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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

ABSTRACT: 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 intermediatefilament 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 the 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hossein Fazelinia

LAB HEAD: Ophir Shalem, Ph.D.

PROVIDER: PXD048693 | Pride | 2024-01-24

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20211022_151143_OS1600_wp_dDIA.sne	Other
	20220531_143059_OS1669.psar	Other
	OS1600_wp_dDIA_Intensity_Qvalue_peptide_report.csv	Csv
	OS1600_wp_dDIA_protein_report.csv	Csv
	OS1669_Peptide_Raw_Report.csv	Csv

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Project description:Pathogenic microorganisms often secrete proteins or nucleic acids that mimic the structure and/or function of molecules expressed within their host counterparts. This molecular mimicry empowers pathogens to target and subvert critical host processes and helps establish their infectious paradigm. We report here that the intracellular bacterium Legionella pneumophila (L.p.) secretes a toxin named SidI (substrate of icm/dot transporter I) which possesses a transfer RNA (tRNA) like fold and functions as a mannosyl transferase enzyme. The 3.1 Å cryo-EM structure of SidI reveals an N-terminal domain that exhibits a characteristic ‘inverted L-shape’ and charge conservation that is present in two known protein mimics of tRNAs, the bacterial elongation factor EF-G and the mammalian release factor eRF1. In addition, SidI’s C-terminal domain adopts a glycosyl transferase B fold and shares considerable homology to a known mannosyl transferase. This molecular coupling of fold and enzymatic function allows SidI to bind to and enzymatically modify components of the host translational apparatus, including the ribosome, resulting in a robust block of protein synthesis that is comparable to the potency exhibited by the toxin ricin. Additionally, we uncovered that the translational pausing activated by SidI elicits a stress response signature that is highly similar to one activated by elongation inhibitors that target the ribosome and induce ribosome collisions. SidI-mediated perturbations to the ribosome activate the stress kinases ZAKα and p38, that in turn drive the accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and subsequently orchestrates the transcription of stress-inducible genes that culminates in the activation of a cell death program. Thus, using Legionella and its effectors as tools, we have unravelled the role of a ribosome to nuclear signaling pathway that regulates cell fate.

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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

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