Functional significance of Hsp70 post-translational modification in prion propagation and cellular function

The term prion (proteinaceous infectious particles) was first coined by Stanley Prusiner while naming the causative agent responsible for a group of invariably fatal neurodegenerative diseases collectively termed transmissible spongiform encephalopathies (TSE). A breakthrough in prion research came with the studies which revealed that yeast species Saccharomyces cerevisiae contains proteins that have the ability to form prions. Sup35 is a S. cerevisiae protein involved in termination of translation. In a prion state referred to as [PSI+], a significant portion of the Sup35 protein in the cell coalesces into non-functional, self-propagating, amyloid-like polymers. Thus, yeast strains that are [PSI+] show increased levels of nonsense suppression. Once present, [PSI+] propagates by recruitment of the soluble form of Sup35 into the aggregate in a manner analogous to that of mammalian prions. A search for genetic factors affecting propagation and maintenance of [PSI+] has identified an essential role for molecular chaperones, namely Hsp70 and Hsp104. The Hsp70 chaperone family and its associated co-chaperones are highly conserved from yeast to mammals. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. In the model eukaryotic S. cerevisiae, to efficiently carry out such functions Hsp70 works in concert with a number of co-chaperones to regulate ATPase hydrolysis cycle of Hsp70, which in-turn dictates the peptide-binding status of Hsp70. While much data has accrued in relation to the ATPase and substrate binding cycles of Hsp70 there is a distinct lack of information regarding the regulation of this important chaperone at the post-translational level. Recent global proteomic studies have demonstrated that in vivo Hsp70 is phosphorylated. Using a simplified yeast system this study systematically assessed a variety of non-phosphorylateable and phosphomimetic Hsp70 mutants for phenotypic alterations in Hsp70 functions. It was found that alteration of Hsp70 phosphorylation status in vivo can impair prion propagation, alter both basal and acquired thermotolerance and in some cases render cells inviable. By looking at analogous mutants in closely related cytosolic Hsp70s this study identified functional similarities and differences between highly homologous Hsp70 species. This study shows a clear link between Hsp70 phosphorylation status and in vivo function. Given Hsp70s central role in a variety of important cellular metabolic pathways and the conservation of these phosphorylatable sites in higher eukaryotes, these findings have far reaching implications

CDK-dependent Hsp70 phosphorylation controls G1 cyclin abundance and cell-cycle progression

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

Rare variants implicate NMDA receptor signaling and cerebellar gene networks in risk for bipolar disorder

Bipolar disorder is an often-severe mental health condition characterized by alternation between extreme mood states of mania and depression. Despite strong heritability and the recent identification of 64 common variant risk loci of small effect, pathophysiological mechanisms remain unknown. Here, we analyzed genome sequences from 41 multiply-affected pedigrees and identified variants in 741 genes with nominally significant linkage or association with bipolar disorder. These 741 genes overlapped known risk genes for neurodevelopmental disorders and clustered within gene networks enriched for synaptic and nuclear functions. The top variant in this analysis - prioritized by statistical association, predicted deleteriousness, and network centrality - was a missense variant in the gene encoding D-amino acid oxidase (DAOG131V). Heterologous expression of DAOG131V in human cells resulted in decreased DAO protein abundance and enzymatic activity. In a knock-in mouse model of DAOG131, DaoG130V/+, we similarly found decreased DAO protein abundance in hindbrain regions, as well as enhanced stress susceptibility and blunted behavioral responses to pharmacological inhibition of N-methyl-D-aspartate receptors (NMDARs). RNA sequencing of cerebellar tissue revealed that DaoG130V resulted in decreased expression of two gene networks that are enriched for synaptic functions and for genes expressed, respectively, in Purkinje neurons or granule neurons. These gene networks were also down-regulated in the cerebellum of patients with bipolar disorder compared to healthy controls and were enriched for additional rare variants associated with bipolar disorder risk. These findings implicate dysregulation of NMDAR signaling and of gene expression in cerebellar neurons in bipolar disorder pathophysiology and provide insight into its genetic architecture

Using steered molecular dynamics to predict and assess Hsp70 substrate-binding domain mutants that alter prion propagation.

Genetic screens using Saccharomyces cerevisiae have identified an array of cytosolic Hsp70 mutants that are impaired in the ability to propagate the yeast [PSI(+)] prion. The best characterized of these mutants is the Ssa1 L483W mutant (so-called SSA1-21), which is located in the substrate-binding domain of the protein. However, biochemical analysis of some of these Hsp70 mutants has so far failed to provide major insight into the specific functional changes in Hsp70 that cause prion impairment. In order to gain a better understanding of the mechanism of Hsp70 impairment of prions we have taken an in silico approach and focused on the Escherichia coli Hsp70 ortholog DnaK. Using steered molecular dynamics simulations (SMD) we demonstrate that DnaK variant L484W (analogous to SSA1-21) is predicted to bind substrate more avidly than wild-type DnaK due to an increase in numbers of hydrogen bonds and hydrophobic interactions between chaperone and peptide. Additionally the presence of the larger tryptophan side chain is predicted to cause a conformational change in the peptide-binding domain that physically impairs substrate dissociation. The DnaK L484W variant in combination with some SSA1-21 phenotypic second-site suppressor mutations exhibits chaperone-substrate interactions that are similar to wild-type protein and this provides a rationale for the phenotypic suppression that is observed. Our computational analysis fits well with previous yeast genetics studies regarding the functionality of the Ssa1-21 protein and provides further evidence suggesting that manipulation of the Hsp70 ATPase cycle to favor the ADP/substrate-bound form impairs prion propagation. Furthermore, we demonstrate how SMD can be used as a computational tool for predicting Hsp70 peptide-binding domain mutants that impair prion propagation

Assessment of Inactivating Stop Codon Mutations in Forty Saccharomyces cerevisiae Strains: Implications for [PSI+] Prion- Mediated Phenotypes

The yeast prion [PSI+] has been implicated in the generation of novel phenotypes by a mechanism involving a reduction in translation fidelity causing readthrough of naturally occurring stop codons. Some [PSI+] associated phenotypes may also be generated due to readthrough of inactivating stop codon mutations (ISCMs). Using next generation sequencing we have sequenced the genomes of two Saccharomyces cerevisiae strains that are commonly used for the study of the yeast [PSI+] prion. We have identified approximately 26,000 and 6,500 single nucleotide polymorphisms (SNPs) in strains 74-D694 and G600 respectively, compared to reference strain S288C. In addition to SNPs that produce non-synonymous amino acid changes we have also identified a number of SNPs that cause potential ISCMs in these strains, one of which we show is associated with a [PSI+]-dependent stress resistance phenotype in strain G600. We identified twenty-two potential ISCMs in strain 74-D694, present in genes involved in a variety of cellular processes including nitrogen metabolism, signal transduction and oxidative stress response. The presence of ISCMs in a subset of these genes provides possible explanations for previously identified [PSI+]-associated phenotypes in this strain. A comparison of ISCMs in strains G600 and 74-D694 with S. cerevisiae strains sequenced as part of the Saccharomyces Genome Resequencing Project (SGRP) shows much variation in the generation of strain-specific ISCMs and suggests this process is possible under complex genetic control. Additionally we have identified a major difference in the abilities of strains G600 and 74-D694 to grow at elevated temperatures. However, this difference appears unrelated to novel SNPs identified in strain 74-D694 present in proteins involved in the heat shock response, but may be attributed to other SNP differences in genes previously identified as playing a role in high temperature growth

Functional significance of Hsp70 post-translational modification in prion propagation and cellular function

The term prion (proteinaceous infectious particles) was first coined by Stanley Prusiner while naming the causative agent responsible for a group of invariably fatal neurodegenerative diseases collectively termed transmissible spongiform encephalopathies (TSE). A breakthrough in prion research came with the studies which revealed that yeast species Saccharomyces cerevisiae contains proteins that have the ability to form prions. Sup35 is a S. cerevisiae protein involved in termination of translation. In a prion state referred to as [PSI+], a significant portion of the Sup35 protein in the cell coalesces into non-functional, self-propagating, amyloid-like polymers. Thus, yeast strains that are [PSI+] show increased levels of nonsense suppression. Once present, [PSI+] propagates by recruitment of the soluble form of Sup35 into the aggregate in a manner analogous to that of mammalian prions. A search for genetic factors affecting propagation and maintenance of [PSI+] has identified an essential role for molecular chaperones, namely Hsp70 and Hsp104. The Hsp70 chaperone family and its associated co-chaperones are highly conserved from yeast to mammals. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. In the model eukaryotic S. cerevisiae, to efficiently carry out such functions Hsp70 works in concert with a number of co-chaperones to regulate ATPase hydrolysis cycle of Hsp70, which in-turn dictates the peptide-binding status of Hsp70. While much data has accrued in relation to the ATPase and substrate binding cycles of Hsp70 there is a distinct lack of information regarding the regulation of this important chaperone at the post-translational level. Recent global proteomic studies have demonstrated that in vivo Hsp70 is phosphorylated. Using a simplified yeast system this study systematically assessed a variety of non-phosphorylateable and phosphomimetic Hsp70 mutants for phenotypic alterations in Hsp70 functions. It was found that alteration of Hsp70 phosphorylation status in vivo can impair prion propagation, alter both basal and acquired thermotolerance and in some cases render cells inviable. By looking at analogous mutants in closely related cytosolic Hsp70s this study identified functional similarities and differences between highly homologous Hsp70 species. This study shows a clear link between Hsp70 phosphorylation status and in vivo function. Given Hsp70s central role in a variety of important cellular metabolic pathways and the conservation of these phosphorylatable sites in higher eukaryotes, these findings have far reaching implications

Functional significance of Hsp70 post-translational modification in prion propagation and cellular function

The term prion (proteinaceous infectious particles) was first coined by Stanley Prusiner while naming the causative agent responsible for a group of invariably fatal neurodegenerative diseases collectively termed transmissible spongiform encephalopathies (TSE). A breakthrough in prion research came with the studies which revealed that yeast species Saccharomyces cerevisiae contains proteins that have the ability to form prions. Sup35 is a S. cerevisiae protein involved in termination of translation. In a prion state referred to as [PSI+], a significant portion of the Sup35 protein in the cell coalesces into non-functional, self-propagating, amyloid-like polymers. Thus, yeast strains that are [PSI+] show increased levels of nonsense suppression. Once present, [PSI+] propagates by recruitment of the soluble form of Sup35 into the aggregate in a manner analogous to that of mammalian prions. A search for genetic factors affecting propagation and maintenance of [PSI+] has identified an essential role for molecular chaperones, namely Hsp70 and Hsp104. The Hsp70 chaperone family and its associated co-chaperones are highly conserved from yeast to mammals. A major function of Hsp70 is to prevent the aggregation of denatured proteins by binding to exposed hydrophobic regions and preventing the accumulation of amorphous aggregates. In the model eukaryotic S. cerevisiae, to efficiently carry out such functions Hsp70 works in concert with a number of co-chaperones to regulate ATPase hydrolysis cycle of Hsp70, which in-turn dictates the peptide-binding status of Hsp70. While much data has accrued in relation to the ATPase and substrate binding cycles of Hsp70 there is a distinct lack of information regarding the regulation of this important chaperone at the post-translational level. Recent global proteomic studies have demonstrated that in vivo Hsp70 is phosphorylated. Using a simplified yeast system this study systematically assessed a variety of non-phosphorylateable and phosphomimetic Hsp70 mutants for phenotypic alterations in Hsp70 functions. It was found that alteration of Hsp70 phosphorylation status in vivo can impair prion propagation, alter both basal and acquired thermotolerance and in some cases render cells inviable. By looking at analogous mutants in closely related cytosolic Hsp70s this study identified functional similarities and differences between highly homologous Hsp70 species. This study shows a clear link between Hsp70 phosphorylation status and in vivo function. Given Hsp70s central role in a variety of important cellular metabolic pathways and the conservation of these phosphorylatable sites in higher eukaryotes, these findings have far reaching implications