BIOLOGICAL OVERVIEW

A screen for gene mutations that extend life-span in Drosophila melanogaster was performed in order to provide a genetic dissection of the processes involved in aging. The mutant line methuselah (mth) displays approximately 35% increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and food supplemented with paraquat, a free-radical generator. The mth gene encodes a protein with homology to several guanosine triphosphate-binding protein-coupled seven-transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span (Lin, 1998).

The effect of genes on life-span in Drosophila has been established by selective breeding. However, the participation of multiple genes with additive, quantitative effects can be difficult to unravel. A direct search for life-extension mutants could identify individual genes that regulate biological aging. Indeed, in the nematode Caenorhabditis elegans, several mutations, for example, age-1, daf-2, and clk-1, have been described that can increase the worm's life-span (see Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene for related literature). The corresponding genes have been cloned and are involved in various aspects of development and metabolism (Lin, 1998 and references therein).

Life-span and stress response are closely associated. In C. elegans, the age-1 mutant displays elevated resistance to thermal exposure and to oxidative stress. In Drosophila, laboratory stocks selected for postponed senescence also show increased tolerance to heat, starvation, desiccation, and oxidative damage. Tandem overexpression of Cu-Zn superoxide dismutase (SOD) and catalase genes in Drosophila increase life-span by 30% (Orr, 1994). Similar observations were made in flies expressing the human SOD1 transgene in motor neurons (Parkes, 1998). However, the physiological and molecular events involved in life-span determination and stress resistance have remained largely elusive (Lin, 1998 and references therein).

A set of P-element insertion lines were generated and screened for lines that outlived a parent strain. methuselah was isolated by its increase in life-span at 29°C. The life extension was confirmed at 25°C. At both temperatures, flies homozygous for the P-element live, on the average, 35% longer than the parent strain (Lin, 1998).

The ability of mth flies to resist stress was assayed. mth mutant flies are more resistant to dietary paraquat, which, upon intake by the cell, generates superoxide anion. At a concentration of 20 mM, paraquat renders normal males sluggish by 12 hours; at 48 hours, nearly 90% are dead. In contrast, mth males are still active at 24 hours, and at 48 hours more than 50% are still alive. In a long-lived strain of Drosophila derived by selection, life-span extension also accompanies increased paraquat resistance (Arking, 1991). Transgenic Drosophila carrying extra copies of SOD and catalase, two primary components of the defense system against reactive oxygen species, also have increased life-span (Orr, 1994). Flies transgenic for the human SOD1 gene display increased life-span and paraquat resistance, the degree of effect correlating with dosage of the transgene (Parkes, 1998). Thus, mth may have a higher capacity of the free-radical defense system (Lin, 1998).

In the starvation test, mth shows a greater than 50% increase in average survival time over the parent strain. Females are consistently more resistant than males, suggesting that their larger body weight may contribute to resistance. Indeed, mth males and females weighed 20% to 30% more than their w¹¹¹⁸ counterparts. In a Drosophila stock selectively bred for postponed senescence, resistance to starvation and lipid content are higher than the baseline stock. In C. elegans, the mutant daf-2, which exhibits marked increase in longevity, has extensive fat accumulation when grown at 25°C, suggesting a coupling of its metabolism with longevity (Lin, 1998 and references therein).

Exposure to high temperature was tested. At 36°C, mth mutants survived longer than the parent strain. Heat shock proteins, a class of molecular chaperones, are thought to counter stress-induced detrimental effects during aging. In a transgenic fly harboring 12 additional copies of the heat-inducible hsp70 gene, there is a positive correlation between life expectancy and elevated Hsp70 protein expression. Correspondingly, in daf-2 and age-1 mutant worms, resistance to thermal stress is higher than in control animals. The increased thermotolerance of mth may result from higher expression of heat shock proteins and related molecular chaperones (Lin, 1998 and references therein).

Because life-span and stress response are closely related, genetic screening by stress resistance provides an effective alternative to the much slower direct screening for lifetime. The ability of the mth fly to resist various kinds of stress is notable because there are likely to exist differences in pathways of response to individual forms of stress (Lin, 1998).

G protein-coupled receptors are involved in a remarkably diverse array of biological activities including neurotransmission, hormone physiology, drug response, and transduction of stimuli such as light and odorants. The data suggest that Mth is a GPCR involved in stress response and biological aging. By regulating an associated G protein and thus its downstream pathway, the normal mth gene may maintain homeostasis and metabolism, playing a central role in modulating molecular events in response to stress. The pre-adult lethality of the null alleles demonstrates that at least some activity of the mth gene is essential for survival. When mutated, the intermediate level of expression of a hypomorphic allele might adjust response to stress in a way that is more favorable for survival, whereas full expression of the normal gene exceeds the optimum value. The delicate balance among the embryonic lethality of a null allele, enhanced longevity of a hypomorphic allele, and the normal wild phenotype, suggests that the level of mth gene expression is an important component of the system controlling life-span (Lin, 1998).

GENE STRUCTURE

cDNA clone length - 1928

Bases in 5' UTR - 108

Exons - 9

Bases in 3' UTR - 295

PROTEIN STRUCTURE

Amino Acids - 514

Structural Domains

The full-length genomic and complementary DNAs of the mth gene were cloned. The cDNA encodes a single open reading frame. The predicted protein sequence has a leader peptide plus seven hydrophobic regions suggestive of transmembrane (TM) domains. A gapped Blast search of this sequence showed homology to a variety of guanosine triphosphate-binding regulatory protein (G protein)-coupled receptors (GPCRs). GPCR was also predicted by the Blocks Search program. The amino acid residues between TM5 and TM6, especially those near the transmembranes, are highly basic, a feature shared by many G protein-linked receptors, and in some cases these residues interact directly with G proteins. Homology was found mainly in the TM regions. The NH₂-terminal segment preceding the first TM domain was not found to share homology with any known sequence, thus diminishing the overall homology scores. The mth gene appears to represent a previously unknown member of the seven-TM protein superfamily. It remains to be seen whether the unique NH₂-terminal sequence is related to the regulation of the MTH protein, and what the identity of its ligand (or ligands) might be (Lin, 1998).

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that to extend average lifespan. Mth mutants show a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. The 2.3-Å resolution crystal structure of the Mth extracellular region is reported, revealing a folding topology in which three primarily ß-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth ectodomain has a relatively compact, ß-sheet-rich fold with five disulfide bonds that does not closely resemble that of any protein of known structure (i.e., no similar folds were found by using the DALI or VAST database search programs). The structure can be divided into three domains. The first domain (D1; residues 1-60) and third domain (D3; residues 123-188) share a similar fold consisting of two three-stranded ß-sheets, whereas the middle domain (D2, residues 61-122), which contains three two-stranded ß-sheets and a short alpha-helix, wraps around the third domain to form a superdomain (D2D3). The D3 portion of the D2D3 superdomain shares a similar folding topology and several common structural features with D1. First, each domain includes two disulfide-linked cysteines at analogous positions (Cys-5 and Cys-59 in D1 and Cys-126 and Cys-185 in D3) that connect the beginning and end of each domain. Second, two of the five potential N-linked glycosylation sites occur at corresponding positions in D1 and D3 (Asn-21 and Asn-146) and ordered carbohydrate is visible at both sites. Although D1 and D3 share these structural features, they are related by <10% sequence identity (based on a structure-based sequence alignment) and superimpose well only over the S2-S3-S4 sheet. One significant difference between D1 and D3 is that the long S5-S6 ß-stand pair in D3 is broken into two segments in D1 (near residues 40 and 53), which causes these strands to bend in such a way as to make contact with the D2D3 superdomain (West, 2001).

Ten homologs of Mth have been predicted from the Drosophila genome (Brody, 2000). Most of these are organized in the same manner as Mth: an NH2-terminal ectodomain followed by a seven-pass transmembrane domain. The NH2-terminal domains of the Mth-related proteins share between 27% and 65% sequence identity with the Mth ectodomain, suggesting that they will fold into similar tertiary structures. The 10 cysteines that form five disulfide bonds in Mth are conserved in all but one Mth homolog (Mth-like 3, in which a leucine substitutes for the cysteine in the strand S5 region of D3). Several of the N-linked carbohydrate sites also are conserved in many of the Mth-related proteins, particularly the analogous sites in D1 and D3 located between strands S2 and S3. A majority of the conserved noncysteine residues are in D1, likely reflecting the greater constraints required to maintain the fold of this small domain. In addition, some of the interdomain interactions that stabilize the Mth fold are conserved in the Mth homologs. For example, conserved residues (D2 Asp-121, D1 His-55, and D1 Thr-36) surround and form hydrogen bonds to D1 Arg-57. The interaction between the side chains of Arg-57 and Asp-121 is one of the most significant between D1 and D2D3, and conservation of this interaction suggests that D1 and D2D2 will have a common arrangement in the Mth homologs. Trp-120 is conserved in only one of the Mth homologs (Mth-like 2), thus the proposal that this residue forms part of the ligand binding site implies that the Mth homologs bind different ligands (West, 2001).

In addition to a ligand interaction surface, another region of the Mth ectodomain that may be functionally important is the region contacting the extracellular face of the seven-pass transmembrane domain, potentially in such a way as to signal ligand binding. In the recently determined crystal structure of the GPCR rhodopsin, the extracellular NH2-terminal region (residues 1-35) makes contact with all three of the extracellular interhelical loops, thus the Mth ectodomain also may contact loops between transmembrane helices. Because there are only seven residues between the C terminus of the Mth ectodomain observed in the structure and the predicted beginning of the first hydrophobic transmembrane region, the region near the Mth ectodomain C terminus has the potential to interact with loop regions between the transmembrane regions. There are three loops in this region: between strands S3 and S4 in D3 (including Phe-153 and conserved residue Asp-154), between S5' and S6' in D1 (including conserved residue Asp-46), and preceding S1 in D3 (around Gly-128). The occurrence of conserved residues within these loops suggests that these regions may be involved in positioning the ectodomain on the transmembrane region and/or signaling ligand binding to the transmembrane region. When the region of Mth containing these loops and the C terminus of the Mth ectodomain is oriented on a model of the seven-pass transmembrane portion, the ectodomain is positioned such that the interdomain cleft containing Trp-120 is oriented away from the membrane, consistent with the suggestion that it represents a ligand binding site (West, 2001).

date revised: 20 September 2001

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