Rahul Samant

Dosage compensation plans: protein aggregation provides additional insurance against aneuploidy.
Samant RS, Masto VB, Frydman J

Gene dosage alterations caused by aneuploidy are a common feature of most cancers yet pose severe proteotoxic challenges. Therefore, cells have evolved various dosage compensation mechanisms to limit the damage caused by the ensuing protein level imbalances. For instance, for heteromeric protein complexes, excess nonstoichiometric subunits are rapidly recognized and degraded. In this issue of , Brennan et al. (pp. 1031-1047) reveal that sequestration of nonstoichiometric subunits into aggregates is an alternative mechanism for dosage compensation in aneuploid budding yeast and human cell lines. Using a combination of proteomic and genetic techniques, they found that excess proteins undergo either degradation or aggregation but not both. Which route is preferred depends on the half-life of the protein in question. Given the multitude of diseases linked to either aneuploidy or protein aggregation, this study could serve as a springboard for future studies with broad-spanning implications.

Genes & development, 33, 1549-5477, 2019

Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae.
Samant RS, Frydman J

Protein misfolding in the cell is linked to an array of diseases, including cancers, cardiovascular disease, type II diabetes, and numerous neurodegenerative disorders. Therefore, investigating cellular pathways by which misfolded proteins are trafficked and cleared ("protein quality control") is of both mechanistic and therapeutic importance. The clearance of most misfolded proteins involves the covalent attachment of one or more ubiquitin molecules; however, the precise fate of the ubiquitinated protein varies greatly, depending on the linkages present in the ubiquitin chain. Here, we discuss approaches for quantifying linkage-specific ubiquitination and clearance of misfolded proteins in the budding yeast Saccharomyces cerevisiae-a model organism used extensively for interrogation of protein quality control pathways, but which presents its own unique challenges for cell and molecular biology experiments. We present a fluorescence microscopy-based assay for monitoring the clearance of misfolded protein puncta, a cycloheximide-chase assay for calculating misfolded protein half-life, and two antibody-based methods for quantifying specific ubiquitin linkages on tagged misfolded proteins, including a 96-well plate-based ELISA. We hope these methods will be of use to the protein quality control, protein degradation, and ubiquitin biology communities.

Methods in enzymology, 619, 1557-7988, 2019

Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control.
Samant RS, Livingston CM, Sontag EM, Frydman J

Protein misfolding is linked to a wide array of human disorders, including Alzheimer's disease, Parkinson's disease and type II diabetes. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin-proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction.

Nature, 563, 1476-4687, 2018