InteractiveFly: GeneBrief

super sex combs: Biological Overview | References

Gene name - super sex combs

Synonyms - Ogt, O-GlcNAc transferase

Cytological map position - 41E4-41E5

Function - enzyme

Keywords - polycomb group - induces Hipk-mediated tumor-like growth - maintains Hipk stability by blocking its degradation - Hipk is O-GlcNAcylated by OGT - cooperates with N-glycanase to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes - controls of synaptic size and synaptic bouton number at the neuromuscular junction/a<> - part of a clock-regulated buffering mechanism that prevents excessive O-GlcNAcylation at non-optimal times of the day-night cycle - O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function - plays a role in habituation learning - O-GlcNAcylation is needed for Polyhomeotic to form functional, ordered assemblies

Symbol - sxc

FlyBase ID: FBgn0261403

Genetic map position - chr2R:5,307,288-5,329,555

Classification - Glycosyl transferase family 41

Cellular location - nuclear and cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

sxc orthologs: Biolitmine

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. This study identified the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT/Super sex combs) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. It was further shown that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, these data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs (Wong, 2020).

Nutrients such as glucose, amino acids, and fatty acids are metabolic fuels that provide energy to cells. They also function as signaling molecules in various nutrient signaling pathways, allowing coordination between nutrient sensing and cellular behaviors like cell growth. One emerging nutrient signaling pathway is the hexosamine biosynthetic pathway (HBP). In the HBP, glutamine:fructose-6-phosphate amidotransferases (GFATs) catalyze the first and rate-limiting step that converts glucose-derived fructose-6-phosphate into glucosamine-6-phosphate. The end product of the HBP is UDP-GlcNAc, the donor nucleotide sugar substrate for various glycosyltransferases, including nucleocytoplasmic O-GlcNAc transferase (OGT). OGT is the only known enzyme that catalyzes the addition of O-GlcNAc to serine and threonine residues of hundreds of target proteins in a UDP-GlcNAc-dependent manner. Notably, O-GlcNAcase (OGA) removes O-GlcNAc from proteins, making O-GlcNAc a dynamic and reversible posttranslational modification (PTM). In this way, cellular UDP-GlcNAc levels and global O-GlcNAcylation are coordinated and are highly responsive to glucose availability, making O-GlcNAc well suited to serve as a nutrient-sensing mechanism (Wong, 2020).

Pathologically, O-GlcNAc has been implicated in cancers. For instance, hyper-O-GlcNAcylation, high levels of OGT or GFAT2, or low OGA levels are positively correlated with poor prognosis of patients with prostate or breast cancers, aggressiveness of bladder tumors, or tumor recurrence of liver cancer. Also, many oncoproteins and tumor suppressors such as c-MYC, p53 , and YAP are O-GlcNAc modified. These observations link O-GlcNAc with multiple hallmarks of cancer, including sustaining proliferative signals and deregulating cellular energetics. Cancer cells are usually metabolically active. In particular they often sustain high rates of glucose uptake, a phenomenon commonly known as the Warburg effect, which is a preferential reliance on aerobic glycolysis to obtain energy. How elevated uptake of glucose triggers O-GlcNAc modification of cancer-related proteins and controls their activities is accordingly a topic of growing interest (Wong, 2020).

Homeodomain-interacting protein kinases (HIPKs; HIPK1-4 in mammals and Hipk in Drosophila [fruit fly]) are protein kinases involved in the regulation of signal transduction, cell proliferation and differentiation, apoptosis, stress response, embryonic development, angiogenesis, adipogenesis, as well as immune homeostasis. The activity of HIPK2 (the most studied member of the mammalian HIPK family) is governed by multiple strategies depending on the environments. HIPK2 is normally maintained at low levels by proteasomal degradation involving various ubiquitin E3 ligases such as Siah1 and Siah2. During hypoxia, HIPK2 degradation is facilitated by the increased association between HIPK2 and Siah2. DNA damage, on the other hand, disrupts the HIPK2-Siah1 interaction, protecting HIPK2 from degradation. Furthermore, high oxidative stresses modulate HIPK2 SUMOylation and acetylation states, which influence HIPK2 localization. Given the capacity for HIPK2 to respond to environmental cues and the growing recognition that nutrient sensing through OGT is a key regulator of cellular homeostasis, the potential for O-GlcNAc modification to serve as a nutritional regulator of HIPK(s) was intreguing (Wong, 2020).

Previous work has established that overexpression of Hipk in Drosophila promotes tissue growth abnormalities and several tumor-like features, including metabolic reprogramming, cell invasion-like behaviors, and cellular changes reminiscent of epithelial-to-mesenchymal transition, including up-regulation of Twist and Matrix metalloproteinase 1 (MMP1) and down-regulation of E-cadherin. Using this in vivo model, it was shown that OGT is not only necessary for Hipk-mediated tissue growth abnormalities, but also sufficient to synergize with mild levels of Hipk to produce tumor-like phenotypes. Furthermore, it was found that HIPKs (both fly Hipk and human HIPK2) are O-GlcNAc-modified proteins. Elevated OGT results in the buildup of HIPK proteins in a posttranscriptional manner. In particular, this study identified that HIPK2 O-GlcNAcylation at residues S852, T1009, and S1147 are responsible for OGT-mediated stabilization (Wong, 2020).

Cancer cells often vigorously consume glucose, aided by metabolic reprogramming involving multiple processes, for example, elevated expression of glucose transporters. Notably, an ample supply of glucose not only facilitates aerobic glycolysis within tumors, but also drives increased flux through parallel pathways, including the HBP. As a consequence, increased levels of UDP-GlcNAc and subsequent hyper-O-GlcNAcylation are found in nearly all cancers examined, including breast, prostate, colon, lung, liver, pancreatic, and leukemias. Indeed, global O-GlcNAcylation is emerging as a node linking glucose availability and cancer progression (Wong, 2020).

The link between dietary sugar and increased tumorigenesis is found to be conserved in Drosophila. Using various fly cancer models, researchers have shown that excess sugar intake affects cancer progression through multiple pathways. For example, high dietary sugar has been shown to drive the malignancy of Ras/Src tumors and neoplasia of EGFR-driven tumors through evasion of insulin resistance and lactate dehydrogenase-dependent aerobic glycolysis, respectively. The involvement of hexosamine signaling in fly tumorigenesis, however, has not been explored. This study reports that the activity of Hipk, a proliferation-promoting protein in flies, is controlled by the HBP-OGT axis in response to metabolic nutrients. Using the fly Hipk tumor-like model, it was demonstrated that OGT is both necessary and sufficient for Hipk-induced growth abnormalities. Thus, this work defines the HBP-OGT axis as a glucose-dependent mechanism regulating Hipk-mediated growth control. This is consistent with several studies of O-GlcNAc-dependent regulation of growth-promoting proteins such as YAP in mammals (Wong, 2020).

In addition, a mild up-regulation of OGT was observed in the Hipk-overexpressing cells, inferring that OGT may be a downstream target of Hipk and that the up-regulation of OGT may sustain ectopic Hipk expression and form a positive feedback loop. Recently, it was reported that fly Hipk tumor-like cells display elevated aerobic glycolysis driven by up-regulation of dMyc (MYC in vertebrates) (Wong, 2019). It is therefore tempting to speculate that the increased glucose metabolism could stimulate HBP flux and generate more UDP-GlcNAc for O-GlcNAcylation, and together with the mild OGT up-regulation, the combined effects may ultimately feedforward to sustain high Hipk protein levels and reinforce tumor-like proliferation. Future studies may shed light on this mechanism (Wong, 2020).

Although Hipk proteins are proposed to be maintained at low levels through degradation by the proteasomal machinery, minimal amounts of Hipk are indispensable for normal growth and development as flies homozygous mutant for hipk are not viable. This hints at the existence of cellular mechanisms that oppose Hipk protein degradation. The current work demonstrates that OGT stabilizes both endogenous and exogenous Hipk and protects Hipk from proteasomal degradation. It was confirmed that fly Hipk is an O-GlcNAc-modified protein. While the fly Hipk O-GlcNAc motifs and their roles remain unknown, this study showed that OGT-mediated stabilization of human HIPK2 relies on direct O-GlcNAcylation of HIPK2 at residues S852/T853, S1008/T1009, and S1147. This report describes HIPK2 O-GlcNAcylation as a molecular mechanism that controls HIPK2 protein stability. Given the nutrient-sensing nature of O-GlcNAc, it is conceivable that HIPK2 O-GlcNAcylation might contribute to the nutrient sensitivity of HIPK2 abundance. Intriguingly, a study shows that HIPK2 protein levels increased in a mouse model of diabetes, and the authors attributed the effect to down-regulation of the E3 ubiquitin ligase Siah-1. Hence, the nutritional regulation of HIPK2 might be achieved by multiple strategies like direct O-GlcNAcylation and impairment of proteasome system, or their cooperative effects (Wong, 2020).

In summary, this work illustrates the conserved regulation of HIPK (fly Hipk and human HIPK2) protein stability by OGT and demonstrates the functional consequences of this regulation in tumor-like events using an in vivo fly model. The roles of human HIPK2 in cancers are context dependent and yet to be fully understood. On the one hand, HIPK2 activates the tumor suppressor p53 after UV irradiation. Also, HIPK2 acts as a tumor suppressor upon induction of two-stage skin carcinogenesis or γ-radiation-induced tumorigenesis . On the other hand, HIPK2 promotes cytoprotection in cancer cells when challenged with chemotherapeutic drugs. Elevation of HIPK2 is also associated with malignancy of pilocytic astrocytomas and cervical carcinogenesis. These results raise concerns that targeting HIPK2 may have paradoxical effects. Elevated HIPK2 is also implicated in fibrosis in kidney, lung, and liver. Thus, depending on the context, it is believed that the dynamic control of HIPK2 abundance by O-GlcNAc modification reported in this study can be of great interest and exploited in the treatment of HIPK2-related disorders with the use of dietary control or metabolic drugs targeting the HBP-OGT axis (Wong, 2020).

Cytosolic O-GlcNAcylation and PNG1 maintain Drosophila gut homeostasis by regulating proliferation and apoptosis

It remains unknown how intracellular glycosylation, O-GlcNAcylation, interfaces with cellular components of the extracellular glycosylation machinery, like the cytosolic N-glycanase NGLY1. This study utilized the Drosophila gut and uncovered a pathway in which O-GlcNAcylation cooperates with the NGLY1 homologue PNG1 to regulate proliferation in intestinal stem cells (ISCs) and apoptosis in differentiated enterocytes. Further, the CncC antioxidant signaling pathway and ENGase, an enzyme involved in the processing of free oligosaccharides in the cytosol, interact with O-GlcNAc and PNG1 through regulation of protein aggregates to contribute to gut maintenance. These findings reveal a complex coordinated regulation between O-GlcNAcylation and the cytosolic glycanase PNG1 critical to balancing proliferation and apoptosis to maintain gut homeostasis (Na, 2022).

Intestinal stem cells regulate tissue homeostasis by balancing self-renewal, proliferation, and differentiation all of which are supported by elevated flux through the hexosamine biosynthetic pathway (HBP). Both N-linked glycosylation and intracellular O-GlcNAc modifications are regulated by the HBP pathway in a nutrient-sensing manner. However, how NGLY1 is utilized to control stem cell homeostasis and differentiation in cells remains largely unknown. This is a critical question as patients with NGLY1-deficiency display global developmental delay, movement disorder and growth retardation. Elevation of NGLY1 was observed in patients' tumor samples, suggesting a function in oncogenic signaling. In Drosophila, PNG1 mutants had severe developmental defects and reduced viability, with the surviving adults frequently sterile (Funakoshi, 2010). This study has identified a pathway by which PNG1 regulates ISC homeostasis in vivo. This study shows that PNG1 levels increased in ISC/EBs concomitant with O-GlcNAc. This interaction between PNG1 and O-GlcNAcylation is critical for maintaining normal ISC proliferation and differentiation. Thus, through their mutual regulation, OGT and PNG1 have key roles in both progenitor (ISCs/EBs) and differentiated cells (ECs) contributing to tissue homeostasis. Previous reports indicated that PNG1 null larvae have specific developmental abnormalities in their midgut that contributes to their lethality. Further, intestinal inflammation in Crohn's disease is associated with increased O-GlcNAc modification. A previous study also showed that increased O-GlcNAc promotes gut dysplasia through regulation of DNA damage (Na, 2020). Thus, PNG1 or O-GlcNAc might still be associated with gut dysfunction in a disease context (Na, 2022).

The regulation of O-GlcNAc by PNG1 and the interaction between PNG1 and O-GlcNAc has been implicated previously. In fact, GlcNAc supplementation partially rescued lethality associated with PNG1 knockdown (Funakoshi, 2010). Although the mechanism by which GlcNAc supplementation rescued these mutant flies has not been fully worked out, Gfat1 transcript levels were downregulated in PNG1 knockdown flies (Funakoshi, 2010). Gfat1 is the enzyme that controls the rate limiting step in the HBP to produce UDP-GlcNAc. Thus, PNG1 through regulation of Gfat1 could impact levels of UDP-GlcNAc and ultimately O-GlcNAc. Additionally, it has been hypothesized that the loss of PNG1 could increase the presence of intracellular N-GlcNAc modification, potentially interfering with O-GlcNAc mediated signaling. Therefore, alterations in UDP-GlcNAc levels or presence of intracellular N-GlcNAc upon PNG1-deficiency can interact with O-GlcNAc to regulate stem cell homeostasis (Na, 2022).

Previous reports have shown that Nrf1 undergoes NGLY1-mediated deglycosylation, followed by proteolytic cleavage and translocation into the nucleus as an active transcription factor. Loss of NGLY1 caused Nrf1 dysfunction, as evidenced by an enrichment of deregulated genes encoding proteasome components and proteins involved in oxidation reduction. Proteasome activity can induce an apoptotic cascade that leads to growth arrest and, subsequently, cell death. The current data indicated that PNG1 or OGT knockdown suppressed ISC proliferation, which was rescued by Oltipraz (CncC activation) treatment in ISCs/EBs. Furthermore, there was increased apoptosis in PNG1 or OGT knockdown with treatment compared to non-treated groups. Interestingly, it was found O-GlcNAc-induced intestinal dysplasia was rescued by knockdown of PNG1 in ISCs/EBs through regulation of ROS levels. Similarly, increases in global O-GlcNAcylation in embryos of diabetic mice caused an overproduction of ROS and subsequent oxidative and ER stress. It is known that activation of SKN-1A/Nrf1 also requires deglycosylation by PNG-1/NGLY1 in C. elegans. Further, SKN-1 is O-GlcNAc modified and translocates to the nucleus in ogt-1(ok430)-null worms. Together, these studies all suggest conserved functional connections between O-GlcNAc and Nrf family transcription factors. This study also showed EC-specific OGT or PNG1 knockdown-induced hyperproliferation and cell death was decreased by CncC activation. This data indicated OGT or PNG1 can be regulated by CncC activity in ISCs/EBs and ECs. CncC has high activity within ISCs/EBs of unstressed as well young ISCs and quiescent ISCs but decreases with age and damage. These data indicated that CncC acts to properly balance between signaling and damage responses necessary for tissue homeostasis. CncC activation increased ISC proliferation in ISCs/EBs and decreased ISC proliferation in ECs of OGT or PNG1 knockdown contributing towards tissue homeostasis. Another study showed that inhibition of NGLY1 resulted in Nrf1 being misprocessed, mislocated, and inactive, thus indicating that functional NGLY1 is essential for Nrf1 processing, nuclear translocation, and transcription factor activity (Tomlin, 2017). Therefore, the data suggests that PNG1 and OGT modulated by CncC activation contribute to ISC proliferation and ultimately regulating tissue homeostasis. Nrf2 activation was able to rescue the developmental growth of NGLY1 deficiency in worm and fly models. In cancer-initiating cells, ER stress-dependent (ROS-independent) CncC induction is an event necessary to maintain stemness. The data showed that PNG1 knockdown-induced Poly-UB accumulation and 26S proteasome expression that was rescued by CncC overexpression and chemical activation. Through functioning as a sensor of cytosolic proteasome activity and an activator of aggresomal formation, Nrf2 alleviates cell damages caused by proteasomal stress. Expression of proteasome subunit genes and mitophagy-related genes were broadly enhanced after sulforaphane (Keap1 inhibitor) treatment and pharmacologically induction of Nrf2 promotes mitophagy and ameliorates mitochondrial defect in Ngly1-/- cells. Thus, it is believed that the sensitized background of the OGT or PNG1 mutant provides an environment where CncC activation promotes proliferation to the normal level through regulation of proteasome activity and protein aggregation (Na, 2022).

In a previously published paper (Ha, 2020), it was shown that OGT overexpression and OGA knockdown in ISCs/EBs both increased O-GlcNAc levels and induced hyperproliferation of the stem cells, whereas OGT knockdown decreased proliferation. However, in differentiated ECs, OGT overexpression and OGA knockdown phenotypes were similar to the normal gut, whereas OGT knockdown elevated proliferation and cell death. In general, EC death promotes proliferation in order to maintain gut homeostasis. Here, NGLY1 knockdown in ISCs/EBs decreased proliferation and clone size but NGLY1 knockdown in ECs induced hyperproliferation and cell death and importantly decreased O-GlcNAc levels. Thus, the phenotypes of OGT and NGLY1 were similar, demonstrating that maintenance of OGT and NGLY1 protein expression is highly interdependent for the maintenance of tissue homeostasis. It is interesting that the progenitor and differentiated cell types within the gut respond differently to changes in O-GlcNAc. It is possible that a certain level of O-GlcNAcylation is needed to maintain stem cells and promote proliferation and self-renewal, however, differentiated cells that do not have the same energy and growth requirements are not as reliant on high levels of O-GlcNAc. On the other hand, both ISC/EBs and ECs require some level of O-GlcNAc and without OGT there is decreased proliferation in progenitor cells and increased cell death of ECs. There are a few possibilities how NGLY1 and OGT can collaboratively work, however, it is unlikely that they share protein targets. First, a previous publication showed that additional deletion of ENGase, another N-deglycosylating enzyme that leaves a single GlcNAc residue, alleviates some of the lethality of Ngly1-deficient mice. Thus, it is possible with the accumulation of aggregation prone intracellular N-GlcNacylated proteins, there is disruption of normal O-GlcNac signaling. These data also showed increased protein aggregation in OGT or NLGY1 knockdown that was rescued by ENGase knockdown. In addition, MYC-OGT protein levels in OGT overexpression fly guts were decreased by PNG1 knockdown. It is possible that loss of NGLY1 disrupts normal OGT degradation and thus impacts levels global of O-GlcNAcylation (Na, 2022).

This study has shown that ENGase levels increased in PNG1 or OGT knockdown ISCs/EBs and ECs. PNGase is involved in the process of endoplasmic reticulum associated degradation (ERAD), acting as a deglycosylating enzyme that cleaves N-glycans attached to ERAD substrates. The small molecule ENGase inhibitors have potential to treat pathogenesis associated with NGLY1 deficiency. Rabeprazole, a proton pump inhibitor, was identified as a potential ENGase inhibitor. It was demonstrated that the consequences of knockdown of OGT or PNG1 on ISC proliferation and ENGase activity was rescued by Rabeprazole treatment in ISCs/EBs or ECs. The data showed that cell death was elevated in ISCs/EBs-specific PNG1/OGT knockdown with Rabeprazole treatment compared to non-treated groups concomitant with an increase in ISC proliferation. On the other hand, cell death decreased in EC-specific PNG1 knockdown treated with Rabeprazole resulting in a decrease in ISC proliferation. It is known that loss of PNG1 function in cells can cause the accumulation of aberrant proteins in the cytosol and the interruption of ERAD. Further, downregulation of ER stress-related genes has been reported in B-cell-specific OGT mutant mice. The protective effects of O-GlcNAc are not limited to mitochondrial function but also rescue injury caused by ER stress. Therefore, NGLY1/OGT seems to be functionally associated with the ERAD machinery. More recently, using a model ERAD substrate, it was reported that the ablation of Ngly1 causes a disruption in the ERAD process in mouse embryonic fibroblast (MEF) cells. Moreover, lethality of mice bearing a knockout of the Ngly1-gene was partially rescued by the additional deletion of the Engase gene. Interestingly, this study showed that OGA knockdown rescued ENGase levels of PNG1 knockdown ISCs/EBs. Hence, these findings suggest that there is a correlation between OGT/PNG1 and ENGase contributing to tissue maintenance (Na, 2022).

Taken together, these findings implicate O-GlcNAc and PNG1 as key regulators of tissue maintenance. PNG1 can impact stem cell homeostasis through regulation of O-GlcNAc both in ISCs/EBs or ECs. Of significance is the finding that PNG1 and OGT phenotypes are rescued by modulating CncC and ENGase activity in ISCs/EBs or ECs. Thus, these findings reveal that nutrient-driven glycosylation contribute towards control of ISC and progenitor cell proliferation and EC cell death via regulation of CncC and ENGase. This study provides a platform for future designs of interventions in which changes in O-GlcNAc can be utilized as a therapeutic for stem-cell-derived diseases like cancer. This study also presents a molecular mechanism and unexpected pathway that can be targeted for treating NGLY1-deificient patients (Na, 2022).

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. This study manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrated an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG (Fenckova, 2022).

Habituation, the brain's response to repetition, is a core element of higher cognitive functions. Filtering out irrelevant familiar stimuli as a result of habituation allows one to focus the cognitive resources on relevant sensory input. Abnormal habituation was observed in a number of neurodevelopmental disorders, including Intellectual Disability (ID) and Autism and characterizes > 100 Drosophila models of ID. To address the role of OGT and its O-GlcNAc transferase activity in this cognition-relevant process, this stud investigated heterozygous sxcH537A/+ flies in light-off jump habituation. It was found that they were not able to suppress their escape behavior as a result of deficient habituation learning. This finding is in line with the recently published habituation deficit of the complete knock-out of OGT ortholog in C. elegans (Ardiel, 2018) and shows that altering O-GlcNAc transferase activity is sufficient to induce this deficit. This study thus demonstrates the importance of O-GlcNAcylation in habituation learning (Fenckova, 2022).

Proper development and maintenance of synapses is an important aspect of neuronal function and cognition. The synaptic connection between motor neurons and muscle cells, termed the neuromuscular junction (NMJ), represents an excellent model system to study the molecular mechanisms of synaptic development in Drosophila. Because NMJ defects were found in several Drosophila disease models with defective habituation, this study investigated the synaptic architecture of the sxcH537A/+ larvae. NMJs of the sxcH537A/+ larvae are characterized by an increased number of synaptic boutons, recognizable structures that contain the synaptic vesicles. Larvae with a stronger homozygous catalytic mutation, sxcsxcH596F/H596F, also show an increase in NMJ length, area, and perimeter. It is concluded that O-GlcNAcylation is important for control of synaptic size and synaptic bouton number (Fenckova, 2022).

Recent work has shown that increased protein O-GlcNAcylation in homozygous Oga knockout flies causes a habituation deficit (Muha, 2020). This study shows that heterozygous Oga knockout can restore the habituation deficit of sxcH537A/+ flies. This indicates that habituation learning depends on O-GlcNAc cycling. Because the loss of one Oga allele does not significantly affect total O-GlcNAc levels, it is presumed that subtle changes in O-GlcNAcylation dynamics rather than gross loss of O-GlcNAc transferase activity inhibits habituation learning (Fenckova, 2022).

It is known that postsynaptic expression of OGT in excitatory synapses is important for synapse maturity in mammals (Lagerlof, 2017). This study shows that presynaptic O-GlcNAc transferase also has role in synapse growth. At the NMJ, the synapses of larvae with neuronal overexpression of sxc are shorter, and the number of synaptic boutons is decreased. Both length and bouton number are normalized when sxc is overexpressed in neurons of the sxcH537A/+ larvae. This phenotype was not observed in Oga knockout larvae with increased O-GlcNAcylation. Knockout of Oga can correct the increased bouton number in larvae with sxcH596F mutation but not the NMJ size (Fenckova, 2022).

These data suggest that the NMJ defects associated with decreased O-GlcNAc transferase function are of neuronal origin and that O-GlcNAcylation controls the number of synaptic boutons and partially also synaptic size. Absence of synaptic size defects in Oga knockout larvae and failure of OgaKO to rescue the NMJ size defects caused by decreased O-GlcNAcylation indicates that other, non-catalytic O-GlcNAc transferase functions may be involved in the control of synaptic size. Levine (1918) recently demonstrated that non-catalytic activities of OGT are necessary for its function in some cellular processes, such as proliferation (Fenckova, 2022).

The sxc catalytic hypomorph mutations (sxcH537A, sxcH596F) as well as the OGT-CDG-patient equivalent mutations (R284P, A319T, L254F) that were introduced with the CRISPR/Cas-9 gene-editing technology in the Drosophila sxc gene (sxcR313P, sxcA348T, sxcL283F), lead to an increase in the number of synaptic boutons, and in some cases also to an increase in synaptic size. NMJ size and the number of synaptic boutons ins this model is determined by the level of sxc activity. Dependence of these parameters on gene activity/dosage was previously established in Fmr1 (the Drosophila model of Fragile X Syndrome) and other Drosophila models of neurodevelopmental or neurological disorders, including Prosap/SHANK mutants (modelling Phelan-McDermid Syndrome caused by mutations in SHANK3, characterized by ID and ASD), Neuroligin 4 (ID and ASD caused by mutations in NLGN4), VAP33 (model of Amyotrophic Lateral Sclerosis caused by mutations in VAP-33A) and highwire (potential therapeutical target in traumatic brain injury). The synaptic phenotypes associated with impaired sxc catalytic activity may be linked to increased microtubule polymerization, since it has been shown that O-GlcNAcylation of tubulin negatively regulates microtubule polymerization and neurite outgrowth in mammalian cell lines and Fmr1 and VAP-33A control synaptic growth and bouton expansion through presynaptic organization of microtubules (Fenckova, 2022).

Increased number of synaptic boutons has been also associated with increased excitability at the NMJ although not consistently. An interesting future direction could involve electrophysiological assessment of NMJ activity to determine whether O-GlcNAc cycling and the patient-related sxc mutations go beyond determining synapse development and affect synapse excitability and/or plasticity. However, these investigations would need to test various aspects of physiology and would still leave the impact of O-GlcNAc on cognition undetermined. For this reason, this study assessed habituation as a highly cognition-relevant paramete (Fenckova, 2022).

The effect of OGT-CDG missense mutations on habituation was assessed. It was found that sxcR313P and sxcA348T inhibit habituation in the light-off jump habituation assay. sxcL283F could not be investigated as these mutants displayed a non-performer phenotype in the light-off jump response. While the full spectrum of ID-related phenotypes in an individual with R284P mutation has been attributed to OGT, the A319T mutation segregates with an uncharacterized missense mutation in another gene implicated in ID, MED12 (G1974H). It was not known which of the mutations is responsible for ID in the affected individuals. Evidence is provided that the Drosophila equivalent of the A319T mutation in the TPR domain of OGT causes a cognitive deficit and support a causal role of A319T in OGT-CDG (Fenckova, 2022).

Consistent with no detectable O-GlcNAc changes in patient samples and cellular models of the non-catalytic OGT mutations, no appreciable reduction in protein O-GlcNAcylation was observed in sxcR313P and sxcA348T flies. However, habituation learning was restored by increasing O-GlcNAcylation through blocking Oga activity. This argues that the mechanism by which sxcR313P and sxcA348T inhibit habituation is defective O-GlcNAc transferase activity, paralleling impaired O-GlcNAc transferase activity and significant reduction of protein O-GlcNAcylation demonstrated in the catalytic OGT-CDG mutations. It is worth noticing that it has been previously shown that mutations in Oga also cause habituation deficits. The finding that genetic combination of loss of OGA with loss of OGT activity rescues the cognitive readout argues that OGA inhibition using available inhibitors may represent a viable treatment strategy. The R284P and A319T reside in the TPR domain, which is responsible for recognition and binding of OGT substrates. All OGT-CDG mutations investigated in this study were shown to impair the substrate interaction properties and the glycosyltransferase kinetics. The observed habituation deficits may thus be caused by impaired O-GlcNAcylation dynamics towards a specific set of substrates that cannot be captured by standard O-GlcNAc detection assays. Identification of these substrates may pinpoint the underlying defective mechanisms and additional treatment targets (Fenckova, 2022).

This explorative analysis found that of 43 established O-GlcNAcylated proteins, nine are orthologs of human proteins implicated in ID: ATP1A2, ATN1, HCF1, LAMA2, PTPN23, CHRNA7, NID1, NUP62 and CENPJ. These proteins represent potential downstream effectors and can be investigated in future studies. Particularly the transcriptional co-regulator HCF1 (Host Cell Factor 1) emerges as a top candidate. In mammals, OGT mediates glycosylation and subsequent cleavage of HCF1, which is essential for its maturation. Recombinant OGT with an R284P amino acid substitution is defective in HCF1 glycosylation and HCF1 processing was shown to be completely abrogated by a catalytic OGT-CDG mutation (Fenckova, 2022).

Drosophila sxc is a member of the polycomb group (PcG), a conserved set of chromatin and transcriptional modifiers that initially have been identified by phenotypic similarity of their mutant phenotypes: homeotic transformations. They are required for maintenance of transcriptional repression (of non-lineage genes) during embryonic development and cell proliferation. Missing O-GlcNAcylation of PcG component Polyhomeotic (Ph) is responsible for misexpression of HOX genes and homeotic transformations in sxc null mutants. A recent study has shown that chromatin redistribution induced by interaction between sxc and PcG member Polycomb like (Pcl) controls plasticity of sensory taste neurons. It is not known whether the regulation of PcG activity by sxc/OGT is important for cognitive function, but it can be noted that a series of PcG genes are associated with ID, and some of them are subject to regulation by OGT in the context of development or cancer. These include PHC1 -human ortholog of Drosophila Ph, RING1B (Drosophila Sce), EZH2 (Drosophila E(z))], YY1 (Drosophila pho) and ASXL1 (Drosophila Asx). In addition, OGT regulates expression of PcG genes by O-GlcNAcylation of PcG transcriptional regulators, for example ATN1 (Drosophila Gug), which was identified in the embryonic O-GlcNAc proteome. The encoded proteins represent interesting candidate targets that may link cognitive deficits of OGT-CDG mutations to PcG function (Fenckova, 2022).

It is proposed that in depth clinical phenotyping of patients with mutations in OGT and the above listed genes may give additional hints to the most crucial downstream targets of OGT-mediated O-GlcNAcylation (Fenckova, 2022).

In summary, this study shows that OGT-CDG mutations in the TPR domain negatively affect habituation learning in Drosophila via reduced protein O-GlcNAcylation. The data support a causal role of A319T in OGT-CDG and demonstrate that Drosophila habituation can be used to analyze the contribution of OGT mutations to cognitive deficits. This important aspect of ID has to date not been addressed for any of the OGT-CDG mutations. Moreover, the genetic approach points to a key role of O-GlcNAc transferase activity in ID-associated cognitive deficits and identifies blocking O-GlcNAc hydrolysis as a treatment strategy that can ameliorate cognitive deficits in OGT-CDG patients. Thanks to its high-throughput compatibility, the light-off jump habituation assay can be used with high efficiency for future identification of the downstream effectors and novel therapeutic targets for OGT-CDG (Fenckova, 2022).

Hexosamine biosynthetic pathway and O-GlcNAc-processing enzymes regulate daily rhythms in protein O-GlcNAcylation

The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, this study shows that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. Strong correlation was observed between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when flies are subjected to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. This buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, a mathematical model was generated to describe the key factors that regulate daily O-GlcNAcylation rhythm (Liu, 2021).

Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes
The reversible posttranslational O-GlcNAc modification of serine or threonine residues of intracellular proteins is involved in many cellular events from signaling cascades to epigenetic and transcriptional regulation. O-GlcNAcylation is a conserved nutrient-dependent process involving two enzymes, with O-GlcNAc transferase (OGT) adding O-GlcNAc and with O-GlcNAcase (OGA) removing it in a manner that's protein- and context-dependent. O-GlcNAcylation is essential for epigenetic regulation of gene expression through its action on Polycomb and Trithorax and COMPASS complexes. However, the important role of O-GlcNAc in adult life and health span has been largely unexplored, mainly due the lack of available model systems. Cataloging the O-GlcNAc proteome has proven useful in understanding the biology of this modification in vivo. In this study, a recently developed oga knockout fly mutant was leveraged to identify the O-GlcNAcylated proteins in adult Drosophila melanogaster. The adult O-GlcNAc proteome revealed many proteins related to cell and organismal growth, development, differentiation, and epigenetics. Many O-GlcNAcylated proteins were identified that play a role in increased growth and decreased longevity, including HCF, SIN3A, LOLA, KISMET, ATX2, SHOT, and FOXO. Interestingly, oga mutant flies are larger and have a shorter life span compared to wild type flies, suggesting increased O-GlcNAc results in increased growth. These results suggest that O-GlcNAc alters the function of many proteins related to transcription, epigenetic modification and signaling pathways that regulate growth rate and longevity. Therefore, these findings highlight the importance of O-GlcNAc in growth and life span in adult Drosophila (Akan, 2021).

O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity

Pathological TDP-43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP); however, how TDP-43 aggregation and function are regulated remain poorly understood. This study shows that O-GlcNAc transferase OGT-mediated O-GlcNAcylation of TDP-43 suppresses ALS-associated proteinopathies and promotes TDP-43's splicing function. Biochemical and cell-based assays indicate that OGT's catalytic activity suppresses TDP-43 aggregation and hyperphosphorylation, whereas abolishment of TDP-43 O-GlcNAcylation impairs its RNA splicing activity. This study further showed that TDP-43 mutations in the O-GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP-43 overexpression in Drosophila motor neurons. This study demonstrates that O-GlcNAcylation of TDP-43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. These findings suggest that O-GlcNAcylation might be a target for the treatment of TDP-43-linked pathogenesis (Zhao, 2021).

OGT Protein Interaction Network (OGT-PIN): A Curated Database of Experimentally Identified Interaction Proteins of OGT>

Interactions between proteins are essential to any cellular process and constitute the basis for molecular networks that determine the functional state of a cell. With the technical advances in recent years, an astonishingly high number of protein-protein interactions has been revealed. However, the interactome of O-linked N-acetylglucosamine transferase (OGT), the sole enzyme adding the O-linked β-N-acetylglucosamine (O-GlcNAc) onto its target proteins, has been largely undefined. To that end, this study collated OGT interaction proteins experimentally identified in the past several decades. Rigorous curation of datasets from public repositories and O-GlcNAc-focused publications led to the identification of up to 929 high-stringency OGT interactors from multiple species studied (including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, and others). Among them, 784 human proteins were found to be interactors of human OGT. Moreover, these proteins spanned a very diverse range of functional classes (e.g., DNA repair, RNA metabolism, translational regulation, and cell cycle), with significant enrichment in regulating transcription and (co)translation. This dataset demonstrates that OGT is likely a hub protein in cells. A webserver OGT-Protein Interaction Network (OGT-PIN) has also been created, which is freely accessible (Ma, 2021).

Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis
Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. This study shows that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. A feedback mechanism was discovered involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). These findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer (Na, 2020).

O-GlcNAcase contributes to cognitive function in Drosophila

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, catalytically inactive and precise knock-out Oga alleles (OgaD133N and OgaKO), respectively) were generated in Drosophila melanogaster. Adult OgaD133N) and OgaKO) flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionary conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, this study reports behavioral and neurodevelopmental phenotypes associated with Oga alleles and shows that Oga contributes to cognition and synaptic morphology in Drosophila (Muha, 2020).

Protein O-GlcNAcylation, a dynamic modification of proteins with GlcNAc on serine/threonine residues, is orchestrated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation maintains cellular homeostasis by modulating translation, protein stability, and subcellular localization of proteins. Furthermore, it plays a key role in regulating transcription and differentiation. Although the mechanism of O-GlcNAcylation is highly evolutionarily conserved, from the early metazoan Trichoplax adhaerens to humans, there are considerable differences in the extent vertebrates and invertebrates tolerate alteration in protein O-GlcNAcylation (Muha, 2020).

OGA, the enzyme that removes the O-GlcNAc modification, is the product of the MGEA5 (meningioma-expressed antigen 5) gene in vertebrates. OGA is indispensable for late embryonic development and postnatal survival of mammals. Mouse pups lacking OGA protein show delayed development, small size, abnormality in lung histology, and perinatal lethality. Drosophila Oga null mutants, however, develop normally to adulthood (Radermacher, 2014; Akan, 2016), making Drosophila an attractive system for uncovering previously unappreciated roles of OGA (Muha, 2020).

A substantial body of evidence indicates that O-GlcNAcylation is crucial for normal development and function of the mammalian nervous system. In mice, increased O-GlcNAcylation induced by a brain-specific knockout of OGA manifested in a delay in brain development, reduced olfactory bulb size, missing anterior pituitary, and enlarged brain ventricles and revealed that OGA is required for neurogenesis. It has recently been established that certain mutations in the human OGT gene cause intellectual disability. The mutations are associated with reduced OGA mRNA and protein levels, suggesting that altered OGA expression may contribute to the diverse developmental and cognitive symptoms in these patients. Furthermore, recently identified SNPs in the intronic sequence of OGA have associated the gene with IQ and intellectual development, together indicating a role for OGA in human cognition (Muha, 2020).

Recent studies have demonstrated that both acute and chronic increases of protein O-GlcNAcylation cause hippocampus-associated learning and memory defects in mice and rats. Despite these studies suggesting a crucial function of OGA in normal learning, knowledge about how OGA affects cognitive ability is limited. Therefore, this study investigated whether the role of OGA in learning is conserved in Drosophila (Muha, 2020).

OGA is a multidomain protein; it consists of an N-terminal O-GlcNAc hydrolase catalytic domain that belongs to the GH84 family of glycoside hydrolases, a middle highly disordered 'stalk' domain, and a C terminus with sequence homology to histone acetyltransferases. The histone acetyltransferase domain lacks key amino acids responsible for acetyl-CoA binding; thus OGA only exhibits O-GlcNAcase enzymatic activity. However, it has never been investigated before whether OGA possesses any nonenzymatic roles. Therefore, tools were developed to dissect enzymatic or nonenzymatic functions of Oga in normal neuronal development and cognition/learning (Muha, 2020).

Rationally designed catalytically inactive Oga (OgaD133N) and novel Oga knockout (OgaKO) alleles were developed by exploiting the CRISPR/Cas9 gene editing toolbox, resulting in elevated levels of protein O-GlcNAcylation in homozygous flies. It was discovered that a loss of O-GlcNAcase activity affects locomotion and causes deficits in habituation learning, thereby demonstrating a conserved role of Drosophila Oga in cognitive function. Additionally, this study showed that synaptic bouton counts at the larval neuromuscular junctions are altered in OgaKO flies, indicating a novel role for Oga in synaptic development. Phenotypic characterization of OgaD133N and OgaKO lines also revealed that the primary role of Oga in these processes is O-GlcNAcase enzyme activity (Muha, 2020).

Earlier studies have uncovered a link between O-GlcNAcase and learning in mouse and rat models. Heterozygous Oga+/- mice with increased O-GlcNAc levels exhibited hippocampal-dependent spatial learning and memory defects, whereas rats treated with an OGA inhibitor, thiamet-G, showed reduced performance in novel object and placement tests. These learning phenotypes were associated with dysregulation of synaptic plasticity, long-term synaptic potentiation, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-dependent long-term synaptic depression. Several O-GlcNAc-modified proteins were found that operate at the mammalian synapse, such as Bassoon, Piccolo, and Synapsin; regulate transcriptional programs relevant to synaptic plasticity in neurons, such as the cAMP-response element binding protein (CREB); control neuronal microtubule dynamics, such as Tau and CRMP2; or mediate synaptic transmission, such as the AMPAR Glu2 subunit. O-GlcNAcylation on these and other proteins together potentially modulates neuronal functions. Although the molecular mechanism behind these learning and synaptic phenotypes are not fully understood, current knowledge indicates that synergic response of multiple voltage-gated ion channels (58) and dysregulation of AMPAR are involved (Muha, 2020).

In contrast to mammalian organisms where MGEA5/OGA is crucial for embryonic development, Oga is not essential in Drosophila melanogaster and Caenorhabditis elegans. However, the fact that the Oga gene is conserved across invertebrates suggests that maintenance of homeostatic O-GlcNAc levels by OGA may provide a considerable advantage to Metazoa. Previous work has shown that knockdown of Oga in the fly leads to altered metabolism through effects on insulin-producing cells. Some of the phenotypes, for example the life-span effects that were observed in this study, could be a manifestation of this. However, OgaKO and OgaD133N mutations led to deficits in habituation, highlighting that the role of OGA in learning is evolutionarily conserved. The data also provide evidence that the Drosophila nervous system is sensitive to an increase of the level of O-GlcNAcylation and absence of OGA, establishing it as a suitable genetic model system to study underlying mechanisms and substrates involved (Muha, 2020).

It has been reported previously that increased protein O-GlcNAcylation caused impaired synaptic plasticity in Oga+/- mice without affecting dendritic spine density in CA1 pyramidal neurons. This study reports that bouton number of the larval NMJ is affected in OgaKO null mutants, showing that synaptic morphology is altered in OgaKO animals (Muha, 2020).

Although OgaD133N and OgaKO caused changes in protein O-GlcNAcylation to the same extent, this study described behavioral and neuronal phenotypes that manifested to a different degree in the two Oga lines. Reduction of total daily activity and an increase in NMJ bouton number was only apparent in OgaKO, whereas this genotype appeared to exhibit less severe habituation deficits. A possible explanation of this lies in the choice of inactivating mutation. Previous work in mammalian and bacterial O-GlcNAcases has shown that the equivalent of the D133N mutation inactivates the enzyme. However, recent work has uncovered that this mutation does not lose the ability to bind O-GlcNAc proteins; indeed this inactive mutant can be used to enrich the O-GlcNAc proteome. Therefore, it is possible that the D133N mutation contributes to the stronger habituation phenotype by binding to (parts of) the O-GlcNAc proteome in the fly, interfering with O-GlcNAc signaling/sites. Thus the OgaD133N potentially behaves as a neomorphic allele. It is possible that the absence or presence of the Oga protein affects the phenotypes. For example, the reduction in daily activity and an increase in NMJ bouton number specific for the OgaKO allele might emerge as a combined effect of lack of Oga activity and absence of the Oga protein. The results thus suggest that such additional functions could modulate the phenotypes arising from a complete loss of O-GlcNAcase activity (Muha, 2020).

In summary, this study has shown that Oga regulates O-GlcNAc homeostasis, thus influencing life span, locomotor, and neuronal performance in D. melanogaster. Further studies are required to define the mechanisms downstream of Oga that affect neuronal development or function, resulting in synaptic morphology and habituation learning defects (Muha, 2020).

High dietary sugar reshapes sweet taste to promote feeding behavior in Drosophila melanogaster

Recent studies find that sugar tastes less intense to humans with obesity, but whether this sensory change is a cause or a consequence of obesity is unclear. To tackle this question, the effects of a high sugar diet on sweet taste sensation and feeding behavior were studied in Drosophila melanogaster. On this diet, fruit flies have lower taste responses to sweet stimuli, overconsume food, and develop obesity. Excess dietary sugar, but not obesity or dietary sweetness alone, caused taste deficits and overeating via the cell-autonomous action of the sugar sensor O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in the sweet-sensing neurons. Correcting taste deficits by manipulating the excitability of the sweet gustatory neurons or the levels of OGT protected animals from diet-induced obesity. This work demonstrates that the reshaping of sweet taste sensation by excess dietary sugar drives obesity and highlights the role of glucose metabolism in neural activity and behavior (May, 2019).

O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix

The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, a protein microarray assay was developed that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing simultaneous assessment of OGT activity across >6000 human proteins. With this assay the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain was examined. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. It is conclude dthat OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain (Levine, 2018).

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, ChIP-chip and microarray analysis were performed after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, a genome-wide increase in the intensity of most O-GlcNAc-occupied regions was seen, including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. A viable null allele of oga (ogadel.1) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. It was found that trithorax (Trx), absent small or homeotic discs 1 (Ash1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga(del.1) mutants. The ogadel.1 mutants displayed altered expression of a distinct set of cell cycle-related genes. These results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1 (Akan, 2016).

Epigenetic regulation of gene expression during development is essential for proper cell fate determination. Epigenetic modifiers act by modifying chromatin and thereby altering chromatin structure. The Polycomb (Pc) repressor and Trithorax (Trx) and Compass activator complexes play major roles in maintaining gene expression profiles required for proper body plan formation. Methylation of several lysine residues of histone 3 are among the best understood epigenetic modifications. The trimethylation of histone 3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) member Enhancer of zeste (E(z)) is a repressive transcription mark. In contrast, histone 3 lysine 4 monomethylation (H3K4me) performed by TRX, histone 3 lysine 36 dimethylation (H3K36me2) by ASH1, and histone 3 lysine 4 trimethylation (H3K4me3) by Compass member SET1 are activating modifications in Drosophila. Pc group member super sex combs (ogt/sxc) encodes the Drosophila O-GlcNAc transferase (OGT), which regulates Pc-mediated repression by post translationally O-GlcNAcylating and stabilizing Polyhomeotic (Ph), a member of PRC1 in Drosophila. Ph forms large protein aggregates and cannot function in ogt mutants, leading to homeotic defects. Moreover, it has been shown that knockdown of mammalian OGT decreases H3K27me3 levels by affecting the stability of E(z) homolog 2 (EZH2) in the MCF7 breast cancer cell line (Akan, 2016).

The hexosamine biosynthetic pathway generates UDP-GlcNAc using glucose, glutamine, acetyl-CoA, and UTP. Therefore changes in the intracellular levels of these nutrition-derived products directly influence the cellular concentration of UDP-GlcNAc making it sensitive to nutrient levels. OGT then catalyzes the addition of O-GlcNAc onto hydroxyl groups of serine/threonine residues of proteins using the nutrient sensor UDP-GlcNAc as a substrate. The O-GlcNAc modification on nucleocytoplasmic proteins is then removed by the enzyme O-GlcNAcase (OGA) in a dynamic fashion, modulating intracellular events ranging from transcription to cell cycle regulation. O-GlcNAcylation, like phosphorylation, can impact protein function, localization, and/or expression levels. The singularity of the OGT and OGA enzymes, the rapidity with which O-GlcNAc cycles, and the diversity of protein substrates poises this post-translational modification to play a critical role in modulating the rapid cellular changes required for proper development (Akan, 2016).

O-GlcNAc was first detected on Drosophila polytene chromosomes and later found at the promoter regions of Caenorhabditis elegans genes that are involved in a wide variety of pathways ranging from metabolism to aging. In addition to its role in Pc repression, OGT is thought to have additional roles in epigenetic regulation in mammals. First, OGT can directly O-GlcNAcylate chromatin remodelers like Sin3A and SET1DA. Although controversial, OGT is argued to directly O-GlcNAc modify histone 2B, thereby altering chromatin structure. Beyond affecting gene expression through directly or indirectly changing chromatin, O-GlcNAc influences transcription by affecting the activity and/or stability of key players including RNA polymerase II (RNA Pol II) and many transcription factors. Furthermore, O-GlcNAc is known to play a role in cell cycle progression with the transcriptional co-regulator host cell factor 1 (HCF1) having been identified as an OGT target. Indeed, HCF1 needs to be O-GlcNAcylated and cleaved by OGT to regulate cell division. O-GlcNAcylation plays a key role in mitosis as overexpression of OGT or inhibition of OGA impairs cell cycle progression. Last, the O-GlcNAc modification of histones in a cell cycle-dependent manner may prime this post-translational modification to influence cell cycle and gene expression (Akan, 2016).

To better understand how O-GlcNAc cycling influences gene expression and which genomic regions are more susceptible to changing O-GlcNAc levels, O-GlcNAc levels were altered by knocking down either OGT or OGA expression by RNAi and performed ChIP-chip for O-GlcNAc and other chromatin associated factors followed by gene expression analysis in Drosophila Schneider 2 (S2) cells. An indicator of active transcription, phosphorylated serine 2 on the carboxyl-terminal tail of RNA polymerase II (RNA Pol II Ser2P) was generally at low levels at sites of O-GlcNAc modified chromatin including the Pho-enriched Hox gene clusters suggesting that the O-GlcNAc modification is mainly associated with transcriptionally silent regions. Interestingly, O-GlcNAc occupies additional sites on chromatin other than Pho co-occupied sites. A number of these Pho independent O-GlcNAc occupied chromatin regions were shared with RNA Pol II Ser2P underscoring that O-GlcNAc plays a role in active transcription as well. Gene expression profiling of these cells revealed that O-GlcNAc levels most significantly affect pathways including cell cycle and metabolism. Intrigued by the presence of O-GlcNAc on transcriptionally silent and active chromatin regions, the consequences of a permanent increase in O-GlcNAc levels in the whole animal were studied by generating a null allele of oga in Drosophila (ogadel.1). In ogadel.1 mutant animals O-GlcNAc cycling on chromatin was globally perturbed when visualized on polytene chromosomes. It was determined that Trithorax members TRX and ASH1, and Compass member SET1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. Furthermore, expression of specific cell cycle-related genes, including host cell factor, were altered in oga mutant ovaries. These findings directly demonstrate that O-GlcNAc cycling is an important part of the epigenetic machinery in Drosophila (Akan, 2016).

Disturbances in proper O-GlcNAcylation can result in defects in embryonic development and complications including tumor formation and insulin resistance. By ChIP-chip analysis, several key features of O-GlcNAc chromatin occupancy were noted upon disruption of either OGT or OGA expression in Drosophila S2 cells. ∼8000 regions of the Drosophila genome were observed where O-GlcNAc resides with a significant overlap with RNA Pol II Ser2P. The bulk of these active regions have a single interval associated with each gene, often at promoters. Over 7500 of these intervals increase in intensity (average 1.4-fold) upon OGA knockdown. A much smaller subset (∼500 genes) either slightly decrease or stay constant upon OGA depletion (ratio of 0.5-1.0). Hox genes and other transcriptional regulators are highly over-represented in these genes that change little in response to OGA knockdown. These are also the major sites of Polycomb repression in Drosophila. The finding that these chromatin regions are insensitive to OGA loss of function is consistent with a model in which these domains are normally unavailable to OGA because of chromatin structure or the lack of a histone signature to recruit OGA to those regions. Intriguingly, the genes showing most dramatic elevation of O-GlcNAc chromatin occupancy upon OGA depletion were those associated with protein degradation (ubiquitin), and rapid gene activation (steroid hormone response) (Akan, 2016).

Whereas it was expected that a significant number of gene expression changes would be seen by whole genome microarray based on the overlap between O-GlcNAc and RNA Pol II Ser2P, only few changes were seen upon OGA knockdown in S2 cells. The knockdown of OGA was demonstrated to be highly efficient although the possibility that it did not reach the threshold required for major changes in gene expression could not be excluded. The knockdown did achieve elevation of total O-GlcNAc levels and increase in O-GlcNAc occupancy at sites across the genome. The minor changes in gene expression after OGA knockdown raises the possibility that a subset of genes may be deregulated in a specialized cell type, or in a tissue that is sensitive to changes in O-GlcNAc levels. Alternatively, redundant bypass mechanisms may act to limit the impact on transcription. It should also be noted that loss of OGA had a more modest effect on transcription than loss of OGT in a previous analysis in C. elegans. Both Ogt and Oga are essential for normal development in mice, and ogt is required for proper body development in Drosophila. It was hypothesized that an oga knock-out in Drosophila would have similar homeotic defects. Surprisinglu, oga knock-out flies are viable with no visible homeotic transformations (Akan, 2016).

Phenotypic analysis suggested that Pc repression is not disturbed in oga mutants. Polytene chromosomes isolated from WT, ogtsxc, and oga mutants were used to further examine the association of O-GlcNAc with polycomb and other chromatin modifiers. Polytenes from WT flies exhibited a banding pattern largely coincident with Pc and Pho; no O-GlcNAc-specific bands were detectable in polytenes prepared from ogtsxc. oga deletion flies showed a polytene banding pattern with many more bands with higher overall intensity. These new bands were in addition to those seen in wild type. These new bands appearing on polytenes derived from oga deletion flies were shown to significantly overlap with members of the Trithorax group (TRX, ASH1) and Compass group (SET1) of transcriptional activators. Interestingly, only a subset of the sites stained positive for TRX, ASH1, or SET1 were also stained positive for O-GlcNAc, suggesting that a subset of their target genes were sensitive to O-GlcNAc levels. Then, whether epigenetic activators in the TRX or Compass complexes were O-GlcNAc modified was examined. Ovary tissue was selected for use in these experiments as Drosophila ovaries have a high number of germ cells that are constantly differentiating from a stem cell to a mature egg making them ideal to study epigenetic machinery. Indeed, it was found that TRX, ASH1, and SET1 are O-GlcNAcylated in ogadel.1 ovaries. However, there were no global changes in the levels of histone modifications these enzymes perform. It was then asked whether this would result in any gene expression changes; a small set of genes whose promoters were highly O-GlcNAc modified in ChIP-chip analysis of S2 cells was selected to analyze. Of the eight genes analyzed, only the expression of HCF and embargoed were changed in ogadel.1 mutant ovaries supporting the hypothesis that the expression of a specific subset of genes in a specific cell or tissue type are likely to be affected by irreversible O-GlcNAc modification of TRX, ASH1, or SET1. HCF, which is necessary for proper cell cycle progression, interacts with both Pc and Trx groups in Drosophila. Moreover, SET1 activity is regulated by HCF1 in mammals and HCF1 activity is regulated by O-GlcNAcylation. In light of these findings, it is speculated that irreversible O-GlcNAcylation of SET1 and increased expression of HCF could alter cell cycle progression in a specific cell type in ogadel.1 mutants (Akan, 2016).

Regulation of gene expression by Pc group is different in male and female germlines in Drosophila. Recent work identified that PRC2 appears to control oogenesis by regulating the expression of cell cycle genes, whereas PRC1 members control sperm development. Similar to sex-specific germline regulation identified with the Pc complexes, TRX, ASH1, and SET1 could also display gender-specific phenotypes. Some evidence suggests that such regulation exists. For example, knockdown of SET1 causes an oogenesis defect, but does not affect neuronal development. It will be interesting to see whether O-GlcNAc plays a role in gender-specific or tissue-specific gene expression. Support for this idea comes from a study showing that OGT plays a critical role in neonatal epigenetic programming (Akan, 2016).

The mammalian and fly OGA molecules show high sequence similarity and are likely to perform similar functions. Mammalian OGA (MGEA5) is suggested to play an O-GlcNAc independent role in activation of gene expression in that the pseudo-histone acetyltransferase domain of MGEA5 may play a role in gene activation. The deletion in ogadel.1 mutants spans the O-GlcNAcase catalytic domain but leaves the C-terminal pseudo-histone acetyltransferase domain intact. The fly system therefore should provide a valuable platform to examine the roles of the domains of OGA in performing its many functions (Akan, 2016).

This study introduces viable ogadel.1 mutant flies as a valuable tool to study in vivo effects of increased O-GlcNAc levels. These mutants have greatly enhanced levels of chromatin-associated O-GlcNAc. Previous work has shown that RNA Pol II and other chromatin factors are substrates for O-GlcNAc on chromatin. This study has shown that increased O-GlcNAc levels correlate with impairment of epigenetic modifications. It was shown that Trx members ASH1 and TRX, and Compass member SET1 histone methyltransferases are O-GlcNAc modified in ovaries, and it is speculated that their stability or functions may be altered when they are irreversibly O-GlcNAcylated, which could ultimately change the expression of a subset of their target genes. The large number of potential O-GlcNAc targets on chromatin, and their increased modification upon interfering with O-GlcNAc cycling suggests a more general role for O-GlcNAcylation in stabilizing and activating epigenetic effectors (Akan, 2016).

Diet-induced podocyte dysfunction in Drosophila and Mammals

Diabetic nephropathy is a major cause of end-stage kidney disease. Characterized by progressive microvascular disease, most efforts have focused on injury to the glomerular endothelium. Recent work has suggested a role for the podocyte, a highly specialized component of the glomerular filtration barrier. This study demonstrates that the Drosophila nephrocyte, a cell analogous to the mammalian podocyte, displays defects that phenocopy aspects of diabetic nephropathy in animals fed chronic high dietary sucrose. Through functional studies, an OGT-Polycomb-Knot-Sns pathway was identified that links dietary sucrose to loss of the Nephrin ortholog Sticks and stones (Sns). Reducing OGT through genetic or drug means is sufficient to rescue loss of Sns, leading to overall extension of lifespan. Upregulation of the Knot ortholog EBF2 is demonstrated in glomeruli of human diabetic nephropathy patients and a mouse ob/ob diabetes model. Furthermore, rescue was demonstrated of Nephrin expression and cell viability in ebf2-/- primary podocytes cultured in high glucose. Therefore, this study provides evidence for a pathway that includes flux through the hexosamine biosynthetic pathway and the Polycomb gene complex, which in turn regulates the transcription factor Knot to regulate Sns expression. In cultured mouse primary podocytes, the Knot ortholog EBF2 similarly mediated response by Nephrin to high dietary sucrose. Finally, it was demonstrated how a chemical inhibitor of hexosamine flux can improve the whole-animal response to high dietary sucrose, providing a guideline for candidate therapeutics (Na, 2015).

Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase

Cells allocate substantial resources toward monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. This study reports a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT; termed super sex combs by FlyBase), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. The mitochondrial motor-adaptor protein Milton was established as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. The GlcNAcylation state of Milton was found to be altered by extracellular glucose, and OGT was found to alter mitochondrial motility in vivo. These findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability (Pekkurnaz, 2014).

O-GlcNAcylation prevents aggregation of the Polycomb group repressor polyhomeotic

The glycosyltransferase Ogt adds O-linked N-Acetylglucosamine (O-GlcNAc) moieties to nuclear and cytosolic proteins. Drosophila embryos lacking Ogt protein arrest development with a remarkably specific Polycomb phenotype, arising from the failure to repress Polycomb target genes. The Polycomb protein Polyhomeotic (Ph), an Ogt substrate, forms large aggregates in the absence of O-GlcNAcylation both in vivo and in vitro. O-GlcNAcylation of a serine/threonine (S/T) stretch in Ph is critical to prevent nonproductive aggregation of both Drosophila and human Ph via their C-terminal sterile alpha motif (SAM) domains in vitro. Full Ph repressor activity in vivo requires both the SAM domain and O-GlcNAcylation of the S/T stretch. Ph mutants lacking the S/T stretch reproduce the phenotype of ogt mutants, suggesting that the S/T stretch in Ph is the key Ogt substrate in Drosophila. It is proposed that O-GlcNAcylation is needed for Ph to form functional, ordered assemblies via its SAM domain (Gambetta, 2014).

A role for O-GlcNAcylation in setting circadian clock speed

Post-translational modifications of one or more central 'clock' proteins, most notably time-of-day-dependent changes in phosphorylation, are critical for setting the pace of circadian (~24 h) clocks. In animals, Period (Per) proteins are the key state variable regulating circadian clock speed and undergo daily changes in abundance and cytoplasmic-nuclear distribution that are partly driven by a complex phosphorylation program. This study identified O-GlcNAcylation (O-GlcNAc) as a critical post-translational modification in circadian regulation that also contributes to setting clock speed. Knockdown or overexpression of Drosophila O-GlcNAc transferase (ogt) in clock cells either shortens or lengthens circadian behavioral rhythms, respectively. The Drosophila Per protein is a direct target of OGT and undergoes daily changes in O-GlcNAcylation, a modification that is mainly observed during the first half of the night, when Per is predominantly located in the cytoplasm. Intriguingly, the timing of when Per translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. These results suggest that O-GlcNAcylation of Per contributes to setting the correct pace of the clock by delaying the timing of Per nuclear entry. In addition, OGT stabilizes Per, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems (E. Y. Kim, 2012).

Circadian clocks operate through negative feedback loops wherein positive elements activate negative elements, which in turn repress the activity of the positive elements until the levels of the negative elements decline, enabling another round of activation by the positive elements. To construct such a type of oscillating system, a lag is required before the negative elements can act to inhibit the positive elements. In Drosophila, the delayed nuclear entry/accumulation of Per contributes to the time delay in feedback repression. A complex web of kinases and phosphatases regulates when in a daily cycle Per participates in repressing Clk-Cyc-mediated transcription by regulating its stability, timing of nuclear entry, and duration in the nucleus. While Dbt is the major kinase driving daily cycles in Per levels, other kinases such as CK2 and GSK-3β/Sgg appear to have preferential effects on regulating the translocation of Per from the cytoplasm to the nucleus. The association of Tim with Per in the cytoplasm not only protects Per from Dbt-mediated degradation, but also enhances, yet is not obligatory for, Per nuclear entry (E. Y. Kim, 2012).

Thus, the regulation of when in a daily cycle Per translocates from the cytoplasm to the nucleus involves numerous factors. This study shows that the timing of Per nuclear entry is more complex than previously thought and identifies O-GlcNAcylation as a critical post-translational modification in setting clock speed (E. Y. Kim, 2012).

This study shows that Per is a direct target of OGT and is modified by O-GlcNAcylation. This modification occurs in a temporally regulated manner, first detected in the early night, peaking in the middle of the night, and declining/disappearing thereafter for the remainder of the Per daily life cycle. At present, it is not clear how O-GlcNAcylation of Per is temporally regulated. Based on studies indicating that expression of either ogt or oga is not under circadian regulation, some other factor(s) might favor O-GlcNAcylation of Per during the first half of the night. For example, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the donor substrate of OGT, is generated from glucose via the hexosamine biosynthetic pathway, indicating that the extent of protein O-GlcNAcylation can be sensitive to nutrient availability. Thus, it is possible that metabolic cues are affecting the activity of OGT in clock cells, leading to rhythmic activity of OGT (E. Y. Kim, 2012).

The physiological significance of O-GlcNAc modification in circadian clock systems was demonstrated by showing that the period of daily activity rhythms is sensitive to the levels of ogt expression in clock-specific cells. Remarkably, the periodicity of behavioral rhythms in ogt knockdown flies is shortened, whereas longer periods are observed in flies overexpressing ogt. This strongly suggests that O-GlcNAcylation of Per is a key variable in setting clock speed. The timing in Per nuclear entry was identified as a key event in the clockworks that is altered by changes in ogt expression in a manner consistent with the changes in overt behavioral rhythms. For example, inhibiting endogenous ogt expression in clock cells advanced the time of Per nuclear entry, likely underlying the shorter behavioral rhythms (E. Y. Kim, 2012).

Alterations in the timing of Per nuclear entry by genetically manipulating ogt levels might also explain the differences in per mRNA abundance cycles. For example, the more rapid nuclear entry of Per in ogt knockdown flies could contribute to the earlier decline in per mRNA levels and its inability to attain maximal peak values. Clearly, the earlier nuclear entry of Per when ogt levels are reduced is not due to an increase in the abundance of Per. Rather, it appears that Per stability is increased by more extensive O-GlyNAcylation. Experiments in S2 cells suggest that OGT has a primary effect on stabilizing Per against DBT-mediated degradation. How this occurs is presently not clear. It is also not established whether OGT-mediated changes in the levels of Per contribute to altering the timing of Per nuclear entry. However, it should be noted that other factors, such as Dbt and Tim, regulate both the stability of Per and its nuclear entry time, so multiple effects of O-GlyNAcylation on both Per levels and nuclear translocation are not unanticipated. Because O-GlyNAcylation of Per is mainly observed when it resides in the cytoplasm, it is proposed that this modification acts as an interval timer to prevent the premature nuclear entry of Per. While future work will be required to better understand the mechanism for how O-GlcNAcylation regulates Per nuclear entry, it is possible that O-GlcNAcylation of Per might attenuate phosphorylation at sites that enhance its translocation to the nucleus. Indeed, there are numerous reports illustrating complex interplays between phosphorylation and O-GlcNAcylation (E. Y. Kim, 2012).

It is intriguing that in plants, Spindly (Spy), which has significant similarity to animal OGT, functions in the same pathways with GIGANTE (GI). Like GI, SPY affects circadian rhythms in Arabidopsis. In loss of spy function mutants, the circadian period in cotyledon movement rhythm is lengthened, whereas overexpressing spy shortens the rhythm. Recent work using cardiomyocytes showed diurnal variations in total protein O-GlcNAcylation and identified Bmal1 as an O-GlcNAcmodified protein in mammals. Together with these results, this implies that O-GlcNAc modification has a conserved role in regulating circadian clock pace (E. Y. Kim, 2012).

Functions of Super sex combs/Ogt orthologs in other species

Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented-animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, behavioural neurogenetic and bioinformatic analyses were used in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. The Caenorhabditis elegans orthologues of Ca(2+)/calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype (i.e. larger mechanosensory responses than wild-type). Overall, this work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates (Ardiel, 2018).

O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix

The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, a protein microarray assay was developed that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing simultaneous assessment of OGT activity across >6000 human proteins. With this assay the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain was examined. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. It is conclude dthat OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain (Levine, 2018).

O-GlcNAc transferase regulates excitatory synapse maturity

Experience-driven synaptic plasticity is believed to underlie adaptive behavior by rearranging the way neuronal circuits process information. Previous work showed that O-GlcNAc transferase (OGT), an enzyme that modifies protein function by attaching beta-N-acetylglucosamine (GlcNAc) to serine and threonine residues of intracellular proteins (O-GlcNAc), regulates food intake by modulating excitatory synaptic function in neurons in the hypothalamus. However, how OGT regulates excitatory synapse function is largely unknown. This study demonstrates that OGT is enriched in the postsynaptic density of excitatory synapses. In the postsynaptic density, O-GlcNAcylation on multiple proteins increased upon neuronal stimulation. Knockout of the OGT gene decreased the synaptic expression of the AMPA receptor GluA2 and GluA3 subunits, but not the GluA1 subunit. The number of opposed excitatory presynaptic terminals was sharply reduced upon postsynaptic knockout of OGT. There were also fewer and less mature dendritic spines on OGT knockout neurons. These data identify OGT as a molecular mechanism that regulates synapse maturity (Lagerlof, 2017).


Search PubMed for articles about Drosophila

Akan, I., Love, D. C., Harwood, K. R., Bond, M. R. and Hanover, J. A. (2016). Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation. J Biol Chem 291(19): 9906-9919. PubMed ID: 26957542

Akan, I., Halim, A., Vakhrushev, S. Y., Clausen, H. and Hanover, J. A. (2021). Drosophila O-GlcNAcase Mutants Reveal an Expanded Glycoproteome and Novel Growth and Longevity Phenotypes. Cells 10(5). PubMed ID: 33925313ƒ

Ardiel, E. L., McDiarmid, T. A., Timbers, T. A., Lee, K. C. Y., Safaei, J., Pelech, S. L. and Rankin, C. H. (2018). Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans. Proc Biol Sci 285(1891). PubMed ID: 30429311

Fenckova, M., Muha, V., Mariappa, D., Catinozzi, M., Czajewski, I., Blok, L. E. R., Ferenbach, A. T., Storkebaum, E., Schenck, A. and van Aalten, D. M. F. (2022). Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila. PLoS Genet 18(5): e1010159. PubMed ID: 35500025

Gambetta, M. C. and Muller, J. (2014). O-GlcNAcylation prevents aggregation of the Polycomb group repressor polyhomeotic. Dev Cell 31: 629-639. PubMed ID: 25468754

Kim, E. Y., Jeong, E. H., Park, S., Jeong, H. J., Edery, I. and Cho, J. W. (2012). A role for O-GlcNAcylation in setting circadian clock speed. Genes Dev 26(5): 490-502. PubMed ID: 22327476

Lagerlof, O., Hart, G. W. and Huganir, R. L. (2017). O-GlcNAc transferase regulates excitatory synapse maturity. Proc Natl Acad Sci U S A 114(7): 1684-1689. PubMed ID: 28143929

Levine, Z. G., Fan, C., Melicher, M. S., Orman, M., Benjamin, T. and Walker, S. (2018). O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix. J Am Chem Soc 140(10): 3510-3513. PubMed ID: 29485866

Ma, J., Hou, C., Li, Y., Chen, S. and Wu, C. (2021). OGT Protein Interaction Network (OGT-PIN): A Curated Database of Experimentally Identified Interaction Proteins of OGT. Int J Mol Sci 22(17). PubMed ID: 34502531

May, C. E., Vaziri, A., Lin, Y. Q., Grushko, O., Khabiri, M., Wang, Q. P., Holme, K. J., Pletcher, S. D., Freddolino, P. L., Neely, G. G. and Dus, M. (2019). High dietary sugar reshapes sweet taste to promote feeding behavior in Drosophila melanogaster. Cell Rep 27(6): 1675-1685.e1677. PubMed ID: 31067455

Muha, V., Fenckova, M., Ferenbach, A. T., Catinozzi, M., Eidhof, I., Storkebaum, E., Schenck, A. and van Aalten, D. M. F. (2020). O-GlcNAcase contributes to cognitive function in Drosophila. J Biol Chem 295(26): 8636-8646. PubMed ID: 32094227

Na, H. J., Akan, I., Abramowitz, L. K. and Hanover, J. A. (2020). Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis. Cell Rep 31(6): 107632. PubMed ID: 32402277

Na, J., Sweetwyne, M. T., Park, A. S., Susztak, K. and Cagan, R. L. (2015). Diet-induced podocyte dysfunction in Drosophila and Mammals. Cell Rep 12: 636-647. PubMed ID: 26190114

Pekkurnaz, G., Trinidad, J. C., Wang, X., Kong, D. and Schwarz, T. L. (2014). Glucose regulates mitochondrial motility via Milton modification by O-GlcNAc transferase. Cell 158: 54-68. PubMed ID: 24995978

Radermacher, P. T., Myachina, F., Bosshardt, F., Pandey, R., Mariappa, D., Muller, H. A. and Lehner, C. F. (2014). O-GlcNAc reports ambient temperature and confers heat resistance on ectotherm development. Proc Natl Acad Sci U S A 111(15): 5592-5597. PubMed ID: 24706800

Wong, K. K. L., Liao, J. Z. and Verheyen, E. M. (2019). A positive feedback loop between Myc and aerobic glycolysis sustains tumor growth in a Drosophila tumor model. Elife 8. PubMed ID: 31259690

Wong, K. K. L., Liu, T. W., Parker, J. M., Sinclair, D. A. R., Chen, Y. Y., Khoo, K. H., Vocadlo, D. J. and Verheyen, E. M. (2020). The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila. Proc Natl Acad Sci U S A 117(4): 2004-2013. PubMed ID: 31932432

Zhao, M. J., Yao, X., Wei, P., Zhao, C., Cheng, M., Zhang, D., Xue, W., He, W. T., Xue, W., Zuo, X., Jiang, L. L., Luo, Z., Song, J., Shu, W. J., Yuan, H. Y., Liang, Y., Sun, H., Zhou, Y., Zhou, Y., Zheng, L., Hu, H. Y., Wang, J. and Du, H. N. (2021). O-GlcNAcylation of TDP-43 suppresses proteinopathies and promotes TDP-43's mRNA splicing activity. EMBO Rep: e51649. PubMed ID: 33855783

Biological Overview

date revised: 14 April 2023

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