Deciphering the role of Hsp31 as a multitasking chaperone
Among different type of protein aggregation, amyloids are biochemically well characterized state of protein aggregation that is commonly associated with a large number of neurodegenerative diseases in mammals and cause heritable traits in Saccharomyces cerevisiae. Among many other neurodegenerative diseases linked with amyloids, Parkinson’s disease is the second most common disorder that is caused by progressive deterioration of dopaminergic neurons in substantia nigra. Cellular stresses such as accumulation of high level of reactive oxygen species, mitochondrial dysfunction and α-syn aggregation lead to toxicity and neuronal cell death in Parkinson’s disease patients. Mutations in certain genes are also involved in the development of a familial form of PD including PARK7 that encodes DJ-1. DJ-1 is a member of ThiJ/DJ-1/PfpI protein superfamily that are the quintessential multitasking or moonlighting protein family as evidenced by their involvement in multiple cellular functions including oxidative stress sensing, protein folding, proteasome degradation, mitochondrial complex stabilization, methylglyoxalase and deglycation enzyme activities. The members of the ThiJ/DJ-1/Pfp1 superfamily appear to have evolved to numerous mechanisms to manage cellular stress. The protein superfamily members are present across the evolutionary spectrum including prokaryotes and the budding yeast, S. cerevisiae, that has four paralogs Hsp31, Hsp32, Hsp33, and Hsp34. Hsp31 consists of 237 amino acids with a MW of 25.5 kDa and forms a homodimer in solution. It possesses the Cys-His-Glu catalytic triad common to ThiJ/DJ-1/PfpI superfamily proteins. Previously, we have shown that Hsp31 possesses chaperone properties with protective effects against α-syn toxicity in yeast. Recently, it is shown that Hsp31 has a methylglyoxalase activity that converts the toxic metabolite methylglyoxal into lactate. Here, we confirmed that Hsp31 is a robust methylglyoxalse that is more potent in activity than its human homolog DJ-1. We demonstrated that Hsp31 chaperone activity to protect the cells from α-syn toxicity is not under the influence of its enzymatic activity or autophagy pathway. Moreover, we confirmed that Hsp31 expression is induced by H2O2 mediated oxidative stress and further showed an increased expression of Hsp31 under α-syn mediated proteotoxic stress. These results establish that Hsp31 molecular chaperone activity is self-sufficient to protect the cells from stress conditions without requiring its enzymatic activities. Another associated class of amyloid aggregation state includes prions, which are self-replicating, misfolded proteins capable of adopting amyloid aggregates in cells. In yeast, [PSI +] prion is the aggregated form of translation termination factor Sup35. Sup35, a translation-termination factor, is one of the original and best-studied prions in yeast. In the present study, we established the role of Hsp31 in preventing Sup35 aggregation both in vivo and in vitro using fluorescence microscopy, flow cytometry and SDD-AGE respectively. In addition, we provide evidence that Hsp31 act early on in the process of protein aggregation as we didn't observe any co-localization of Hsp31 with larger Sup35 prion aggregates. Moreover, Hsp31 transiently prevents prion induction with no significant reduction over a prolonged induction of Sup35 aggregation indicating that Hsp31 acts prior to the formation of larger aggregates. This was further confirmed, as an elevated level of Hsp31 by itself was unable to cure [PSI+] prion with formerly present large aggregates. We establish that Hsp31 inhibit Sup35 [ PSI+] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 inhibits Sup35 aggregates formation and potentiates [PSI+] prion curing by overexpression of Hsp104. The absence of Hsp31 reduces the rate of [PSI+] prion curing by Hsp104 without influencing its ability to rescue the cell by thermotolerance. We also showed that Hsp31 physically interact with Hsp104 and together they prevent Sup35 prion toxicity to greater extends than if they were expressed individually in the yeast. These results elucidate a mechanism of Hsp31 on prion modulation that could have implication in many neurodegenerative diseases. Taken together, the results show that Hsp31 is a stress-inducible protein with chaperone and glyoxylase activity that acts on a wide spectrum of misfolded proteins including α-syn and Sup35. These studies set the stage for further mechanistic insight in the biological roles of the Hsp31/DJ-1 chaperone family.
Hazbun, Purdue University.
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