Rahul Samant

The accumulation of misfolded or otherwise non-native proteins in the cell is linked to an array of ageing-related disorders, including cancers and neuro-degenerative diseases. Healthy cells limit the toxicity of misfolded proteins by promoting their clearance and maintaining proteome balance: a process we call ‘proteostasis’.

The importance of discovering the two major pathways for misfolded protein clearance—the ubiquitin-proteasome system and autophagy—was highlighted by their recognition with Nobel Prizes in 2004 and 2016, respectively. How they are integrated to maintain proteostasis, however, is poorly understood. Addressing this question is the central scientific driver of our lab. Given that loss of proteostasis—including decline in both proteasomal and autophagic degradation—is a major hallmark of ageing, investigating the co-ordination between protein clearance pathways in young and aged cells will provide insights into improving health and well-being across the life-course.

We use a multi-disciplinary approach with an emphasis on mass spectrometry-based proteomic methods together with cutting-edge cell and molecular biology tools for probing ubiquitin-mediated protein clearance pathways. By performing studies in a range of model systems—from single-celled budding yeast, to humans—we hope to unravel underlying rules governing proteostasis conserved throughout evolution, development, and ageing.

Our current focus is on the use of drugs targeting the molecular chaperone HSP90—a key regulator of proteostasis—to investigate the plasticity of protein clearance pathways in young, aged, and diseased cells.

Oncoproteins

Drugs targeting the molecular chaperone HSP90 in cancer cells trigger clearance of cancer-causing proteins. Here, the oncoprotein HER2/ERBB2 (green)—normally at the cell surface (left) gets internalised for clearance following 8 hours of HSP90 inhibitor treatment (middle). By 24 hours, the protein is undetectable (right). Cell nuclei shown in blue.
From Samant, Clarke & Workman (2014).

Latest Publications

Alternative systems for misfolded protein clearance: life beyond the proteasome.
Johnston HE, Samant RS

Protein misfolding is a major driver of ageing-associated frailty and disease pathology. Although all cells possess multiple, well-characterised protein quality control systems to mitigate the toxicity of misfolded proteins, how they are integrated to maintain protein homeostasis ('proteostasis') in health-and how their dis-integration contributes to disease-is still an exciting and fast-paced area of research. Under physiological conditions, the predominant route for misfolded protein clearance involves ubiquitylation and proteasome-mediated degradation. When the capacity of this route is overwhelmed-as happens during conditions of acute environmental stress, or chronic ageing-related decline-alternative routes for protein quality control are activated. In this review, we summarise our current understanding of how proteasome-targeted misfolded proteins are re-trafficked to alternative protein quality control routes such as juxta-nuclear sequestration and selective autophagy when the ubiquitin-proteasome system is compromised. We also discuss the molecular determinants of these alternative protein quality control systems, attempt to clarify distinctions between various cytoplasmic spatial quality control inclusion bodies (e.g., Q-bodies, p62-bodies, JUNQ, aggresomes, and aggresome-like induced structures 'ALIS'), and speculate on emerging concepts in the field that we hope will spur future research-with the potential to benefit the rational development of healthy ageing strategies.

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The FEBS journal, 1, 1, 01 Nov 2020

PMID: 33135311

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.

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Genes & development, 33, 1549-5477, 2019

PMID: 31371460

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.

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Methods in enzymology, 619, 1557-7988, 2019

PMID: 30910025