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CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly [1]

['Katelyn M. Sweeney', 'Center For Cellular', 'Molecular Therapeutics', 'Children S Hospital Of Philadelphia', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Department Of Genetics', 'Perelman School Of Medicine', 'University Of Pennsylvania']

Date: 2024-02

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

The presence of large protein inclusions is a hallmark of many neurodegenerative diseases, yet the precise mechanisms by which cells compartmentalize unfolded proteins is still not completely understood. Here we used a novel screening approach that enables FACS-based inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. In follow up experiments, using both proximity labeling and imaging, we show that SRRD is involved in the regulation of intermediate filaments (IF) spatial organization. Loss of SRRD results in fragmented IF network and the reduced formation of aggresomes, which are transient compartments to which cells transfer unfolded proteins under cellular stress. Interestingly, without SRRD, aggresomes completely lack vimentin cages, which are typical structures that encapsulate aggresomes and have been associated with the recruitment of protein degradation and chaperones machinery to these structures. Altogether, we present a novel screening approach for the identification of gene associated with cellular proteostasis. We reveal several novel protein factors, including SRRD, which our data suggests has a broad and previously overlooked role cytoskeletal organization and protein quality control.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: OS and KMS have filed a patent application for the use of SRRD fragments through the Children’s Hospital of Philadelphia. J.S. is a consultant for Dewpoint Therapeutics, ADRx, and Neumora. J.S. is a shareholder and advisor for Confluence Therapeutics. The authors declare no other conflicts of interest relevant to this publication.

Funding: This work was supported by the following grants: NIH/NIGMS DP2GM137416 (OS, GC and ML, TL), PA DoH SAP#4100083086 (OS and SC, TL), NINDS/NIH R03NS111447-01 (OS), iAward Sanofi (OS), NINDS - F31NS116999 to KMS, The Packard Center (JS), TargetALS (JS), The Association for Frontotemporal Degeneration (JS), the Amyotrophic Lateral Sclerosis Association (JS), Office of the Assistant Secretary of Defense for Health Affairs through the Amyotrophic Lateral Sclerosis Research Program W81XWH-20-1-0242 and W81XWH-17-1-0237 (JS) G. Harold and Leila Y. Mathers Foundation (JS), NIH R01GM099836 (JS), NIH R21AG065854 (JS), EMB from (Milton Safenowitz Post-Doctoral Fellowship from the ALSA, NIH; BP from (AHA and the BrightFocus Foundation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We used a fluorescent TDP-43 aggregation reporter that, when coupled to Pulse Shape Analysis (PulSA), an approach that uses flow cytometry to distinguish between a diffuse and punctate fluorescent signal in cells [ 26 , 27 ], enabled us to quantify TDP-43 aggregation at the single cell level. We leveraged this reporter with a genome-wide CRISPR-Cas9 KO screen which revealed both canonical proteostasis machinery and novel modifiers of TDP-43 aggregation. Our screen identified SRRD, a protein of unknown function, as a top positive regulator of TDP-43 inclusion formation. We further describe a role for SRRD as a regulator of intermediate filaments (IF) dynamics and aggresome formation. Using APEX proximity labeling we found that SRRD resides in close proximity to proteins IFs including multiple keratin proteins and vimentin, which has a well established role in aggresome formation. Interestingly, two molecular functions were highly enriched in the set of SRRD interactors: Protein disulfide-isomerase (PDI) and calcium binding. A calcium dependent formation and breakage of disulfide bonds has been shown to be essential for the regulation of IF structures [ 28 ], suggesting that SRRD may act as a regulator of IF spatial dynamics. Indeed, loss of SRRD results impaired vimentin organization in cells, lower protein expression of several cytoskeletal proteins, and impaired aggresome assembly characterized by lower aggresome formation and an almost complete lack of vimentin cages, under stress conditions. We also examined the localization of SRRD under proteotoxic stress and found that it localizes to both aggresomes and unfolded proteins, mediated by an N-terminal unstructured region suggesting it may also have a direct effect on unfolded proteins. Altogether, our work suggests a previously unappreciated role for SRRD in the regulation of cellular organization and proteostasis.

In this study we used a unique approach to screen for novel regulators of protein inclusion formation directly, rather than the downstream protein toxicity. As a model for perturbed proteostasis, we used TDP-43, an essential RNA-binding protein (RBP), which can be found in cytoplasmic aggregates that are a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [ 18 – 21 ]. Mutations in TDP-43 are rare, explaining less than ~5% of ALS and FTD cases, and yet ~97% of ALS patients and ~50% of FTD patients present with TDP-43 pathology [ 22 ]. TDP-43 pathology can also be observed in cases of Alzheimer’s disease and Parkinson’s disease [ 21 , 23 – 25 ]. These findings suggest that aggregation of TDP-43 is heavily affected by many other proteins in the cell, making it a candidate to screen for modifiers of protein aggregation.

The loss of proteostasis is a hallmark of many human diseases such as cancer, and neurodegenerative diseases and is thought to be directly related to the aging process [ 3 – 5 ]. In the case of neurodegenerative diseases, proteins that are prone to misfolding or contain intrinsically disordered regions misfold, mislocalize, and are unable to be cleared by degradation machinery. These proteinopathies are often associated with cellular toxicity, and thus to date many groups have employed genetic screens in yeast, Drosophila, and mammalian cells to find regulators of proteotoxicity [ 13 – 17 ]. These approaches will frequently capture downstream mechanisms of proteotoxicity that connect aggregation to cell death rather than providing mechanistic insight into aggregate formation.

Cellular proteostasis refers to an array of cellular mechanisms that maintain the proteome in a folded and functioning state [ 1 ]. Cells harbor specialized molecular mechanisms to deal with the presence of misfolded proteins including regulation of protein translation, compartmentalization, folding, and degradation [ 2 ]. During unfolded protein stress, a number of quality control pathways are activated, such as chaperones and degradation machinery to alleviate protein misfolding and overabundance [ 3 – 5 ]. Additionally, synthesis of new proteins is paused, and the translation machinery is sequestered into transient cellular structures like stress granules. If chaperones or degradation machinery are overwhelmed, misfolded proteins are often sequestered into subcellular compartments to i) prevent their deleterious interactions with other cellular components, ii) enhance clearance at local sites with enriched chaperones, proteasomes, and autophagy machinery, or iii) terminally sequester insoluble proteins to promote asymmetric inheritance after cell division [ 4 , 5 ]. One example of such a subcellular compartment is the mammalian aggresome, a perinuclear body consisting of unfolded proteins that are actively recruited to the centrosome via HDAC6-coupled dynein-mediated trafficking [ 6 – 10 ]. Aggresomes are enriched in ubiquitinated proteins and chaperones [ 7 , 11 ], as well as in degradation machinery such as proteasomes and the autophagy adapter SQSTM1 (Johnston and Samant 2021), and they are encircled by cage-like structures formed from the intermediate filament protein Vimentin (VIM) [ 7 , 8 , 12 ].

Results

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011138

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