Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

AlacatDesigner─Computational Design of Peptide Concatamers for Protein Quantitation.

Protein quantitation via mass spectrometry relies on peptide proxies for the parent protein from which abundances are estimated. Owing to the variability in signal from individual peptides, accurate absolute quantitation usually relies on the addition of an external standard. Typically, this involves stable isotope-labeled peptides, delivered singly or as a concatenated recombinant protein. Consequently, the selection of the most appropriate surrogate peptides and the attendant design in recombinant proteins termed QconCATs are challenges for proteome science. QconCATs can now be built in a "a-la-carte" assembly method using synthetic biology: ALACATs. To assist their design, we present "AlacatDesigner", a tool that supports the peptide selection for recombinant protein standards based on the user's target protein. The user-customizable tool considers existing databases, occurrence in the literature, potential post-translational modifications, predicted miscleavage, predicted divergence of the peptide and protein quantifications, and ionization potential within the mass spectrometer. We show that peptide selections are enriched for good proteotypic and quantotypic candidates compared to empirical data. The software is freely available to use either via a web interface AlacatDesigner, downloaded as a Desktop application or imported as a Python package for the command line interface or in scripts.

The cytoprotective sequestration activity of small heat shock proteins is evolutionarily conserved.

The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family.

Yeast Protein Kinase A Isoforms: A Means of Encoding Specificity in the Response to Diverse Stress Conditions?

Eukaryotic cells have developed a complex circuitry of signalling molecules which monitor changes in their intra- and extracellular environments. One of the most widely studied signalling pathways is the highly conserved cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which is a major glucose sensing circuit in the yeast Saccharomyces cerevisiae. PKA activity regulates diverse targets in yeast, positively activating the processes that are associated with rapid cell growth (e.g., fermentative metabolism, ribosome biogenesis and cell division) and negatively regulating the processes that are associated with slow growth, such as respiratory growth, carbohydrate storage and entry into stationary phase. As in higher eukaryotes, yeast has evolved complexity at the level of the PKA catalytic subunit, and Saccharomyces cerevisiae expresses three isoforms, denoted Tpk1-3. Despite evidence for isoform differences in multiple biological processes, the molecular basis of PKA signalling specificity remains poorly defined, and many studies continue to assume redundancy with regards to PKA-mediated regulation. PKA has canonically been shown to play a key role in fine-tuning the cellular response to diverse stressors; however, recent studies have now begun to interrogate the requirement for individual PKA catalytic isoforms in coordinating distinct steps in stress response pathways. In this review, we discuss the known non-redundant functions of the Tpk catalytic subunits and the evolving picture of how these isoforms establish specificity in the response to different stress conditions.

Tolerance to nascent protein misfolding stress requires fine-tuning of the cAMP/PKA pathway.

Protein aggregation is the abnormal association of misfolded proteins into larger, often insoluble structures that can be toxic during aging and in protein aggregation-associated diseases. Previous research has established a role for the cytosolic Tsa1 peroxiredoxin in responding to protein misfolding stress. Tsa1 is also known to downregulate the cAMP/protein kinase A (PKA) pathway as part of the response to hydrogen peroxide stress. However, whether the cAMP/PKA pathway is involved in protein misfolding stress is not known. Using transcriptomics, we examined the response to protein misfolding stress and found upregulation of numerous stress gene functions and downregulation of many genes related to protein synthesis and other growth-related processes consistent with the well-characterized environmental stress response. The scope of the transcriptional response is largely similar in wild-type and tsa1 mutant strains, but the magnitude is dampened in the strain lacking Tsa1. We identified a direct protein interaction between Tsa1 and the Bcy1 regulatory subunit of PKA that is present under normal growth conditions and explains the observed differences in gene expression profiles. This interaction is increased in a redox-dependent manner in response to nascent protein misfolding, via Tsa1-mediated oxidation of Bcy1. Oxidation of Bcy1 causes a reduction in cAMP binding by Bcy1, which dampens PKA pathway activity, leading to a targeted reprogramming of gene expression. Redox regulation of the regulatory subunit of PKA provides a mechanism to mitigate the toxic consequences of protein misfolding stress that is distinct to stress caused by exogenous sources of reactive oxygen species.