Dataset Information

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes

ABSTRACT: The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD039649 | Pride | 2023-09-02

REPOSITORIES: Pride

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Publications

Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.

Henneberg Lukas T LT Singh Jaspal J Duda David M DM Baek Kheewoong K Yanishevski David D Murray Peter J PJ Mann Matthias M Sidhu Sachdev S SS Schulman Brenda A BA

Nature chemical biology 20230831 12

The cullin-RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conform ...[more]

PMID: 37653169

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Project description:The COP9 signalosome (CSN) is a conserved protein complex occurring in all eukaryotes. The mammalian CSN consists of eight subunits (CSN1 – CSN8) and possesses an intrinsic metalloprotease JAMM motif localised to CSN5. In addition, it is associated with kinases such as protein kinase CK2 and D and the ubiquitin (Ub) specific protease 15. It regulates cullin-RING Ub ligases (CRLs) that label proteins for proteolysis with poly-Ub chains in the Ub proteasome system (UPS). CRLs contain one of the scaffolding cullins 1 – 7, a RING domain protein e. g. Rbx1, and one of hundreds of substrate binding units such as F-box or BTB proteins that specifically target proteins for ubiquitination. The CSN forms functional supercomplexes with CRLs. It removes the Ub-like protein Nedd8 from cullins by its metalloprotease activity and thereby inactivates CRLs. On the other hand, it is essential for CRL function by protecting components and allowing reassembly of the complexes. As a regulator of the UPS the CSN is involved in cell cycle10, DNA repair and development. Overexpression of CSN subunits has been reported in tumour cells. Up to date nothing is known about CSN biogenesis and its regulation. Ten years ago we have observed that overexpression of selected CSN subunits caused a coordinated increase of all CSN subunits and a de novo biosynthesis of the entire complex. Here we show that overexpression of CSN1 mediates an increase of c-Myc which coordinates CSN subunit expression via microRNAs (miRNAs). miRNAs are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides that function as posttranscriptional gene regulators. We found that inhibitors of let-7 family miRNAs or upregulation of c-Myc, which represses let-7 miRNAs, resulted in a coordinated increase of CSN subunits and de novo CSN biogenesis. Keywords: Gene expression analysis of human RNA samples using topic-defined PIQOR™ Ubiquitin-PS Microarrays. Two different platforms were used (these are two different microarray batches which are identical in terms of the spotted cDNAs, but differ in the position of some cDNAs) Gene expression analysis of Hela cells overexpressing either CSN2 or CSN1 was performed to study CSN biogenesis and its regulation. Curcumin, Nr. 8 (Curcumin analogon) and Piceatannol were used for the treatment of Hela cells since they inhibit the CSN associated kinases. The aim was to investigate the effect on the signalosome. For each treatment 50 µM Curcumin, Nr. 8 and Piceatannol were used.

Dataset Information

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes

Publications

Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.

Similar Datasets

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