Hsp83 interaction. As determined by immunohistochemistry, Met is mainly distributed in the cytoplasm of the larval fat body cells when the JH titer is low and JH induces Met nuclear import. Hsp83 was also accumulated in the cytoplasm area adjunct to the nucleus in the presence of JH and Met/Gce. Loss-of-function of Hsp83 attenuates JH binding and JH-induced nuclear import of Met, resulting in a decrease in the JHRR-driven reporter activity leading to the reduction of Kr-h1 expression. These data show that Hsp83 facilitates the JH-induced nuclear import of Met that induces Kr-h1 expression through the JHRR (He, 2014).
Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).
This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).
LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).
What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).
Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).
This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012).
It was somewhat surprising that the sgt1 pins double mutants had increased numbers of brain neuroblasts, because each single mutant had reduced neuroblast numbers. The double mutant phenotype may be due to loss of both Pins and cortical Dlg/Scrib, as the sgt1 pins double mutant phenotype is similar to the dlg pins double mutant phenotype. It could also be due to a change in an unknown downstream effector of both Sgt1 and Pins. A not mutually exclusive possibility is that the sgt1 pins double mutant phenotype is due to loss of all Par/Pins cortical polarity. This model is consistent with the observation that sgt1 or pins single mutants retain some neuroblast cortical polarity, whereas the sgt1 pins double mutants lack all known neuroblast cortical polarity. It is proposed that the apolar double mutant neuroblasts partition cell fate determinants equally to both siblings, and that both siblings frequently assume a neuroblast identity. This is supported by the recent finding that when the neuroblast spindle is aligned orthogonal to a normal apical/basal polarity axis, such that both siblings inherit equal amounts of apical cortical proteins, the siblings always acquire a neuroblast identity. Thus, equal partitioning of apical/basal cell fate determinants (in spindle orientation mutants) or failure to establish any cortical polarity (sgt1 pins mutants) may result in neuroblast/neuroblast siblings and an expansion of the neuroblast population (Anderson, 2012).
Canalization, also known as developmental robustness, describes an organism's ability to produce the same phenotype despite genotypic variations and environmental influences. In Drosophila, Hsp90, the trithorax-group proteins and transposon silencing have been previously implicated in canalization. Despite this, the molecular mechanism underlying canalization remains elusive. Using a Drosophila eye-outgrowth assay sensitized by the dominant Kr(irregular facets-1)(Kr(If-1)) allele, this study shows that the Piwi-interacting RNA (piRNA) pathway, but not the short interfering RNA or micro RNA pathway, is involved in canalization. Furthermore, a protein complex was isolated composed of Hsp90, Piwi and Hsp70/Hsp90 organizing protein homolog (Hop) and the function of this complex in canalization was demonstrated. The data indicate that Hsp90 and Hop regulate the piRNA pathway through Piwi to mediate canalization. Moreover, they point to epigenetic silencing of the expression of existing genetic variants and the suppression of transposon-induced new genetic variation as two major mechanisms underlying piRNA pathway-mediated canalization (Gangaraju, 2011).
The mechanism of canalization has been a subject of great debate. The findings of Rutherford (1998) indicate that Hsp90 acts as a capacitor for phenotypic variation; however, a complex gene network model generated by Bergman (2003) predicts that a mutation in any one gene can result in expression of cryptic genotypes. Yet another report argues that expression of cryptic genotypes is not caused by canalization and no particular mechanism is needed to prevent expression of the cryptic phenotypes. The finding of piwi and Hop mutations as enhancers for expression of cryptic genotypes validates the existence of a piRNA-pathway dependent mechanism for preventing phenotypic variation. Piwi is a piRNA-binding protein that is required for silencing of transposons and epigenetic regulation. Hence, post-translational regulation of Piwi by Hsp90 and Hop may allow Piwi both suppress the generation of new genotypes and epigenetically silence the expression of existing genetic variants. Both mechanisms can be 'fixed' and inherited in subsequent generations (Gangaraju, 2011).
This study also shows that Piwi acts at two distinct phases of fly development in mediating phenotypic capacitance. First, maternal Piwi plays a direct role in canalization and/or suppresses transposon-induced mutagenesis during embryogenesis. This allows the inheritance of correct epigenetic and genetic codes from parental cells to daughter cells, thereby ensuring the robustness of the developmental programs. Subsequently, zygotic Piwi is required for maintaining the inherited developmental programs during subsequent stages of development. This Piwi function likely represents the piRNA pathway, since Aubergine, a Piwi homolog also involved in the piRNA pathway, has similar function. Furthermore, canalization appears to involve only the piRNA pathway, but not miRNA or siRNA pathway, since Dicer 1 and 2 deficiency does not lead to increased eye outgrowth (Gangaraju, 2011).
Then, what could be the roles for Hsp90 and Hop in Piwi-mediated canalization? As an essential component in canalization, Hsp90 likely ensures proper function of its clients involved in canalization, such as Piwi, by mediating their proper post-translational modification, such as phosphorylation, that is required for their molecular activities. A salient feature of Hsp90-mediated chaperoning, unlike Hsp70, is that Hsp90 predominantly binds to metastable states of proteins instead of hydrophobic stretches (Sangster, 2004). The Hsp90-bound metastable state of Piwi may be a necessary step for its phosphorylation at proper sites, which may then be required to form active complexes with piRNAs and/or epigenetic factors to promote epigenetic and transposon silencing, leading to canalization (Gangaraju, 2011).
Triosephosphate isomerase (TPI) deficiency is a severe glycolytic enzymopathy that causes progressive locomotor impairment and neurodegeneration, susceptibility to infection, and premature death. The recessive missense TPIsugarkill mutation in Drosophila melanogaster exhibits phenotypes analogous to human TPI deficiency such as progressive locomotor impairment, neurodegeneration, and reduced life span. This study shows that the TPIsugarkill protein is an active stable dimer; however, the mutant protein is turned over by the proteasome reducing cellular levels of this glycolytic enzyme. As proteasome function is often coupled with molecular chaperone activity, it is hypothesized that TPIsugarkill is recognized by molecular chaperones that mediate the proteasomal degradation of the mutant protein. Coimmunoprecipitation data and analyses of TPIsugarkill turnover in animals with reduced or enhanced molecular chaperone activity indicate that both Hsp90 and Hsp70 are important for targeting TPIsugarkill for degradation. Furthermore, molecular chaperone and proteasome activity modified by pharmacological or genetic manipulations resulted in improved TPIsugarkill protein levels and rescue some but not all of the disease phenotypes suggesting that TPI deficiency pathology is complex. Overall, these data demonstrate a surprising role for Hsp70 and Hsp90 in the progression of neural dysfunction associated with TPI deficiency (Hrizo, 2010).
Studies of TPI deficiency disease pathogenesis have previously focused on dimer stability and catalytic activity of the mutant enzyme. Several labs have shown that some, but not all of the human disease causing TPI alleles, have reduced dimerization and isomerase function using in vitro assays. However, previously published data suggests that TPIsugarkill dimer stability is not compromised. As the TPIsugarkill protein does not appear to aggregate and increased levels of the TPIsugarkill protein rescues the mutant phenotypes, these data cumulatively suggest that the mutant protein can conform to a functional shape and likely retains significant activity (Hrizo, 2010 and references therein).
Hsp70 and Hsp90 are molecular chaperones that have been implicated in the progression and amelioration of other neurodegenerative diseases. However, TPIsugarkill is a unique cytosolic model protein for the study of these chaperones and their role in mutant protein turnover and disease pathogenesis as it is a soluble protein that is not aggregation prone. Other neurodegenerative models can be rescued with increased activity of the molecular chaperones as they help target the misfolded protein for degradation before toxic cellular protein aggregates can form. The role of Hsp90 and Hsp70 in TPIsugarkill pathogenesis is distinct from the previously mentioned examples, as decreased chaperone activity reduces pathogenesis for some mutant phenotypes and TPIsugarkill protein does not appear to cause the toxic cellular aggregates observed in other neurodegenerative diseases. In the case of TPIsugarkill the molecular chaperones are targeting a protein for degradation before it can function within the cell, thus reducing the function of the enzyme and contributing to the disease states. TPI is folded in the cytoplasm and previous labs have studied TPI protein folding rates in vitro and have found them to be rather rapid. The mechanism of chaperone identification and targeting of TPIsugarkill for degradation is not known. For example, the cytosolic TPIsugarkill protein may be recognized by molecular chaperones and targeted for degradation due to a slower folding rate than the wildtype protein, as is the case with the ER secretory CFTRΔF508 mutant protein. Overall, while other studies have shown that up-regulation of molecular chaperones may prove beneficial for reducing neurodegeneration caused by formation of toxic aggregates, the current study corresponds with previously published work that also suggests that excessive up-regulation or unregulated chaperone and proteasome activity may lead to undesirable side-effects and that a balance of chaperone and proteasome activity may be required for neuronal function and health (Hrizo, 2010).
This study observed that modulation of molecular chaperone activity alone is not sufficient to restore TPIsugarkill protein to wildtype levels. The results are in line with what is expected for modest hypomorphic or increased expression conditions examined. Transgenic overexpression of mutant TPIsugarkill protein also rescued TPIsugarkill phenotypes, consistent with the interpretation that the mutant protein retains function. Thus far any means examined of altering steady state TPIsugarkill protein, either by increasing rate of synthesis or decreasing proteasomal-dependent degradation, results in the predicted affect on TPIsugarkill phenotypes. These data demonstrate that TPIsugarkill degradation is not a protective mechanism, as has been seen with aggregation prone protein models, but rather leads to a loss of functional protein that underlies pathogenesis (Hrizo, 2010).
While the proteasome and molecular chaperones have been shown to be involved in TPIsugarkill turnover, it is not clear how TPIsugarkill is recognized and targeted for degradation. The majority but not all proteasomal-targeted proteins are polyubiquitinated prior to recognition and subsequent degradation by the proteasome. Further studies will be needed to clarify the recognition and targeting mechanisms (Hrizo, 2010).
Overall, these experiments suggest that TPIsugarkill retains activity and that by modulating TPI turnover disease pathogenesis can be altered. In addition, a new mutant substrate that interacts with molecular chaperones and is degraded by the proteasome has been identified for study. Previous studies of cytosolic protein targeting for protein degradation have focused on ER proteins and exogenous mutant proteins such as VHL tumor suppressor. TPIsugarkill is a unique protein quality control substrate, the further study of which will yield important insight into the mechanisms of identification and targeting of cytosolic proteasomal substrates (Hrizo, 2010).
The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins: Pih1D1, RPAP3 and the AAA+ ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, snoRNP (small nucleolar RiboNucleoParticles) and PIKK (Phosphatidyl-Inositol 3-Kinase-related Kinases). This study characterized the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, it was shown that spaghetti is necessary for (1) the stabilization of snoRNP core proteins, (2) TOR (Target Of Rapamycin) activity, and likely, the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients, and further shows that Spag, unlike S. cerevisae Tah1, performs essential functions in Metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90, to facilitate their assembly into macro-molecular complexes (Benbahouche, 2014).
The canalization concept describes the resistance of a developmental process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways, indicate that it is a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. This study offers an additional, perhaps alternative, explanation for proposals of a concrete mechanism underlying canalization. This study shows that, in Drosophila, functional alterations of Hsp90 affect the Piwi-interacting RNA (piRNA; a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the induction of morphological mutants. This indicates that Hsp90 mutations can generate new variation by transposon-mediated 'canonical' mutagenesis (Specchia, 2010).
In Drosophila, primary spermatocytes of Hsp90 mutant males exhibit crystalline aggregates, usually absent in wild-type testes, the formation of which is due to the transcriptional activation, in male germ line, of the repeated Stellate (Ste)
The silencing of the Stellate sequences is mediated by RNA interference (RNAi) mechanism and mutations in genes involved in RNAi, such as aubergine, armitage and spindle E (also called homeless), activate Stellate in testes of mutant males. It has been shown that Stellate is repressed by a piRNA-mediated mechanism that is specific for repetitive sequences and transposon silencing. Consistently, it was found that Hsp90 mutations affect the biogenesis of piRNAs specific for Stellate and transposons. These results prompted a test, in ovaries and testes, for possible effects of Hsp90 mutations on the expression of long terminal repeat (LTR) springer, opus, roo and aurora, non-LTR I elements that transpose by an RNA intermediate, and invert repeat (IR) Bari1, an element that transposes by a DNA intermediate. Homozygous hsp83scratch and trans-heterozygous hsp83scratch/hsp83e4A mutants were analyzed. Hsp83 is the denomination of Hsp90 in Drosophila; hsp83scratch is a viable hypomorphic male sterile mutation and hsp83e4A is a lethal amorphic mutation. The expression of the same transposons was also tested in ovaries and testes of wild-type Oregon-R (Ore-R) flies treated with geldanamycin. In mutant ovaries and testes the amount of transcripts increases significantly, although differentially, for all the transposons. The increase is more abundant in trans-heterozygous than in homozygous mutants and in ovaries than in testes. Treatment with geldanamycin induces a significant increase of all the transposon transcripts, except for the I element, but only in testes (Specchia, 2010).
Southern blot analysis was used to look at the effect of Hsp90 mutations on transposon mobility. To compare genomes homogeneously, DNA was extracted from single heterozygous flies (the parents) and from single homozygous flies of F1 progeny (males and females); all the DNA genomic samples were digested with HindIII. Hybridization patterns were examined with aurora, I and Bari1, where significant differences between parents and F1 progeny are evident for each element. New bands in the DNA of the F1 progeny were found compared to the parental DNA, thus suggesting a mutation-induced mobilization of these elements. Similar results were obtained with springer, opus and roo. To ensure that the effects on transposons are due exclusively to the homozygosis of the hsp83scratch mutation and not to the genetic background of the mutagenized III chromosome, the same experiments were done with aurora, I and Bari1 in trans-heterozygous hsp83scratch/hsp83e4A mutants with similar results. In addition, no significant mobilization or increased transcription of the same elements were found in a homozygous hsp83rev22 revertant strain (Specchia, 2010).
Because active transposons are mutagenic, these data suggest that the phenotypic variation observed in Hsp90 mutants could be due to de novo mutations produced by activated transposons. To test this possibility, 3,220 hsp83scratch homozygous flies were screened and 30 flies were found with morphological abnormalities, corresponding to a frequency of about 1%. This is similar to the frequency previously reported in some mutant stocks. Also 3,220 flies from an Ore-R stock were analyzed without finding any morphological abnormalities. Among the abnormalities observed, there was a fly resembling the dominant mutation Scutoid. Because the Scutoid phenotype is caused by mutations in the no ocelli (noc) gene, this gene was molecularly analysed in the phenotypic variant. Two pairs of primers were chosed that in the wild-type gene amplify two DNA fragments of 2,100 bp and 3,400 bp. The 2,100-bp fragment is present in both the Sco-like mutant and in Ore-R, whereas the 3,400-bp fragment is detected only in the wild type. Inverse polymerase chain reaction (PCR) analysis showed that the noc gene is interrupted at nucleotide 1394 of the cDNA sequence by an I element-like sequence. Consistent with this observation is the amplification of a DNA fragment obtained by using an upper primer (the position of the noc gene) and a lower primer corresponding to the I element at the 1243 nucleotide of the M14954 sequence. After sequencing it was found that this fragment encodes a Noc truncated protein, which cannot function as a transcriptional factor owing to the loss of the zinc-finger domain. The noc sequence was also analyzed in the DNA extracted from about 1,000 phenotypically wild-type flies collected during the screening, and only the normal noc sequence was found. This indicates that the Scutoid phenotype that was found was caused by a de novo mutation instead of being the expression of a pre-existing cryptic mutation. The impairment of piRNA biogenesis by Hsp83 indicated that other mutations affecting transposon activity might also induce phenotypic variation. To test this possibility, a screen was made for morphological variants in a stock carrying spindle Ec00786, a male sterile mutation at the spindle E gene. It has helicase activity, de-represses Stellate sequences and transposon, and is involved in piRNA biogenesis. 1,500 flies were screened, and 19 morphological variants were found, a similar rate to that found in some Hsp90 mutants strains (1%-2%). The transposons are activated in the germ line of male and female spindle E mutants. This further indicates that the expression of morphological variability could be related to the disruption of the piRNA silencing mechanism (Specchia, 2010).
These data clearly show a novel function of Hsp90 with important implications for the current hypothesis about this protein as a capacitor of morphological evolution. Hsp90 is involved in stress responses and the expression of wide ranging morphological changes in Drosophila and other organisms. A reduced amount of Hsp90 can induce abnormal developmental phenotypic variations; these morphogenetic changes can become fixed and stably transmitted even if wild-type Hsp90 function is restored in subsequent generations. The current interpretation is that Hsp90 buffers pre-existing genetic variation that is not expressed and accumulates in neutral conditions; its mutations will then induce the expression of this variation (Rutherford, 1998). This stress-sensitive storage and release of genetic variation by Hsp90 would favour adaptive evolution. The hypothesis of pre-existing genetic variation has been based on the observation that, when flies carrying Hsp83 alleles were outcrossed to different laboratory stocks, the observed defects were typical for each outcross, thus suggesting that the defects depended on specific genetic backgrounds (Rutherford, 1998). Those data could be also explained in the light of the current results. There is evidence that different genetic backgrounds may induce different transposon insertions (Specchia, 2010).
The demonstration that Hsp90 is involved in the control of transcription and mobilization of transposable elements in germ cells by affecting piRNA biogenesis strongly suggests that the reduction of Hsp90 causes a stress-response-like activation and transposition of mobile elements affecting piRNA silencing. This in turn would induce de novo gene mutations that affect development pathways and that can be expressed and fixed across subsequent generations. This explanation agrees with the suggestion that transposable element activity could be a response to stress. The results do not exclude, however, that Hsp90 could be both a buffering factor as well as a suppressor of transposable element (TE)-induced mutations (Specchia, 2010).
These data indicate an additional, if not alternative, mechanism to the canalization and assimilation hypothesis based on a link between stress and transposon activity through piRNA-mediated silencing. This mechanism also potentially provides another molecular interpretation with respect to the vague capacitor concept. It is not yet known if Hsp90 also has a direct role in piRNA biogenesis, or is only involved in triggering the stress response leading to transposon activation (Specchia, 2010).
Heat-shock proteins (Hsps) are molecular chaperones that control protein folding and function. Argonaute 2 (Ago2), the effector in RNA interference (RNAi), is associated with Hsp90; however, its function in RNAi remains elusive. This study shows that Hsp90 is required for Ago2 to receive the small interfering RNA (siRNA) duplex from the RNA-induced silencing complex-loading complex in RNAi, suggesting a model where Hsp90 modifies Ago2 conformation to accommodate the siRNA duplex (Miyoshi, 2010).
In RNA interference (RNAi), small interfering RNA (siRNA) is associated with Argonaute 2 (Ago2) and guides the protein to its target mRNAs for silencing. In Drosophila melanogaster, siRNAs are processed from long double-stranded RNA precursors by Dicer2. Upon processing, siRNAs are still in a duplex form and are associated with Dicer2 and R2D2, which are major components of the RNA-induced silencing complex (RISC)-loading complex (RLC). siRNA duplexes are then transferred to Ago2 from the RLC to form the precursor of the RISC, or pre-RISC. It has been shown that pre-RISC formation occurs in an ATP-dependent manner. However, it remains unclear why ATP is needed for this process (Miyoshi, 2010).
In the pre-RISC, siRNA duplexes are 'unwound' by Ago2 endonuclease or Slicer activity; the passenger strand of the duplexes is cleaved by Slicer and displaced from Ago2. This step is known to occur in an ATP-independent manner. The guide strand of the duplex remains associated with Ago2. The resultant Ago2-siRNA complex is termed RISC and is now active and ready to bind and cleave target RNAs. In this way, genes targeted by the RNAi machinery are effectively silenced. Ago2 frequently co-purifies with heat-shock protein (Hsp) 90, a chaperone whose activity depends on ATP. Thus, Hsp90 may have a role in RNAi. However, the functional involvement of Hsp90 at the molecular level in RNAi remains unclear (Miyoshi, 2010).
To determine for which step Hsp90 is required in RNAi, the RNAi pathway was dissected into multiple steps and assays were performed for the individual steps in the presence or absence of geldanamycin, the specific Hsp90 inhibitor that mimics ATP binding with the protein. In siRNA processing assays, Flag-tagged Dicer2 isolated from S2 cells was incubated with 32P-labeled dsRNA precursors with or without geldanamycin. The ability of Dicer2 to excise siRNA duplexes from the precursors was not affected by geldanamycin. Thus, Hsp90 is not necessary for siRNA excision from the precursor (Miyoshi, 2010).
Next, whether the siRNA-unwinding step in RNAi requires Hsp90 function was examined. siRNA duplexes, which were labeled with 32P at the 5′ end of the guide siRNA within the duplexes, was incubated in S2 lysates with or without geldanamycin. In the presence of geldanamycin, siRNA duplexes remained as duplexes even after 1 h incubation. DMSO, an organic solvent used for dissolving geldanamycin in the lysates, did not affect the activity. Thus, Hsp90 is required for unwinding siRNA duplexes into single-stranded siRNAs (Miyoshi, 2010).
It was then speculated that the RISC would not be assembled in S2 lysates when Hsp90 was inhibited. To test this, RISC formation assays were performed. Whereas DMSO alone did not show any obvious effect, geldanamycin severely inhibited RISC formation. RLC formation was not affected by geldanamycin treatment. These results indicate that Hsp90 has an important role in the particular step(s) necessary for converting RLC into RISC. A recent study has shown that geldanamycin significantly reduces the levels of Argonaute proteins in mammalian cells and thus reduces the programming of the RISC. However, the stability of Ago2 was barely changed in S2 lysates even after geldanamycin was added. Thus, the reduced RISC-forming activity was likely not due to reduced Ago2 stability (Miyoshi, 2010).
siRNA duplexes remained as duplexes in the presence of geldanamycin, implying that geldanamycin inhibits Ago2 Slicer activity. To test this, target RNA cleavage assays were performed. After expressing Flag-tagged Ago2 or Flag-tagged enhanced green fluorescent protein (EGFP) in S2 cells, the cells were lysed and the RISC in the lysates was assembled by adding siRNA duplexes. Flag-Ago2 and Flag-EGFP were isolated from the lysates and target RNAs were added to the complexes in the presence or absence of geldanamycin. The Flag-tagged Ago2 complex cleaved the target RNA in both cases. Thus, Hsp90 was not required for Ago2 to cleave the target RNAs once a functional RISC was properly formed. An earlier report showed that Hsp90 inhibition impairs RNAi in Drosophila ovary extracts (Pare, 2009). However, in these experiments, siRNAs and geldanamycin were added simultaneously to the lysates to examine the inhibitory effect of the functional loss of Hsp90 (Miyoshi, 2010).
Conversion of the RLC into the pre-RISC requires siRNA duplexes to be transferred from the RLC to Ago2. At this step, Dicer2, the main component of the RLC, should interact with Ago2, although this interaction may be transient. Thus,whether inhibition of Hsp90 activity interferes with the association between Dicer2 and Ago2 was examined. Flag-tagged Ago2 interacted with Dicer2 similarly either with or without geldanamycin. This indicated that, at least in Drosophila cells Hsp90 has little or no effect on the Dicer2-Ago2 association. This is in contrast to mammalian cells, where Hsp90 activity was required for the Argonaute-Dicer interaction (Miyoshi, 2010).
Two models regarding Hsp90 function in RNAi can now be proposed: the first is that Hsp90 functions in displacing the siRNA duplex from the RLC, whereas the second is that Hsp90 is required for Ago2 to receive the siRNA duplex from the RLC, after the siRNA duplex has been properly displaced from the RLC. To examine which model is correct, siRNA-protein interaction experiments were performed using three siRNA duplexes, siRNA1, siRNA2 and siRNA3, each of which was composed of the same sequence but contained an iodine-uridine at different positions within the duplex. siRNA duplexes individually incubated in S2 lysates for different time periods and then the mixtures were exposed to UV light to cross-link the siRNA duplexes with the proteins physically associated with them. Previous studies have indicated that Dicer2 and R2D2, the second known component of the RLC, are mainly cross-linked at the 3' end of the guide and passenger strands of the duplex, respectively. Ago2 was shown to be cross-linked predominantly at the 5' end of the guide strand of the duplex. The association of R2D2 and Dicer2 with siRNA1 and siRNA2, respectively, was not significantly altered by Hsp90 inhibition. However, Hsp90 inhibition drastically affected siRNA duplex association with Ago2; in the presence of geldanamycin, Ago2 barely interacted with siRNA3. The inhibitory effect was observed when geldanamycin was added at a concentration of 10 μM or higher. Radicicol, another Hsp90 inhibitor, also interfered with siRNA duplex association with Ago2. A siRNA duplex mutant (siRNA3 mutant), in which the passenger strand was modified with 2'-O-methyl groups at the 9th and 10th nucleotides from the 5' end and thus interferes with Ago2 Slicer activity, behaved similarly to siRNA3. In addition, it was shown that Hsp90 was present in immunoprecipitated Ago2 complex. These results suggest that Hsp90 is required for Ago2 to receive siRNA duplexes from the RLC in the RNAi pathway. In siRNA-Ago2 binding assays, the guide siRNAs were barely associated with Ago2 when geldanamycin was added first to the lysates prior to the addition of the siRNA duplexes. These results further support the idea that Hsp90 is needed for Ago2 to bind the siRNA duplex. Structural analysis of the eubacterial Ago protein has revealed that the cavity in the Ago protein that accommodates the siRNA duplex is too small to be bound by an RNA molecule, and a conformational change in the Ago protein would be required for binding. In the current study, the results suggest that Hsp90 acts as the driving force for changing the conformation of Ago2, likely by hydolyzing ATP as an energy source, as geldanamycin is known to inhibit the ATPase activity of Hsp90 by occupying the N-terminal ATP binding pocket of the protein, enabling it to accommodate siRNA duplex from the RLC . This step is crucial in RNAi, and so, without Hsp90, the pre-RISC is not formed and RNAi is impaired (Miyoshi, 2010).
Molecular chaperone heat-shock protein 90 kDa (Hsp90) is known to facilitate the conformational maturation of a diverse range of proteins involved in different signal transduction pathways during development. Recent studies have implicated Hsp90 in transcriptional regulation and an important role for Hsp90 in epigenetic processes has been proposed. Importantly, genetic and pharmacological perturbation of Hsp90 was shown to reveal heritable phenotypic variation and Hsp90 was found to play an important role in buffering genetic and epigenetic variation whose expression led to altered phenotypes. The underlying molecular mechanism remains elusive, however. This study shows a direct molecular interaction between Hsp90 and Trithorax (Trx). Trx is a member of the TrxG chromatin proteins controlling, together with the members of the Polycomb group, the developmental fate of cells by modulating epigenetic signals. Hsp90 cooperates with Trx at chromatin for maintaining the active expression state of targets like the Hox genes. Pharmacological inhibition of Hsp90 results in degradation of Trx and a concomitant down-regulation of homeotic gene expression. A similar effect is observed with the human orthologue mixed-lineage leukemia. Connecting an epigenetic network controlling major developmental and cellular pathways with a system sensing external cues may explain the rapid fixation and epigenetic inheritance of phenotypic variation as a result of impaired Hsp90 (Tariq, 2009).
This study has established a well-defined link between Hsp90 and the TrxG by demonstrating the genetic and molecular interaction between Hsp90 and Trx that is required for maintaining active gene expression in Drosophila. Evidence is provided that genetic manipulation or pharmacological inhibition of Hsp90 strongly affects Trx target genes and results in their reduced expression because of depletion of Trx. This illustrates that Hsp90 facilitates Trx in maintaining active gene expression indicates how epigenetic gene expression maintained by the TrxG can be quickly modulated (Tariq, 2009).
Ectopic activation of homeotic genes in PcG mutants relies on the TrxG, leading to morphological transformations. Appearance of additional sex combs on the first tarsal segment of the second and third leg pairs in Pc heterozygous males is one of such transformations. However, a reduced dose of TrxG proteins counteracts the reduced dose of Pc, restoring normal regulation of homeotic genes and suppressing the sex comb phenotype. Strong suppression of extra sex combs phenotype by Hsp90 mutants illustrates that Hsp90 interacts with the TrxG in counteracting silencing maintained by PcG genes. In corroboration, genetic or pharmacological manipulation of Hsp90 resulted in reduced Ubx and Dfd gene expression whose active expression is maintained by Trx. A molecular link between Hsp90 and Trx was validated by the evidence that pharmacological inhibition of Hsp90 specifically leads to depletion of Trx, which alleviates chromatin association of Trx and consequently affects expression of Dfd within 4 h of drug treatment. This provides strong evidence for a rapid Hsp90-dependent transcriptional modulation of active chromatin (Tariq, 2009).
There is convincing evidence that Hsp90 regulates transcription processes (Freeman, 2002; Hung, 2005; Floer, 2008) and involvement of Hsp90 in epigenetic processes has been proposed (Zhao, 2005; Sollars, 2003). However, molecular evidence for involvement of Hsp90 in heritable transcriptional regulation remained elusive. For example, the role of Hsp90 in steroid hormone receptor-mediated transcription activation is well characterized (Picard, 1990; Kimura, 1995). In particular, Hsp90 is known to hold glucocorticoid receptors (GR) in a conformation competent for ligand binding and activation of transcription. Importantly, Hsp90 is also proposed to interact with GR at chromatin where it facilitates the dissociation of GR from its targets and recycles the ligand-free GR back to chromatin in an Hsp90-dependent chaperone cycle (Stavreva, 2004; DeFranco, 2000). Inhibition of Hsp90 is known to severely impair GR-mediated gene activation (Picard, 1990). Evidence for association of Hsp90 with Trx described in this study now strengthens a recently proposed role for Hsp90 in epigenetic processes where molecular evidence was lacking. The mechanistic link between Hsp90 and Trx may be similar to a reported interaction of Hsp90 with a histone H3 lysine 4-specific methyltransferase, SMYD3, which is up-regulated in cancer cells. In colorectal and hepatocellular carcinoma cells, SMYD3 was shown to interact with Hsp90, which dramatically enhanced catalytic activity of SMYD3 in vitro. Importantly, pharmacological inhibition of Hsp90 also significantly reduced SMYD3-mediated activation of a homeobox gene (Hamamoto, 2004). The consequential effect of restrained Hsp90-SMYD3 association on homeobox genes appears comparable to reduced Dfd expression because of impaired Hsp90 activity described in this study. In mammals, Hsp90 was also shown to regulate the activity and stability of tumor suppressor P53 under physiological and elevated temperatures (Walerych, 2004). After heat shock, Hsp90 was found to associate with Trx at chromatin in polytene chromosomes, which may indicate that Hsp90 ensures stability and activity of Trx not only under normal conditions but also in times of stress. Because heat-shock puffs visualize active transcription, it also indicates that Hsp90 may facilitate Trx in maintaining active gene expression, which is also validated by the presence of Hsp90 at the active Abd-B gene in SF4 cells. The overlap between Hsp90 and Trx in non-heat-shocked chromosomes is limited to interband regions, which are also often active sites of transcription. The lack of overlap on some developmental puffs might be a sensitivity problem, because these are expressed at a lower level than the heat-shock genes. A non-chromatin-bound role of Hsp90 in stabilizing Trx and subsequently affecting gene expression cannot be ruled out because Hsp90 was also found to be prevalent in the nucleoplasm (Muller, 2004). The molecular interaction between Hsp90 and Trx could be analogous to the GR–Hsp90 partnership. Hsp90 may hold Trx in a stable, ready to be activated state before Trx receives a signal for gene activation. Further, Hsp90 may also play a crucial role in recycling Trx proteins, which may undergo a dynamic process of association and dissociation at their target sites. It is plausible to envisage that Hsp90-stabilized Trx may have a higher affinity for its interacting partners in chromatin-associated or soluble TAC1 complexes (Tariq, 2009).
This study has uncovered how Hsp90 can directly influence an important, evolutionary highly conserved epigenetic network that supervises the appropriate cellular identities during development and homoeostasis of an organism. Because PcG and TrxG gene control has such a fundamental and evolutionary conserved role, its modulation by factors influenced by external signals could have substantial implications for the process of transcriptional memory and thus on phenotype. Interplay between Hsp90 and Trx supports the notion that chromatin regulators play an important role in developmental canalization and signifies the likelihood that different canalization factors may be linked to further ensure developmental stability. Because of the reversible nature of epigenetic processes, Hsp90- and Trx-mediated switches may enable cells and organisms to adapt to environmental conditions more easily. The observed phenotypic variation in Drosophila because of genetic and environmental manipulation of Hps90 can also be explained as a consequence of reduction in Hsp90-Trx interactions affecting homeotic and other developmental genes. However, PcG and TrxG proteins not only maintain gene expression patterns heritable during mitosis but also can in certain conditions transmit epigenetically controlled states through meiosis to the next generations. Thus, Hsp90-dependent phenotypic variations might also become fixed and inherited through the action of the PcG/TrxG system providing an epigenetic basis for transgenerational inheritance of Hsp90-dependent traits. The notion that Hsp90-dependent phenotypic variations have a combined genetic and epigenetic origin is supported by this study, and it is suggested that epigenetic inheritance mediated by TrxG or PcG proteins may significantly contribute in rapid fixation of heritable phenotypes (Tariq, 2009).
In metazoa, the spatio-temporal translation of diverse mRNAs is essential to guarantee proper oocyte maturation and early embryogenesis. The eukaryotic translation initiation factor 4E (eIF4E), which binds the 5' cap structure of eukaryotic mRNAs, associates with either stimulatory or inhibitory factors to modulate protein synthesis. In order to identify novel factors that might act at the translational level during Drosophila oogenesis, a functional proteomic approach was undertaken, and the product of the Hsp83 gene, the evolutionarily conserved chaperone Hsp90, was isolated as a specific component of the cap-binding complex. This study reports that Hsp90 interacts in vitro with the translational repressor Cup. In addition, it was shown that Hsp83 and cup interact genetically, since lowering Hsp90 activity enhances the oogenesis alterations linked to diverse cup mutant alleles. Hsp90 and Cup co-localize in the cytoplasm of the developing germ-line cells within the germarium, thus suggesting a common function from the earliest stages of oogenesis. Taken together, these data start elucidating the role of Hsp90 during Drosophila female germ-line development and strengthen the idea that Cup has multiple essential functions during egg chamber development (Pisa, 2009).
In order to discover novel proteins involved in translational regulation during Drosophila oogenesis, a cap-binding analysis was performed. Whole protein extracts from adult ovaries were used in affinity chromatography on m7GTP-Sepharose beads and approximately 30 candidates were isolated. These proteins were separated by SDS/PAGE and identified by liquid chromatography-tandem mass spectrometry (LC-MSMS) (Pisa, 2009).
Several already known components of the active cap-binding complex could be detected, namely: eukaryotic translation initiation factor 4E (eIF4E-1 and eIF4E-2; eukaryotic translation initiation factor 4G (eIF4G); Poly(A) binding protein (PABP); eukaryotic translation elongation factor 2b (EF-2); Suppressor of variegation 3-9 (eIF-2γ); eukaryotic translation initiation factor 2α (eIF-2α); Trip1 (eIF-3β); Ribosomal protein S3 (RpS3); Ribosomal protein S3A (RpS3A); Ribosomal protein L5 (RpL5). Cap-binding protein 80 (Cbp80), which appears to replace eIF4E for binding to the cap structure during pre-mRNA maturation and nonsense-mediated mRNA decay processes, was also isolated (Pisa, 2009).
Specific factors were identified whose involvement in translational repression mechanisms and/or localization of select mRNAs, during egg chamber development, had been either previously demonstrated or predicted: Cup; Maternal expression at 31B (Me31B); Trailer hitch (Tral); Ypsilon schachtel (Yps); TER94; Rasputin; Heterogeneous nuclear ribonucleoprotein at 87F (Hrb87F); Heterogeneous nuclear ribonucleoprotein at 98DE (Hrb98DE) (Pisa, 2009).
Furthermore, three members of the heat shock protein superfamily were identified: Glycoprotein 93 (Gp93; homolog to human Endoplasmin, a member of the Hsp90 family); Heat shock protein 83 (Hsp83, hereafter called Hsp90); and Heat shock protein cognate 4 (Hsc70-4) (Pisa, 2009).
The biochemical and genetic interactions observed between Hsp90 and Cup suggest that the protein complex formed by these two proteins might mediate localization/translation of select mRNAs during egg chamber development. This hypothesis is corroborated by the finding that Hsp90 is required for mRNA localization during embryogenesis (Song, 2007). However, it cannot be excluded that Hsp90 could also act as a hub molecular chaperone contributing to the proper structure and/or function of other proteins involved in localization/translation of specific mRNAs during oogenesis (Pisa, 2009).
Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs' 5' untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species (Duncan, 2008).
During mitosis, cyclin B is extremely dynamic and although it is concentrated at the centrosomes and spindle microtubules (MTs) in organisms ranging from yeast to humans, the mechanisms that determine its localisation are poorly understood. To understand how cyclin B is targeted to different locations in the cell, proteins were isolated that interact with cyclin B in Drosophila embryo extracts. Cyclin B interacts with the molecular chaperone Hsp90 and with the MT-associated protein (MAP) Mini spindles. Both Hsp90 and Msps are concentrated at centrosomes and spindles, and Hsp90, but not Msps, is required for the efficient localisation of cyclin B to these structures. Unlike what happens with other cell cycle proteins, Hsp90 is not required to stabilise cyclin B or Msps during mitosis. Thus, it is proposed that Hsp90 plays a novel role in regulating the localisation of cyclin B and Msps during mitosis (Basto, 2007; full text of article).
How might Hsp90 function in recruiting cyclin B to centrosomes and spindles? As Hsp90 is itself located at centrosomes and can bind to tubulin, it is possible that Hsp90 binds cyclin B and directly targets it to these locations. It is suspected that this is not how Hsp90 targets cyclin B to MTs, since only a small fraction of the total cyclin B is bound to Hsp90 in embryo extracts. Virtually all of the cyclin B in an embryo extract is capable of binding to MTs in MT spin-down experiments. Thus, it seems unlikely that Hsp90 could act as an essential co-factor that directly mediates the interaction between cyclin B and MTs. Similarly, it is suspected that Hsp90 does not directly target cyclin B to centrosomes, since the initial recruitment of cyclin B to centrosomes during prophase is only mildly disrupted in Drosophila cells (in HeLa prophase recruitment is not disrupted) when Hsp90 has been perturbed. Rather, Hsp90 seems to be involved in maintaining the centrosomal localisation of cyclin B during prometaphase and metaphase. Perhaps Hsp90 is essential for the proper folding or function of a specific domain of cyclin B that is required for the localisation of cyclin B on centrosomes and spindles. In such a scenario Hsp90 could even act indirectly to allow cyclin B to associate with other proteins that target it to centrosomes and MTs (Basto, 2007).
If the assumption that Hsp90 does not directly target cyclin B to centrosomes or MTs is correct, it raises the intriguing question of what targets cyclin B to these locations? It has previously been shown that cyclin B can interact with XMAP215 and it has been proposed that this interaction could target cyclin B to centrosomes and MTs. It was also found that cyclin B can interact with Msps/XMAP215 in Drosophila embryo extracts, suggesting that this interaction is conserved between frogs and flies. In msps mutant cells, or in human cells partially depleted of ch-TOG, however, it was found that cyclin B was still localised to centrosomes and MTs. Thus, it is concluded that Msps is not directly responsible for targeting cyclin B to centrosomes or MTs. Msps family members play an important role in regulating MT dynamics during the cell cycle so the interaction between cyclin B and Msps may simply reflect the fact that cyclin B/Cdc2 regulates Msps activity during the cell cycle. Indeed, it has been shown that Msps/XMAP215 is phosphorylated by cyclin B/Cdc2 in vitro (Vasquez, 1999; Basto, 2007 and references therein).
If Msps and Hsp90 do not directly target cyclin B to centrosomes and MTs, it remains unclear what does. A priori, it is expected that cyclin B in Drosophila embryo extracts would exist in a tight complex with any factor that would target it to MTs, since the vast majority of cyclin B binds to MTs in embryo extracts. Since cyclin B appears to localise at centrosomes and MTs in virtually all systems, it remains possible that cyclin B can directly bind to centrosomes and MTs. Indeed, bacterially expressed MBP-CBFL interacts strongly with purified MTs in MT-pelleting assays. In light of these results it seems that cyclin B is capable of interacting directly with MTs, although caution should b taken in interpretation of this in vitro experiment since fusion proteins containing cyclin B could have a tendency to aggregate in solution (Basto, 2007).
Nevertheless, the fact that no one has identified a factor that directly mediates the interaction between cyclin B and MTs or centrosomes, despite many years of effort in identifying cyclin B interacting proteins, suggests that there may be no other protein directly required for these interactions. In the favoured hypothesis, Hsp90 would serve simply to ensure that cyclin B was correctly folded to allow it to directly interact with MTs and with centrosomes. Interestingly it has been proposed that Hsp90 also contributes to increasing the association efficiency of Tau with MTs. Tau is a MAP with an important role in Alzheimer's disease. In the absence of Hsp90, Tau tends to aggregate and therefore less soluble Tau is available to bind to MTs. In this study, although it was also found that cyclin B and Msps require Hsp90 for their efficient recruitment to the spindle it is not thought that their activity, outside the spindle, is compromised (Basto, 2007).
Finally, it was found that Hsp90 was not only required to allow cyclin B to localise efficiently to centrosomes and MTs, it was also required to allow Msps to localise properly, and it was shown that the endogenous Hsp90 can interact with the endogenous Msps. Importantly, Hsp90 is not required for the localisation of several other proteins to centrosomes or MTs, demonstrating that its function in localising cyclin B and Msps is specific. Like cyclin B, the levels of Msps protein were not decreased in cells where Hsp90 function had been perturbed, suggesting that Hsp90 is not simply required to stabilise Msps protein. Thus, it is proposed that Hsp90 may act on several MT-associated proteins to ensure that specific domains of these proteins are in the correct conformation to allow these proteins to be targeted to different locations within the cell (Basto, 2007).
Localization of maternal nanos mRNA to the posterior pole is essential for development of both the abdominal segments and primordial germ cells in the Drosophila embryo. Unlike maternal mRNAs such as bicoid and oskar that are localized by directed transport along microtubules, nanos is thought to be trapped as it swirls past the posterior pole during cytoplasmic streaming. Anchoring of nanos depends on integrity of the actin cytoskeleton and the pole plasm; other factors involved specifically in its localization have not been described to date. This study used genetic approaches to show that the Hsp90 chaperone (encoded by Hsp83 in Drosophila) is a localization factor for two mRNAs, nanos and pgc. Other components of the pole plasm are localized normally when Hsp90 function is partially compromised, suggesting a specific role for the chaperone in localization of nanos and pgc mRNAs. Although the mechanism by which Hsp90 acts is unclear, this study found that levels of the LKB1 kinase are reduced in Hsp83 mutant egg chambers and that localization of pgc (but not nos) is rescued upon overexpression of LKB1 in such mutants. These observations suggest that LKB1 is a primary Hsp90 target for pgc localization and that other Hsp90 partners mediate localization of nos (Song, 2007).
The specific role that maternal Hsp90 plays in localization of a subset of mRNAs to the pole plasm is somewhat surprising, given the number of proteins that are thought to require the activity of this chaperone. Although ubiquitously distributed, Hsp90 is enriched in the testes and ovaries and the male germ line is particularly sensitive to a reduction in Hsp90 activity (Yue, 1999). The experiments reported in this study define a role for maternal Hsp90 in the localization of nos and pgc mRNAs. The apparent integrity of the pole plasm in Hsp83stc/Hsp83E317K ovaries and embryos and the rescue of pgc localization upon overexpression of a single kinase are consistent with the idea that the defects in pgc (and perhaps also nos) localization arise from the reduction in activity of a few discrete Hsp90 clients. Hsp83 mRNA is itself concentrated in the pole plasm (Ding, 1993) and Hsp90 is found at particularly high levels in the germ line precursors of other organisms, which may reflect a conserved role in mRNA localization (Song, 2007).
It is not known whether Hsp90 acts directly or indirectly to stabilize LKB1. Mammalian LKB1 binds directly to Hsp90 and Cdc37, a cochaperone for kinase clients (Boudeau, 2003). However, a direct interaction between Drosophila Hsp90 and LKB1 has not been observed, either by co-immunoprecipitation or in yeast interaction experiments in which the DNA-binding domain was fused to the Hsp90 C terminus to avoid interfering with dimerization. It is currently unknown whether Hsp90 binding to LKB1 is ephemeral (and thus difficult to detect) or whether Hsp90 acts indirectly to stabilize LKB1 (Song, 2007).
How might LKB1 act to localize pgc mRNA? Despite its conserved role in regulation of cellular polarity, no LKB1 substrate that plays a direct role in mRNA localization has been described. Loss of LKB1 function in germ line clones of presumptive null alleles prevents the reorganization of the oocyte microtubule network at stage 7 that is required for posterior localization of osk mRNA and affects epithelial polarity in the ovarian follicle cells. LKB1 colocalizes with cortical actin in the oocyte, integrity of which is required for anchoring of pole plasm components and nos mRNA. It is therefore attractive to speculate that LKB1 might act at the cortex, where actin and microtubule filaments meet, phosphorylating a currently unknown substrate to promote the trapping of pgc-containing RNPs. The results suggest that the level of LKB1 in Hsp83stc/Hsp83E317K flies is insufficient for pgc localization but sufficient for viability as well as proper polarization of microtubules during oogenesis and localization of osk. According to this idea, LKB1 hypomorphs might exhibit many of the defects observed in flies with reduced Hsp90 function (Song, 2007).
he differential effects observed on localization of nos, pgc, and CycB suggest that each mRNA is localized by a somewhat different mechanism. CycB mRNA localization is normal in Hsp83stc/Hsp83E317K embryos and thus appears to be relatively Hsp90 independent; pgc mRNA localization requires normal levels of Hsp90 activity, primarily to stabilize LKB1; and nos mRNA localization requires normal levels of Hsp90 activity, presumably to stabilize or activate other (currently unknown) factors. Studies of the polar granule component Tudor support the idea that different mechanisms underlie localization of nos, pgc, and gcl mRNAs, each of which is concentrated at the posterior pole late in oogenesis. Among this class of mRNAs, only nos has been studied in detail. Similar detailed studies of pgc, gcl, and CycB localization might reveal some of the mechanistic differences (Song, 2007).
The genetic screen employed to identify Hsp90 appears to constitute a promising approach for the identification of additional nos mRNA localization factors. One key aspect of the screen was reliance on the identification of dominant modifier mutations. Such mutations may be relatively easy to isolate in the case of Hsp83, as the encoded protein is a multidomain dimer that forms large complexes with other factors, including its cochaperones (Pearl, 2001). Nevertheless, a scaled-up version of the screen that surveys the entire genome and characterization of resulting mutants should yield additional components of the nos mRNA localization machinery (Song, 2007).
The molecular chaperone protein Hsp90 has been widely discussed as a candidate gene for developmental buffering. This study used the methods of geometric morphometrics to analyze its effects on the variation among individuals and fluctuating asymmetry of wing shape in Drosophila melanogaster. Three different experimental approaches were used to reduce Hsp90 activity. In the first experiment, developing larvae were reared in food containing a specific inhibitor of Hsp90, geldanamycin, but neither individual variation nor fluctuating asymmetry was altered. Two further experiments generated lines of genetically identical flies carrying mutations of Hsp83, the gene encoding the Hsp90 protein, in heterozygous condition in nine different genetic backgrounds. The first of these, introducing entire chromosomes carrying either of two Hsp83 mutations, did not increase shape variation or asymmetry over a wild-type control in any of the nine genetic backgrounds. In contrast, the third experiment, in which one of these Hsp83 alleles was introgressed into the wild-type background that served as the control, induced an increase in both individual variation and fluctuating asymmetry within each of the nine genetic backgrounds. No effect of Hsp90 on the difference among lines was detected, providing no evidence for cryptic genetic variation of wing shape. Overall, these results suggest that Hsp90 contributes to, but is not controlling, the buffering of phenotypic variation in wing shape (Debat, 2006).
An evolutionary capacitor buffers genotypic variation under normal conditions, thereby promoting the accumulation of hidden polymorphism. But it occasionally fails, thereby revealing this variation phenotypically. The principal example of an evolutionary capacitor is Hsp90, a molecular chaperone that targets an important set of signal transduction proteins. Experiments in Drosophila and Arabidopsis have demonstrated three key properties of Hsp90: (1) it suppresses phenotypic variation under normal conditions and releases this variation when functionally compromised; (2) its function is overwhelmed by environmental stress; and (3) it exerts pleiotropic effects on key developmental processes. But whether these properties necessarily make Hsp90 a significant and unique facilitator of adaptation is unclear. This study used numerical simulations of complex gene networks, as well as genome-scale expression data from yeast single-gene deletion strains, to present a mechanism that extends the scope of evolutionary capacitance beyond the action of Hsp90 alone. This study illustrates that most, and perhaps all, genes reveal phenotypic variation when functionally compromised, and that the availability of loss-of-function mutations accelerates adaptation to a new optimum phenotype. However, this effect does not require the mutations to be conditional on the environment. Thus, there might exist a large class of evolutionary capacitors whose effects on phenotypic variation complement the systemic, environment-induced effects of Hsp90 (Bergman, 2003).
Hsp90 is gaining increasing importance as a protein involved in controlling the normal functioning of the cell. To do this it apparently interacts with a battery of co-chaperone proteins that are involved in both substrate recognition and the progression of the Hsp90 catalytic pathway. This report identified the Drosophila Dpit47 protein (DNA polymerase interacting tpr containing protein of 47 kDa) through its interaction with the DNA polymerase alpha. This protein is a predominantly nuclear protein, which forms a tight and stoichiometric interaction with Hsp90 and shows interaction with Hsp70. It also has substantial homology to other known Hsp90 co-chaperones, e.g., CNS1 and hop1, making it likely that this protein also functions as an Hsp90 co-chaperone. The interaction with the DNA polymerase alpha is not related to the special situation in early embryos where there are large amounts of maternal protein stockpiles of the polymerase, as it occurs to the same level in early and late embryos and also in proliferating cell culture. However, it does not occur in quiescent cells, making it likely that the protein is related to proliferation. This is also consistent with Dpit47 expression being higher in proliferating cells. The interaction between the Dpit47 and the polymerase takes place predominantly in the nucleoplasm, and seems to involve several subunits of the polymerase in comparable amounts, making it unlikely that it is solely required for the assembly of the polymerase complex. The polymerase can also be seen to interact with Hsp90, and the interaction between Dpit47 and the polymerase is increased by the specific Hsp90 inhibitor geldanamycin. This suggests that a complex of the Dpit47, Hsp90 and DNA polymerase exists in the cell. The interaction between DNA polymerase alpha and Dpit47 completely inhibits the activity of the polymerase. These results suggest that Hsp90 acts as a chaperone for DNA polymerase alpha and that this interaction is mediated through the novel co-chaperone Dpit47. This provides the first suggestion of a role for chaperones in DNA replication in higher eukaryotes (Crevel, 2001).
This study has identified the Drosophila Dpit47 protein as a novel Hsp90 co-chaperone protein that interacts with the DNA polymerase α. Like other Hsp90 co-chaperone proteins, Dpit47 possesses a TPR domain. TPR domains are found in a wide variety of proteins and are thought to be involved in protein:protein interactions. In the Hsp90 co-activators they are thought to be involved in the interaction with Hsp90 (Scheufler, 2000), and the co-chaperone TPR domains form a distinct subset of TPR motif containing proteins that are more closely related to each other than to other TPR proteins, e.g. the APC proteins. Quite a large number of proteins have now been identified as Hsp90 co-chaperones; however, this blanket term covers a number of different subgroups. A more detailed analysis of the sequence of Dpit47 suggests that it falls into that class of co-chaperones that contains proteins such as Hop. These are thought to be involved in the early part of the reaction, and in recognition of the client proteins, although how closely these proteins are related to each other functionally and whether they also possess other biochemical activities remains to be determined. The suggestion of a co-chaperone role for Dpit47 is strengthened by isolation of tight and stoichiometric complex of Dpit47 with Hsp90 in extracts, and further supported by the observed interaction between Dpit47 and Hsp70, another protein which has been shown to act in the Hsp90 pathway. The isolation of a complex containing only these two proteins, the relative levels of the Hsp90 and 70 in the complex and the effects of the addition of ATP or the specific Hsp90 inhibitor geldanamycin on these interactions is also consistent with Dpit47 being involved in the early stages of the Hsp90 pathway (Crevel, 2001).
The recent release of the Drosophila genome sequence has further allowed the determinatopm that Dpit47 is a member of a family of proteins in Drosophila. Seven other homologues can be identified from their genome sequence, although in most cases the homology is largely confined to the TPR region. One of these proteins has already been defined as the Drosophila Hop homologue; the others are as yet uncharacterised (Crevel, 2001).
Database searching has also allowed identification of Dpit47 homologues in other species from budding yeast to mammals. The closest of these is a human protein TTC4 (accession number NM_004623.1), which was first identified as a putative tumour suppressor gene frequently deleted in breast cancer. Since this is closely related to Dpit47 it would be interesting to determine if TTC4 has retained functional characteristics of the Dpit47 protein (Crevel, 2001).
Most of the work presented in this study centers around the study of the interaction of the Dpit47 protein with the DNA polymerase α. Although the interaction was first seen in the two hybrid system, this study also confirmed that the interaction can be detected in extracts from various types of cells. The interaction was detected in both cytoplasmic and nucleoplasmic fractions, suggesting the interaction may occur in both compartments. However, the possibility cannot be completely ruled that Dpit47 associates with DNA polymerase α predominantly in the nucleoplasmic fraction, and that the interactions observed in cytoplasmic fractions are caused by leakage from the nucleus during the nuclear preparation, particularly from cells in mitosis (Crevel, 2001).
Although both proteins are quite abundant, the interaction only involves about 10%-20% of the polymerase and 1%-2% of the Dpit47. This is consistent with the Dpit47 having other substrates. For the DNA polymerase α this could represent an instability of the complex to the isolation conditions, alternatively only a subpopulation of the DNA polymerase α molecules may bind to Dpit47 (Crevel, 2001).
In addition to showing the interaction between Dpit47 and DNA polymerase α, it was possible to immunoprecipitate Hsp90 directly with DNA polymerase α antibodies. Although the trimeric complex has not been completely ruled, this observation, taken together with the observed effect of geldanamycin on the Dpit47:polymerase interaction, strongly suggests that the DNA polymerase α:Dpit47 complex is likely to also include Hsp90 (Crevel, 2001).
The data is suggestive of a role for the Hsp90 pathway in DNA polymerase α function. Interestingly, it is also consistent with a much earlier report in which geldanamycin was shown to inhibit DNA polymerase α activity. However, an important question is the function of this interaction in a cellular context. Among Hsp90 client proteins the purpose of the interaction with Hsp90 varies depending on the protein concerned. For a large group of proteins, including the widely studied glucocorticoid receptor (Buchner, 1999) and telomerase, it seems that the interaction is involved in the conversion of the protein from the inactive to the active state. For other proteins it is involved in holding the protein in a particular configuration so that it can interact with protein partners or be modified in some way (e.g., phosphorylated). The interaction with the DNA polymerase that was studied in this paper causes a severe inhibition of the polymerase activity in the complex (the specific activity is at least 50x less than that normally seen). This suggests that the most likely role of the interaction in this case is to sequester polymerase in an inactive form. What still remains to be determined is the role this plays in the normal functioning of the cell. It is likely to be involved in proliferation, as the interaction does not occur in quiescent cells. The presence of the interaction in late embryos/larvae and cell culture also suggests that it is not just related to the unusual situation in early embryos in Drosophila where excessive amounts of maternal proteins are present (Crevel, 2001).
In the normal progression of the cell cycle there are a number of places where such an activity could be useful. Inactive polymerase must be maintained prior to initiation of DNA replication, or after the completion of one complete round, therefore Dpit47 could function at either of these stages. Because the observed location of Dpit47 is predominantly nucleoplasmic but not chromatin bound, any observed effect must take place prior to or subsequent to the association of the polymerase with the replication complex, rather than actually in the complex itself. Therefore, Dpit47 could function by facilitating rapid binding of the polymerase to the chromatin during initiation of DNA replication by concentrating the polymerase in an inactive form close to its potential substrate. Alternatively, it could function by allowing rapid sequestration of the polymerase after it has finished synthesis (Crevel, 2001).
A sequestration role for the Dpit47 DNA polymerase α interaction does not preclude the possibility that the interaction may have other additional effects on the polymerase that are more in line with the effects that the Hsp90 pathway has been seen to have on other client proteins. It cannot be ruleed out completely that passage through the Hsp90 complex is required for activation of all polymerase molecules (as for the glucocorticoid receptor). However, the high level of activity of the polymerase isolated from early embryos when only small amounts need to have been activated owing to the small number of genomes to replicate, and the observation that polymerases from other organisms can be overproduced and are active, makes it less likely (Crevel, 2001).
Equally, the observation that the Dpit47 immunoprecipitates contain all four subunits in equal amounts makes it unlikely that interaction is required for the assembly of the complex. However, it is still possible that the Dpit47 interaction is required for modification of the polymerase, or for allowing its interaction with other components of the replication complex (Crevel, 2001).
Hsp90 is an essential abundant molecular chaperone involved in the folding, assembly and activation of number of proteins involved in signal transduction, cell cycle control, telomerase activity Hsp90 (Holt, 1999) or transcriptional regulation (Pratt, 1998). This study has presented evidence that links the Hsp90 pathway with a mainstream replication protein, thereby providing the first report of a possible role for chaperones in the process of DNA replication in higher eukaryotes. However, the amount of Dpit47 is far greater than that of DNA polymerase α, making it very likely that Dpit47 has other client partners. Sensitive Coomassie analysis of the fractions where DNA polymerase α is detected eluting from the anti-Dpit47 antibody reveal the presence of about a dozen bands of comparable intensity to DNA polymerase α. It would therefore be interesting to identify these proteins to determine if any others are involved in DNA replication and to see what other cellular processes might involve Dpit47 (Crevel, 2001).
To determine the molecular composition of the centrosome of a higher eukaryote, a systematic nano-electrospray tandem or MALDI mass spectrometry analysis was carried out of the polypeptides present in highly enriched preparations of immunoisolated Drosophila centrosomes. One of the proteins identified is Hsp83, a member of the highly conserved Hsp90 family including chaperones known to maintain the activity of many proteins but suspected to have other essential, unidentified functions. This study found that a fraction of the total Hsp90 pool is localized at the centrosome throughout the cell cycle at different stages of development in Drosophila and vertebrates. This association between Hsp90 and the centrosome can be observed in purified centrosomes and after treatment with microtubule depolymerizing drugs, two criteria normally used to define core centrosomal components. Disruption of Hsp90 function by mutations in the Drosophila gene or treatment of mammalian cells with the Hsp90 inhibitor geldanamycin, results in abnormal centrosome separation and maturation, aberrant spindles and impaired chromosome segregation. This suggests that another role of Hsp90 might be to ensure proper centrosome function (Lange, 2000).
The relationship between thermal tolerance and environmental conditions has been extensively studied in Drosophila. However, comparisons of thermal tolerance of laboratory-bred flies derived from distinct geographic locations have produced puzzling results. The differential expression of heat shock protein (HSP) after heat (34°C) and cold (-4°C) temperature treatments in two species of Drosophila flies, with distinct biogeographic origins (tropical = D. melanogaster and Andean = D. gaucha), previously exposed to sublethal acclimation temperatures (10, 20 and 30°C). The relationship between thermal acclimation and survival value was also evaluated as a proxy of fitness. A positive relationship was found between thermotolerance and the patterns of hsp90 transcript expression was found in both species. Nevertheless, in the cases in which hsp90 mRNA expression does not match thermotolerance induction, the biogeographic origin of the species could explain such mismatches. Survival at upper and lower experimental temperatures were also related with species origin (Boher, 2012).
The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. Eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. It is also reported that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, these are suggested to be indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves (Yue, 1999).
The heat-shock protein Hsp90 supports diverse but specific signal transducers and lies at the interface of several developmental pathways. When Drosophila Hsp90 is mutant or pharmacologically impaired, phenotypic variation affecting nearly any adult structure is produced, with specific variants depending on the genetic background and occurring both in laboratory strains and in wild populations. Multiple, previously silent, genetic determinants produced these variants and, when enriched by selection, they rapidly became independent of the Hsp90 mutation. Therefore, widespread variation affecting morphogenic pathways exists in nature, but is usually silent; Hsp90 buffers this variation, allowing it to accumulate under neutral conditions. When Hsp90 buffering is compromised, for example by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored. This provides a plausible mechanism for promoting evolutionary change in otherwise entrenched developmental processes (Rutherford, 1998).
Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. This study shows that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules -- cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, this study showed that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. The possible developmental significance of this complex control of Hsp83 transcript distribution is considered (Ding, 1993).
The heat shock protein HSP90, which is mainly cytoplasmic, has recently been reported to be present in the nucleus. This study has found a specific chromosomal localization of HSP90 in different species of Drosophila and Chironomus using immunocytochemical techniques with different mono- and polyclonal antibodies for this hsp. HSP90 was found associated with heat shock-induced puffs at 93D and 48B in salivary gland chromosomes of Drosophila melanogaster and Drosophila hydei, respectively. The localization of HSP90 to locus 93D occurred rapidly after the onset of heat shock and disappeared during recovery, concomitant with puff regression. The association of HSP90 with the 93D locus was strictly heat shock dependent as shown by the absence of HSP90 in puff 93D induced by either benzamide or colchicine. No specific nuclear staining was observed in unstressed control cells. HSP90 was also found in the temperature-induced telomeric Balbiani ring puffs (T-BRs) in Chironomus thummi and in one heat shock puff at I-1C in Chironomus tentans. Other heat shock puffs also appeared lightly stained with the HSP90 polyclonal antibody in both species of Chironomus. HSP90 was absent from the T-BRs when RNA synthesis was inhibited with Actinomycin D suggesting that the localization of HSP90 is dependent on transcription. Inhibition of protein synthesis did not prevent association of this hsp with the T-BRs, indicating that pre-existing HSP90 can associate with this locus. HSP90 did not associate with any telomeric chromosomal regions of unstressed cells. The present observations suggest that heat shock gene products such as HSP90 may somehow be involved in the regulation at the chromosomal level of other members of the heat shock gene family. Puffs 93D (D. melanogaster) and 48B (D. hydei) are equivalent and correspond to homologous gene loci (hsr omega) that have unusual features that distinguish them from other heat shock puffs. The binding of HSP90 at T-BRs and at puff I-1C in the genus Chironomus is the first demonstration, albeit indirect, of the existence of hsr omega analogous loci in species other than Drosophila (Morcillo, 1993).
Search PubMed for articles about Drosophila Hsp93
Andersen, R. O., Turnbull, D. W., Johnson, E. A. and Doe, C. Q. (2012). Sgt1 acts via an LKB1/AMPK pathway to establish cortical polarity in larval neuroblasts. Dev Biol 363: 258-265. Pubmed: 22248825
Benbahouche, N. E., Iliopoulos, I., Torok, I., Marhold, J., Henri, J., Kajava, A. V., Farkas, R., Kempf, T., Schnolzer, M., Meyer, P., Kiss, I., Bertrand, E., Mechler, B. M. and Pradet-Balade, B. (2014). Drosophila Spag is the homolog of RNA Polymerase II Associated Protein 3 (RPAP3), and recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries. J Biol Chem. [Epub ahead of print] PubMed ID: 24394412
Bandura, J. L., Jiang, H., Nickerson, D. W. and Edgar, B. A. (2013). The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila. PLoS Genet 9: e1003835. PubMed ID: 24086162
Bansal, P. K., Mishra, A., High, A. A., Abdulle, R. and Kitagawa, K. (2009). Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361. J Biol Chem 284: 18692-18698. Pubmed: 19398558
Basto, R., et al. (2007). Hsp90 is required to localise cyclin B and Msps/ch-TOG to the mitotic spindle in Drosophila and humans. J. Cell Sci. 120(Pt 7): 1278-87. Medline abstract: 17376965
Bergman, A. and Siegal, M. L. (2003). Evolutionary capacitance as a general feature of complex gene networks. Nature 424: 549-552. PubMed ID: 12891357
Boher, F., Trefault, N., Piulachs, M. D., Belles, X., Godoy-Herrera, R. and Bozinovic, F. (2012). Biogeographic origin and thermal acclimation interact to determine survival and hsp90 expression in Drosophila species submitted to thermal stress. Comp Biochem Physiol A Mol Integr Physiol 162: 391-396. PubMed ID: 22561660
Boudeau, J., Deak, M., Lawlor, M. A., Morrice, N. A. and Alessi, D. R. (2003). Heat-shock protein 90 and Cdc37 interact with LKB1 and regulate its stability. Biochem J 370: 849-857. PubMed ID: 12489981
Buchner, J. (1999). Hsp90 & Co. - a holding for folding. Trends Biochem Sci 24: 136-141. PubMed ID: 10322418
Crevel, G., Bates, H., Huikeshoven, H. and Cotterill, S. (2001). The Drosophila Dpit47 protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase alpha. J Cell Sci 114: 2015-2025. PubMed ID: 11493638
Debat, V., Milton, C. C., Rutherford, S., Klingenberg, C. P. and Hoffmann, A. A. (2006). Hsp90 and the quantitative variation of wing shape in Drosophila melanogaster. Evolution 60: 2529-2538. PubMed ID: 17263114
DeFranco, D. B. and Csermely, P. (2000). Steroid receptor and molecular chaperone encounters in the nucleus. Sci STKE 2000: pe1. PubMed ID: 11752599
Ding, D., Parkhurst, S. M., Halsell, S. R. and Lipshitz, H. D. (1993). Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis. Mol Cell Biol 13: 3773-3781. PubMed ID: 7684502
Duncan, R. F. (2008). Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in Drosophila: implications for the mechanisms of Hsp mRNA translation. Cell Stress Chaperones 13: 143-155. PubMed ID: 18418733
Floer, M., Bryant, G. O. and Ptashne, M. (2008). HSP90/70 chaperones are required for rapid nucleosome removal upon induction of the GAL genes of yeast. Proc Natl Acad Sci U S A 105: 2975-2980. PubMed ID: 18287040
Freeman, B. C. and Yamamoto, K. R. (2002). Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296: 2232-2235. PubMed ID: 12077419
Gangaraju, V. K., Yin, H., Weiner, M. M., Wang, J., Huang, X. A. and Lin, H. (2011). Drosophila Piwi functions in Hsp90-mediated suppression of phenotypic variation. Nat Genet 43: 153-158. PubMed ID: 21186352
Guertin, M. J. and Lis, J. T. (2010). Chromatin landscape dictates HSF binding to target DNA elements. PLoS Genet 6: e1001114. PubMed ID: 20844575
Hamamoto, R., Furukawa, Y., Morita, M., Iimura, Y., Silva, F. P., Li, M., Yagyu, R. and Nakamura, Y. (2004). SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol 6: 731-740. PubMed ID: 15235609
He, Q., Wen, D., Jia, Q., Cui, C., Wang, J., Palli, S. R. and Li, S. (2014). Heat shock protein 83 (Hsp83) facilitates Methoprene-tolerant (Met) nuclear import to modulate juvenile hormone signaling. J Biol Chem 289(40):27874-85. PubMed ID: 25122763
Holt, S. E., Aisner, D. L., Baur, J., Tesmer, V. M., Dy, M., Ouellette, M., Trager, J. B., Morin, G. B., Toft, D. O., Shay, J. W., Wright, W. E. and White, M. A. (1999). Functional requirement of p23 and Hsp90 in telomerase complexes. Genes Dev 13: 817-826. PubMed ID: 10197982
Hrizo, S. L. and Palladino, M. J. (2010). Hsp70- and Hsp90-mediated proteasomal degradation underlies TPIsugarkill pathogenesis in Drosophila. Neurobiol Dis 40: 676-683. PubMed ID: 20727972
Huang, J. and Wang, H. (2018). Hsp83/Hsp90 physically associates with Insulin receptor to promote neural stem cell reactivation. Stem Cell Reports 11(4): 883-896. PubMed ID: 30245208
Hung, J. J., Wu, C. Y., Liao, P. C. and Chang, W. C. (2005). Hsp90alpha recruited by Sp1 is important for transcription of 12(S)-lipoxygenase in A431 cells. J Biol Chem 280: 36283-36292. PubMed ID: 16118214
Jarosz, D. F. and Lindquist, S. (2010). Hsp90 and environmental stress transform the adaptive value of natural genetic variation. Science 330: 1820-1824. PubMed ID: 21205668
Kimura, Y., Yahara, I. and Lindquist, S. (1995). Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways. Science 268: 1362-1365. PubMed ID: 7761857
Lange, B. M., Bachi, A., Wilm, M. and Gonzalez, C. (2000). Hsp90 is a core centrosomal component and is required at different stages of the centrosome cycle in Drosophila and vertebrates. EMBO J 19: 1252-1262. PubMed ID: 10716925
Luo, S., Wang, T., Qin, H., Lei, H. and Xia, Y. (2011). Obligatory role of heat shock protein 90 in iNOS induction. Am J Physiol Cell Physiol 301: C227-233. PubMed ID: 21430289
Miyoshi, T., Takeuchi, A., Siomi, H. and Siomi, M. C. (2010). A direct role for Hsp90 in pre-RISC formation in Drosophila. Nat Struct Mol Biol 17: 1024-1026. PubMed ID: 20639883
Mollapour, M., Tsutsumi, S. and Neckers, L. (2010). Hsp90 phosphorylation, Wee1 and the cell cycle. Cell Cycle 9: 2310-2316. PubMed ID: 20519952
Morcillo, G., Diez, J. L., Carbajal, M. E. and Tanguay, R. M. (1993). HSP90 associates with specific heat shock puffs (hsr omega) in polytene chromosomes of Drosophila and Chironomus. Chromosoma 102: 648-659. PubMed ID: 8306827
Muller, L., Schaupp, A., Walerych, D., Wegele, H. and Buchner, J. (2004). Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures. J Biol Chem 279: 48846-48854. PubMed ID: 15358771
O'Keeffe, B., Fong, Y., Chen, D., Zhou, S. and Zhou, Q. (2000). Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription. J Biol Chem 275: 279-287. PubMed ID: 10617616
Pare, J. M., Tahbaz, N., Lopez-Orozco, J., LaPointe, P., Lasko, P. and Hobman, T. C. (2009). Hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and P-bodies. Mol Biol Cell 20: 3273-3284. PubMed ID: 19458189
Pearl, L. H. and Prodromou, C. (2001). Structure, function, and mechanism of the Hsp90 molecular chaperone. Adv Protein Chem 59: 157-186. PubMed ID: 11868271
Pearl, L. H., Prodromou, C. and Workman, P. (2008). The Hsp90 molecular chaperone: an open and shut case for treatment. Biochem J 410: 439-453. PubMed ID: 18290764
Picard, D., Khursheed, B., Garabedian, M. J., Fortin, M. G., Lindquist, S. and Yamamoto, K. R. (1990). Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348: 166-168. PubMed ID: 2234079
Pisa, V., Cozzolino, M., Gargiulo, S., Ottone, C., Piccioni, F., Monti, M., Gigliotti, S., Talamo, F., Graziani, F., Pucci, P. and Verrotti, A. C. (2009). The molecular chaperone Hsp90 is a component of the cap-binding complex and interacts with the translational repressor Cup during Drosophila oogenesis. Gene 432: 67-74. PubMed ID: 19101615
Pratt, W. B. (1998). The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med 217: 420-434. PubMed ID: 9521088
Rutherford, S. L. and Lindquist, S. (1998). Hsp90 as a capacitor for morphological evolution. Nature 396: 336-342. PubMed ID: 9845070
Sangster, T. A., Lindquist, S. and Queitsch, C. (2004). Under cover: causes, effects and implications of Hsp90-mediated genetic capacitance. Bioessays 26: 348-362. PubMed ID: 15057933
Sawarkar, R., Sievers, C. and Paro, R. (2012). Hsp90 globally targets paused RNA polymerase to regulate gene expression in response to environmental stimuli. Cell 149: 807-818. PubMed ID: 22579285
Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F. U. and Moarefi, I. (2000). Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101: 199-210. PubMed ID: 10786835
Sollars, V., Lu, X., Xiao, L., Wang, X., Garfinkel, M. D. and Ruden, D. M. (2003). Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution. Nat Genet 33: 70-74. PubMed ID: 12483213
Song, Y., Fee, L., Lee, T. H. and Wharton, R. P. (2007). The molecular chaperone Hsp90 is required for mRNA localization in Drosophila melanogaster embryos. Genetics 176: 2213-2222. PubMed ID: 17565952
Specchia, V., Piacentini, L., Tritto, P., Fanti, L., D'Alessandro, R., Palumbo, G., Pimpinelli, S. and Bozzetti, M. P. (2010). Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons. Nature 463: 662-665. PubMed ID: 20062045
Stavreva, D. A., Muller, W. G., Hager, G. L., Smith, C. L. and McNally, J. G. (2004). Rapid glucocorticoid receptor exchange at a promoter is coupled to transcription and regulated by chaperones and proteasomes. Mol Cell Biol 24: 2682-2697. PubMed ID: 15024059
Tariq, M., Nussbaumer, U., Chen, Y., Beisel, C. and Paro, R. (2009). Trithorax requires Hsp90 for maintenance of active chromatin at sites of gene expression. Proc Natl Acad Sci U S A 106: 1157-1162. PubMed ID: 19144915
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Vozzolo, L., Loh, B., Gane, P. J., Tribak, M., Zhou, L., Anderson, I., Nyakatura, E., Jenner, R. G., Selwood, D. and Fassati, A. (2010). Gyrase B inhibitor impairs HIV-1 replication by targeting Hsp90 and the capsid protein. J Biol Chem 285: 39314-39328. PubMed ID: 20937817
Walerych, D., Kudla, G., Gutkowska, M., Wawrzynow, B., Muller, L., King, F. W., Helwak, A., Boros, J., Zylicz, A. and Zylicz, M. (2004). Hsp90 chaperones wild-type p53 tumor suppressor protein. J Biol Chem 279: 48836-48845. PubMed ID: 15358769
Workman, P., Burrows, F., Neckers, L. and Rosen, N. (2007). Drugging the cancer chaperone HSP90: combinatorial therapeutic exploitation of oncogene addiction and tumor stress. Ann N Y Acad Sci 1113: 202-216. PubMed ID: 17513464
Yue, L., Karr, T. L., Nathan, D. F., Swift, H., Srinivasan, S. and Lindquist, S. (1999). Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis. Genetics 151: 1065-1079. PubMed ID: 10049923
Zou, J., Guo, Y., Guettouche, T., Smith, D. F. and Voellmy, R. (1998). Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94: 471-480. PubMed ID: 9727490
Zhao, R., Davey, M., Hsu, Y. C., Kaplanek, P., Tong, A., Parsons, A. B., Krogan, N., Cagney, G., Mai, D., Greenblatt, J., Boone, C., Emili, A. and Houry, W. A. (2005). Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120: 715-727. PubMed ID: 15766533
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date revised: 15 April 2020
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