Biology subjects

Pincus, D.

Publications and source records attributed to Pincus, D..

7 recordsLinked to original sources

bioRxiv2018

Transient Hsp90 suppression promotes a heritable change in protein translation

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a unique role promoting the evolution of new heritable traits. To investigate the role of Hsp90 in modulating protein synthesis, we screened more than 1200 proteins involved in mRNA regulation for physical interactions with Hsp90 in human cells. Among the top hits was CPEB2, which strongly binds Hsp90 via its prion domain, reminiscent of the prion-like regulation of translation of Aplysia CPEB. In a yeast model of CPEB prion-dependent translation regulation, transient inhibition of Hsp90 amplified CPEB2 prion activity and resulted in persistent translation of the CPEB reporter. Remarkably, inhibition of Hsp90 was sufficient to induce a heritable change in protein translation that persisted for 30 generations, even in the absence of exogenous CPEB. Although we identified a variety of perturbations that enhanced translation of the reporter, only Hsp90 inhibition led to persistent activation. Thus, transient loss of Hsp90 function leads to the non-genetic inheritance of a novel translational state. We propose that, in addition to sculpting the conformational landscape of the proteome, Hsp90 promotes phenotypic variation by modulating protein synthesis.

cell biology↗

bioRxiv2018

Genetic and Epigenetic Determinants Establish a Continuum of Hsf1 Occupancy and Activity Across the Yeast Genome

Heat Shock Factor 1 (Hsf1) is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting element - Heat Shock Element (HSE) - across the eukaryotic lineage. In budding yeast, Hsf1 drives transcription of ~20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here we combine Hsf1 ChIP-seq, nascent RNA-seq and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. Hsf1 binds 74 loci during acute heat shock, 46 of which are linked to genes with strong Hsf1-dependent transcription. Most of these targets show detectable Hsf1 binding under basal conditions, but basal occupancy and heat shock-inducible binding both vary over two orders of magnitude. Notably, Hsf1s induced DNA binding leads to a disproportionate (up to 50-fold) increase in nascent transcription. While variation in basal Hsf1 occupancy poorly correlates with the strength of the HSE, promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of pioneer factors. Such accessible chromatin may be critical for Hsf1 occupancy of its genomic sites as the activator is incapable of binding HSEs embedded within a stable nucleosome in vitro. In response to heat shock, however, Hsf1 is able to gain access to nucleosomal sites and promotes chromatin remodeling with the RSC complex playing a key role. We propose that the interplay between nucleosome occupancy, HSE strength and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

genomics↗

bioRxiv2017

Hierarchical organization endows the kinase domain with regulatory plasticity

The functional diversity of kinases enables specificity in cellular signal transduction. Yet general rules for how the kinase domain allows the more than 500 members of the human kinome to receive specific regulatory inputs and convey information to appropriate substrates - all while using the common signaling currency of phosphorylation - remain enigmatic. Here, using co-evolution analysis and quantitative live-cell assays, we reveal a deep hierarchical organization of the kinase domain that facilitates the orthogonal evolution of regulatory inputs and substrate outputs while maintaining catalytic function. Three quasi-independent functional units in the kinase domain (known as protein sectors) encode for catalysis, substrate specificity and regulation, and these distinct subdomains are differentially exploited by somatic cancer mutations and harnessed by allosteric inhibitors. We propose that this functional architecture endows the kinase domain with inherent regulatory plasticity.

systems biology↗

bioRxiv2017

Evolution and Engineering of Allosteric Regulation in Protein Kinases

Allosteric regulation - the control of protein function by sites far from the active site - is a common feature of proteins that enables dynamic cellular responses. Reversible modifications such as phosphorylation are well suited to mediate such regulatory dynamics, yet the evolution of new allosteric regulation demands explanation. To understand this, we mutationally scanned the surface of a prototypical kinase to identify readily evolvable phosphorylation sites. The data reveal a set of spatially distributed \"hotspots\" that coevolve with the active site and preferentially modulate kinase activity. By engineering simple consensus phosphorylation sites at these hotspots we successfully rewired in vivo cell signaling. Beyond synthetic biology, the hotspots are frequently used by the diversity of natural allosteric regulatory mechanisms in the kinase family and exploited in human disease.\n\nONE SENTENCE SUMMARYCell signaling is easily rewired by introducing new phosphoregulation at latent allosteric surface sites.

systems biology↗

bioRxiv2017

Hsf1 phosphorylation generates cell-to-cell variation in Hsp90 levels and promotes phenotypic plasticity

Clonal populations of cells exhibit cell-to-cell variation in the transcription of individual genes. In addition to this \"noise\" in gene expression, heterogeneity in the proteome and the proteostasis network expands the phenotypic diversity of a population. Heat shock transcription factor (Hsf1) regulates chaperone gene expression, thereby coupling transcriptional noise to proteostasis. Here we show that cell-to-cell variation in Hsf1 activity is an important determinant of phenotypic plasticity. Budding yeast cells with high Hsf1 activity were enriched for the ability to acquire resistance to an antifungal drug, and this enrichment depended on Hsp90 - a known \"phenotypic capacitor\" and canonical Hsf1 target. We show that Hsf1 phosphorylation promotes cell-to-cell variation, and this variation - rather than absolute Hsf1 activity - promotes antifungal resistance. We propose that Hsf1 phosphorylation enables differential tuning of the proteostasis network in individual cells, allowing populations to access a wide range of phenotypic states.

systems biology↗

bioRxiv2017

Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides

Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation - a post-translational protein modification that has emerged as a powerful modulator of HSP70 activity - influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in C.elegans directly affect aggregation properties and associated toxicity of amyloid-{beta} (A{beta}), of a polyglutamine (polyQ)- extended polypeptide and of -synuclein (-syn). Expression of a constitutively active C. elegans AMPylase Fic-1(E274G) under its own promoter expedites aggregation of A{beta} and -syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Over-expression of Fic-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cyto-protective, rather than a cytotoxic role for such protein aggregates.

cell biology↗

bioRxiv2017

Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response

Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone encoded by an Hsf1 target gene. Previously, we identified Hsp70 as the chaperone responsible for Hsf1 repression in Saccharomyces cerevisiae and constructed a mathematical model based on Hsp70-mediated negative feedback that recapitulated the dynamic activity of Hsf1 during heat shock. The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validated these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next we mapped a discrete Hsp70 binding site on Hsf1 to a motif in the C-terminal activation domain known as conserved element 2 (CE2). Removal of CE2 resulted in increased Hsf1 activity under non-heat shock conditions and delayed deactivation kinetics. In addition, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the feedback loop charged with maintaining cytosolic proteostasis.

biochemistry↗