Biochemistry

In this Letter, we described a shift in lysosome activity and mitochondrial metabolism that triggers the removal of protein aggregates in Caenorhabditis elegans oocytes when their maturation is stimulated by signals from sperm. An early step in this process is the acidification of lysosomes. We postulated that signals from sperm trigger lysosome acidification by stimulating the proteasomal destruction of the GLD-1 translational repressor, which in turn enables the synthesis of lysosomeacidifying V-ATPase subunits (Fig. 3 of our Letter). In support of this conclusion, the GFP::GLD-1-expressing transgene we used to visualize GLD-1 (axIs1498[Ppie-1::gfp::gld-1::gld-1 3'UTR, unc-119(+)]) 1 resulted in GFP expression in proximal oocytes of females (which lack sperm) but not in those of hermaphrodites (which have sperm) (Fig. 3 of our Letter). After our Letter was published, D. Greenstein informed us that this GLD-1 transgene probably produced a misleading GFP signal, because neither a different GLD-1::GFP transgene nor a GLD-1 antibody produced a detectable signal in the proximal oocytes of females (see ref. 2 for data and a discussion of these transgenes). His results prompted us to take an approach that was less likely to generate artefacts; namely, to tag the endogenous gld-1 gene with GFP using CRISPR-Cas9 technology. Using this strain, we were also unable to detect a GLD-1::GFP signal in the proximal oocytes of females (see Fig. 1, Supplementary Fig. 1 and Supplementary Methods of this Addendum). This suggests that the proteasomal activity that degrades GLD-1 in the proximal oocytes of hermaphrodites (further explored by ref. 2) may also degrade GLD-1 in the proximal oocytes of females, and something other than GLD-1 may prevent lysosomes from being acidified in the absence of sperm. This mechanism remains unresolved, and is under investigation in our laboratory. Figure 2 of this Amendment describes our revised model, in which the fundamental conclusions of the study remain intact: signals from sperm trigger the destruction of protein aggregates in female oocytes by stimulating lysosome acidification (see Figs. 1 and 2 of our Letter). Lysosome acidification enables mitochondria to transit from what appears to be a poised state (with a high membrane potential) to an active state (with a lower, hermaphrodite-like membrane potential). This shift in mitochondrial physiology is required for the acidified lysosomes to engulf protein aggregates (Fig. 4 of our Letter), probably by the process of microautophagy (Fig. 2 of our Letter). We thank D. Greenstein for bringing this matter to our attention. Authors J.G. and M.S., both members of the Kenyon laboratory at Calico Life Sciences, carried out the analyses described in this Amendment and

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated with...

Parkinson's disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with 'mitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can "make or break" mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotidebinding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

shows distinct state 2, 3 and 4 respiration, which can be mimicked in E9.5 WT hearts by incubation with 200 nM CsA. By E11.5 mitochondrial oxygen consumption in WT hearts is not significantly different when compared to E11.5 CypD KO mice or E13.5 hearts. In addition, supercomplexes were not detectable in WT hearts of E9.5 and E11.5 embryos by native electrophoresis or immunocapturing. This may explain why NADH-cytochrome c reductase activity is not detectable in WT embryos at E9.5 but in E11.5 or E13.5 WT mice. Furthermore, Cx-1 is in its de-active form at E9.5 and becomes activated around E11.5. Our data suggests that at E11.5 the PTP is closed and Cx-1, Cx-2 and Cx-3 of the ETC are present, but are not yet arranged in supercomplexes and a coordinated electron transfer is still developing.

Background: ThiJ/DJ-1/PfpI protein family members possess glutathione-independent glyoxalase activity. Results: S. cerevisiae Hsp31, an efficient glyoxalase among all paralogs is essential for cytoprotection under oxidative stress conditions. Conclusion: The robust glyoxalase activity of Hsp31 aids in the maintenance of glutathione levels, and its mitochondrial localization provides protection against oxidative stress. Significance: This is the first demonstration of the mechanisms of ThiJ/DJ-1/PfpI family in combating oxidative stress in yeast.

Somatic cells age and die, but the germ-cell lineage is immortal. In C. elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte-maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in ER activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal...

Somatic cells age and die, but the germ-cell lineage is immortal. InCaenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic lin...

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Δpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Mitochondria biogenesis requires the import of several precursor proteins that are synthesized in the cytosol. The mitochondrial heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of the mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 toward mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5 peptide as compared with the other Hsp70s. We observed that the two human mitochondrial matrix J-protein splice variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hsp70 escort protein (Hep) possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C terminus of mtHsp70 in a full-length context and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thus promoting a similar conformational change that leads to ATPase stimulation. Additionally, we highlight the biochemical defects of the mtHsp70 mutant (G489E) associated with a myelodysplastic syndrome.

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Dpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Nature and % of student contribution Co-author name(s) Nature and % of Coauthor's contribution* Coauthor(s), Monash student Y/N* 3 The single disulfidedirected βhairpin fold. dynamics, stability and engineering Accepted 60%. Concept and collecting data and writing first draft 1. Krishnarjuna Bankala, Collecting data, input into manuscript 10% No 2. Rodrigo A. V. Morales, intellectual input, manuscript preparation, input into manuscript 5% No 3.ChristopherA.MacRaild, intellectual input,data analysis, manuscript preparation, input into manuscript 5% No 4. Maiada Sadek, Collecting data, input into manuscript 2% Yes 5. Eleanor W. W. Leung, Collecting data, input into manuscript 2% No 6. Samuel D. Robinson, Collecting data, input into manuscript 2% No 7. Michael W. Pennington, Collecting data, input into manuscript 2 % No 8. Raymond S. Norton, 6.3.1 Identification of contryphan-Vc1 homologous sequences 6.3.2 Disulfide bond is critical for the folding and maintenance of SDH fold 6.3.3 Full-length oyster-Vc1 is unfolded 6.3.4 Analogues contryphan-cg11-22(M9Y, P18I) and contryphan-cg11-22(R4Q, M9Y, R15I, P18I] restore the SDH fold 6.4 Discussion Chapter 7. Conclusions and Future Directions Chapter 8. References Appendix I. A naturally occurring peptide with an elementary single disulfidedirected β-hairpin fold. Appendix II. Structure and activity of contryphan-Vc2: Importance of the D-amino acid residue Peptide Sequence References 40-44

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

Certain peptide folds, owing to a combination of intrinsic stability and resilience to amino acid substitutions, are particularly effective for the display of diverse functional groups. Such "privileged scaffolds" are valuable as starting points for the engineering of new bioactive molecules. We have identified a precursor peptide expressed in the venom gland of the marine snail Conus victoriae, which appears to belong to a hitherto undescribed class of molluscan neuropeptides. Mass spectrometry matching with the venom confirmed the complete mature peptide sequence as a 31-residue peptide with a single disulfide bond. Solution structure determination revealed a unique peptide fold that we have designated the single disulfide-directed β hairpin (SDH). The SDH fold is highly resistant to thermal denaturation and forms the core of several other multiple disulfide-containing peptide folds, including the inhibitor cystine knot. This elementary fold may offer a valuable starting...

In natural proteins and peptides, amino acids exist almost invariably as l-isomers. There are, however, several examples of naturally-occurring peptides containing d-amino acids. In this study we investigated the role of a naturally-occurring d-amino acid in a small peptide identified in the transcriptome of a marine cone snail. This peptide belongs to a family of peptides known as contryphans, all of which contain a single d-amino acid residue. The solution structure of this peptide was solved by NMR, but further investigations with molecular dynamics simulations suggest that its solution behaviour may be more dynamic than suggested by the NMR ensemble. Functional tests in mice uncovered a novel bioactivity, a depressive phenotype that contrasts with the hyperactive phenotypes typically induced by contryphans. Trp3 is important for bioactivity, but this role is independent of the chirality at this position. The d-chirality of Trp3 in this peptide was found to be protective against ...

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed β-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc11-22[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone (15)N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structur...

Nature and % of student contribution Co-author name(s) Nature and % of Coauthor's contribution* Coauthor(s), Monash student Y/N* 3 The single disulfidedirected βhairpin fold. dynamics, stability and engineering Accepted 60%. Concept and collecting data and writing first draft 1. Krishnarjuna Bankala, Collecting data, input into manuscript 10% No 2. Rodrigo A. V. Morales, intellectual input, manuscript preparation, input into manuscript 5% No 3.ChristopherA.MacRaild, intellectual input,data analysis, manuscript preparation, input into manuscript 5% No 4. Maiada Sadek, Collecting data, input into manuscript 2% Yes 5. Eleanor W. W. Leung, Collecting data, input into manuscript 2% No 6. Samuel D. Robinson, Collecting data, input into manuscript 2% No 7. Michael W. Pennington, Collecting data, input into manuscript 2 % No 8. Raymond S. Norton, 6.3.1 Identification of contryphan-Vc1 homologous sequences 6.3.2 Disulfide bond is critical for the folding and maintenance of SDH fold 6.3.3 Full-length oyster-Vc1 is unfolded 6.3.4 Analogues contryphan-cg11-22(M9Y, P18I) and contryphan-cg11-22(R4Q, M9Y, R15I, P18I] restore the SDH fold 6.4 Discussion Chapter 7. Conclusions and Future Directions Chapter 8. References Appendix I. A naturally occurring peptide with an elementary single disulfidedirected β-hairpin fold. Appendix II. Structure and activity of contryphan-Vc2: Importance of the D-amino acid residue Peptide Sequence References 40-44

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy to enhance their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed β-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analog (rCon-Vc1 1-22 [Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone 15 N relaxation measurements for rCon-Vc1[Z1Q] indicate that N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc1 1-22 [Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited remarkable proteolytic stability against trypsin, chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase (iNOS). We show that sCon-Vc1 1-22 [NNN 12-14 ] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 µM while maintaining the SDH fold. This study serves as a starting point in utilising the SDH fold as a promising peptide scaffold.

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock proteins (HSPs), namely, Hsp70, Hsp40, Hsp60, Hsp90, Hsp100 and small HSP. The HSPs are essential for the survival of all living organisms, as they protect the conformations of proteins on exposure to various stress conditions. They are a highly conserved group of proteins involved in diverse physiological functions, including de novo folding, disaggregation and protein trafficking. Moreover, their critical role in the control of disease progression made them a prime target of research. Presently, limited information is available on HSPs in reference to their identification and structural classification across genera. To that extent, HSPIR provides manually curated information on sequence, structure, classification, ontology, domain organization, localization and possible biological functions extracted from UniProt, GenBank, Protein Data Bank and the literature. The database offers interactive search with incorporated tools, which enhances the analysis. HSPIR is a reliable resource for researchers exploring structure, function and evolution of HSPs.