Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensu... more Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bin...
Protein function is often regulated by posttranslational modifications (PTMs), and recent advance... more Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.
CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be ... more CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]nand [CGG]nwith more than seven to nine units were rare (1/2000), and perfect [CAG]n9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n> 9 and [CGG]n> 7 were characterized. The comparison of our data with other [CAG]nand [CGG]nresources suggests that the screening of reference cDNAs leads to unique sources of new [CAG]nand [CGG]nand will enhance the study of enlarged triplet repeats in human genetic diseases.
Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensu... more Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bin...
Protein function is often regulated by posttranslational modifications (PTMs), and recent advance... more Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.
CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be ... more CAG and CGG expansion is associated with 10 inherited neurological diseases and is thought to be involved in other human genetic diseases. To identify new candidate genes, we have undertaken a large-scale screening project for CAG/CTG ([CAG]n) and CGG/GCC ([CGG]n) repeats in human brain reference cDNAs. Here, we present the final classification for 597 cDNAs selected by CAG and CGG hybridization from two libraries (100,128 clones) and the updated characterization of [CAG]n- and [CGG]n-positive cDNAs (repeat polymorphism and cDNA localization). We have selected 124 CAG and 83 CGG hybridization-positive clones representing new genes, from which 49 CAG and 7 CGG repeats could be identified. New [CAG]nand [CGG]nwith more than seven to nine units were rare (1/2000), and perfect [CAG]n9 were more likely polymorphic. Overall, highly polymorphic to monomorphic new [CAG]n> 9 and [CGG]n> 7 were characterized. The comparison of our data with other [CAG]nand [CGG]nresources suggests that the screening of reference cDNAs leads to unique sources of new [CAG]nand [CGG]nand will enhance the study of enlarged triplet repeats in human genetic diseases.
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