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Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

ABSTRACT: Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the trafficking of proteins to the centrosome. During aggresome assembly, satellites were required for the growth of the aggresomal structure from an initial ring of phosphorylated HSP27 deposited around the centrioles. The seeding of this phosphorylated HSP27 ring depended on the centrosomal proteins CP110, CEP97 and CEP290. Owing to limiting amounts of CP110, senescent cells, which are characterized by the accumulation of protein aggregates, were defective in aggresome formation. Furthermore, satellites and CP110-CEP97-CEP290 were required for the aggregation of mutant huntingtin. Together, these data reveal roles for CP110-CEP97-CEP290 and satellites in the control of cellular proteostasis and the aggregation of disease-relevant proteins.

SUBMITTER: Prosser SL

PROVIDER: S-EPMC9033585 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

Prosser Suzanna L SL Tkach Johnny J Gheiratmand Ladan L Kim Jaeyoun J Raught Brian B Morrison Ciaran G CG Pelletier Laurence L

Nature cell biology 20220411 4

Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the ...[more]

PMID: 35411088

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Project description:Generation of Flp-In T-REx 293 cells for miniTurbo proximity labelling Proximity labelling was carried out utilizing miniTurbo. Full-length CP110 cDNA was cloned into a pcDNA5-FRT/TO-miniTurbo-FLAG destination vector via the Gateway cloning system. Using the Flp-In system, HEK293 cells stably expressing miniTurbo-FLAG (control) and miniTurboFLAG-CP110 were generated. Sample preparation for mass spectrometry (MS) Each cell pellet was resuspended in 10 mL of RIPA lysis buffer and streptavidin sepharose beads (GE Healthcare) was added. Beads were washed and washed twice more with ammonium bicarbonate buffer and tryptic digestion was performed by incubating the beads with TPCK-treated modified trypsin (Promega, Madison, WI). Supernatants were transferred to a fresh Eppendorf tube. The samples were lyophilized and resuspended in buffer A (0.1% formic acid). Mass spectrometry High-performance liquid chromatography was conducted using a 2 cm pre-column and 50 cm analytical column was performed, running a 120-min reversed-phase buffer gradient at 225 nl/min on a Proxeon EASY-nLC 1000 pump in-line with a Thermo Q Exactive HF quadrupole-Orbitrap mass spectrometer. A parent ion scan was performed using a resolving power of 60,000, then up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1,000 for activation), using higher energy collision-induced dissociation (HCD) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 10 ppm; exclusion list size = 500) detected twice within 5 s was excluded from analysis for 15 s. For protein identification, Thermo. RAW files were converted to the .mzXML format using ProteoWizard, then searched using X!Tandem and Comet against the human Human RefSeq Version 45 database (containing 36,113 entries). Search parameters specified a parent ion mass tolerance of10 ppm and an MS/MS fragment ion tolerance of 0.4 Da, with up to 2 missed cleavages allowed for trypsin. Variable modifications of +16(M W), +32 (M and W), +42 (Nterminus), and +1 (N and Q) were allowed.

Dataset Information

Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

Publications

Aggresome assembly at the centrosome is driven by CP110-CEP97-CEP290 and centriolar satellites.

Similar Datasets

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