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1.
Mol Cell ; 79(2): 332-341.e7, 2020 07 16.
Article in English | MEDLINE | ID: mdl-32521225

ABSTRACT

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.


Subject(s)
Aspartic Acid Proteases/metabolism , Nuclear Respiratory Factor 1/metabolism , Ubiquitin/metabolism , Aspartic Acid Proteases/genetics , Binding Sites , CRISPR-Cas Systems , Cell Line , Gene Knockout Techniques , HEK293 Cells , Humans , Protein Biosynthesis , Proteolysis
2.
J Am Chem Soc ; 141(6): 2703-2712, 2019 02 13.
Article in English | MEDLINE | ID: mdl-30657686

ABSTRACT

Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.


Subject(s)
Enzyme Inhibitors/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Drug Evaluation, Preclinical , Enzyme Inhibitors/metabolism , HEK293 Cells , Humans , Ligands , MCF-7 Cells , Models, Molecular , Protein Structure, Secondary , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/chemistry
3.
Biochem J ; 475(1): 329-340, 2018 01 11.
Article in English | MEDLINE | ID: mdl-29229763

ABSTRACT

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.


Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , NF-kappa B p50 Subunit/antagonists & inhibitors , Peptides/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Anti-Inflammatory Agents/chemical synthesis , Gene Expression , HEK293 Cells , High-Throughput Screening Assays , Humans , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , Peptide Library , Peptides/chemical synthesis , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity
4.
Mol Biosyst ; 13(1): 92-105, 2016 Dec 20.
Article in English | MEDLINE | ID: mdl-27824369

ABSTRACT

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein-protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.


Subject(s)
Protein Interaction Mapping/methods , Protein Interaction Maps , Proteome , Proteomics , Cell Fractionation , Cell Line , Chromatography, Liquid , Cluster Analysis , Humans , Intracellular Space/metabolism , Mass Spectrometry , Protein Binding , Protein Transport , Proteomics/methods , Sensitivity and Specificity , Subcellular Fractions
5.
Elife ; 5: e12994, 2016 Jan 14.
Article in English | MEDLINE | ID: mdl-26765561

ABSTRACT

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.


Subject(s)
Carcinogenesis , Cell Proliferation , rho-Associated Kinases/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Gene Knockout Techniques , Melanoma/pathology , Mice , rho-Associated Kinases/genetics
6.
Dev Cell ; 35(3): 344-57, 2015 Nov 09.
Article in English | MEDLINE | ID: mdl-26555054

ABSTRACT

Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a key determinant of protein localization to protrusions. Accordingly, inhibition of local translation destabilizes protrusions and inhibits mesenchymal-like morphology. Interestingly, many mRNAs localized to protrusions are translationally repressed. Specific cis-regulatory elements within mRNA UTRs define whether mRNAs are locally translated or repressed. Finally, RNAi screening of RNA-binding proteins (RBPs) enriched in protrusions revealed trans-regulators of localized translation that are functionally important for protrusions. We propose that by deciphering the localized mRNA UTR code, these proteins regulate protrusion stability and mesenchymal-like morphology.


Subject(s)
Cell Movement/genetics , Cell Surface Extensions/metabolism , Protein Biosynthesis/genetics , RNA, Messenger/genetics , Protein Binding/genetics , Protein Transport , RNA-Binding Proteins/genetics , Sequence Analysis, RNA/methods
7.
Cancer Cell ; 19(6): 695-7, 2011 Jun 14.
Article in English | MEDLINE | ID: mdl-21665143

ABSTRACT

The tumor environment consists of tumor-associated cells, such as macrophages, fibroblasts, and extracellular matrix, and has an important impact on tumor progression. In this issue of Cancer Cell, Samuel et al. show that ROCK-driven actomyosin contractility increases tissue stiffness affecting epidermal homeostasis, as well as tumor growth and progression.

8.
PLoS One ; 5(7): e11801, 2010 Jul 27.
Article in English | MEDLINE | ID: mdl-20668551

ABSTRACT

BACKGROUND: Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, beta-catenin and alpha-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn. METHODOLOGY/PRINCIPAL FINDINGS: We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, beta-catenin and alpha-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, beta-catenin and alpha-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins. CONCLUSIONS/SIGNIFICANCE: Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, beta-catenin or alpha-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.


Subject(s)
Cadherins/metabolism , Catenins/metabolism , Cell Movement/physiology , Adherens Junctions/genetics , Adherens Junctions/metabolism , Cadherins/genetics , Catenins/genetics , Cell Line, Tumor , Cell Movement/genetics , Fluorescent Antibody Technique , Humans , Immunoblotting , Male , RNA, Small Interfering , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , Delta Catenin
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