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Oxygen-dependent regulation of E3(SCF)ubiquitin ligases and a Skp1-associated JmjD6 homolog in Dictyostelium

ABSTRACT: The SCF (Skp1/cullin-1/F-box protein) class of E3 polyubiquitin ligases generates a signal for proteasomal degradation of thousands of proteins in animals. The evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged elucidation of these complexes in protists. Using bioinformatics and orthogonal Skp1 interactome methods, we expand the list of putative FBPs in the social amoeba Dictyostelium and show that Skp1 interactions are highly remodeled between growth and development. In addition, a subset of FBPs was less represented when the posttranslational hydroxylation and glycosylation of the Skp1 subunit was abrogated by deletion of the O2-dependent Skp1 prolyl hydroxylase PhyA. The significance of Skp1 modification for SCF activity was indicated by partial rescue of phyA-KO cell development with proteasomal inhibitors.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Dictyostelium Discoideum (slime Mold)

SUBMITTER: Christopher West

LAB HEAD: Christopher Mark West

PROVIDER: PXD033864 | Pride | 2022-07-22

REPOSITORIES: Pride

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The phosphatase DUSP22/JKAP inhibits UBR2-mediated K63-ubiquitination and activation of Lck in TCR signaling ( Sequential regulation of Lck K63-ubiquitination and autophosphorylation by UBR2 and DUSP22/JKAP in TCR signaling)

Project description:DUSP22 (also named JKAP) is a dual-specificity phosphatase that inhibits T cell activation. Here we identified the E3 ubiquitin ligase UBR2 as an upstream activator of Lck during T-cell activation. JKAP dephosphorylated UBR2 at two residues, leading to ubiquitin-mediated UBR2 degradation. The SCF (SKP1-CUL1-βTrCP) complex induced UBR2 Lys48-linked ubiquitination at three lysine residues. Moreover, single-cell RNA sequencing analysis and UBR2 knockout showed that UBR2 increased proinflammatory cytokines. Remarkably, UBR2 induced Lys63-linked ubiquitination of Lck at two lysine residues and subsequent Lck Tyr394phosphorylation/activation in TCR signaling. Conversely, TCR-induced Lck activation and JKAP knockout-enhanced inflammatory phenotypes were attenuated by UBR2 knockout. Consistently, the UBR2-Lck interaction and Lck Lys63-linked ubiquitination were induced in peripheral blood T cells of human SLE patients. Collectively, UBR2 protein stability and UBR2-induced Lck ubiquitination/activation are inhibited by JKAP, leading to attenuation of T-cell activation and T-cell-mediated inflammation.

2024-01-04 | PXD043454 | Pride

Regulatory role of E3 ubiquitin ligase Smurf2 on transcriptional corepressor KAP1

Project description:An E3 ubiquitin ligase Smurf2 and transcriptional co-repressor KAP1 have been documented to play crucial roles in similar cellular pathways including cell cycle, DNA damage response, chromatin compaction, senescence and cell death. Smurf2 and KAP1, through regulation of these cellular processes either drive or suppress tumorigenesis. Studies till date confer Smurf2 with a dual role in carcinogenesis, an oncogene and a tumor suppressor, depending on the cellular context. However, the molecular mechanisms governing Smurf2 functions remain unclear. Furthermore, studies have demonstrated significantly altered KAP1 protein levels in many human malignancies, despite which, the mechanisms operating in and regulating KAP1 stability and activity are obscure. In this study, we obtained evidence which indicates a strong and dynamic association between Smurf2 and KAP1. We found that Smurf2 directly binds KAP1 through its C2, WW1 and HECT domains. Further mechanistic studies also revealed that Smurf2 multi(mono)ubiquitinates KAP1 in E3 ligase-dependent manner. Moreover, Smurf2 differentially alters KAP1 protein levels in different types of mammalian and cancer cells and tissues, and significantly alters KAP1 interactome. Based on these findings, we propose a mechanism to explain the dual role and differential regulation of Smurf2 on KAP1 in a cell-context dependent manner. Further investigations of the identified Smurf2/KAP1 module could potentially lead to development of new, more efficient diagnostics tools and treatment paradigms.

2022-04-07 | PXD029642 | Pride

Proteomic profiling of the extracellular matrix (slime sheath) of Dictyostelium discoideum

Project description:Dictyostelium discoideum has historically served as a model system for cell and developmental biology, but recently it has gained increasing attention as a model for the study of human diseases. The extracellular matrix (ECM) of this eukaryotic microbe serves multiple essential functions during development. It not only provides structural integrity to the moving multicellular pseudoplasmodium, or slug, it also provides components that regulate cell motlity and differentiation. An LC/MS/MS analysis of slug ECM revealed the presence of a large number of proteins in two wild-type strains, NC4 and WS380B. GO annotation identified proteins involved in binding, as well as proteins that modulate metabolic processes, cell movement, and multicellular development. In addition, this proteomic analysis identified numerous expected, as well as unexpected components.

2015-07-22 | PXD002040 | Pride

Deeply conserved gene-regulatory functions of carboxy-terminal domains in dictyostelid C-module-binding factors

Project description:We describe the gene expression pattern of a Dictyostelium discoideum mutant depleted in the C-module-binding factor (CbfA) and autonomous gene-regulatory activities of CbfA carboxy-terminal domains (CbfA-CTDs) Comparison of gene expression in growing cells of a CbfA mutant compared to wild-type

2012-06-03 | E-GEOD-38418 | biostudies-arrayexpress

Molecular connection between the TUTase URT1 and decapping activators

Project description:Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators including DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6. A conserved Helical Leucine-rich Motif (HLM) within an intrinsically disordered region of URT1 binds to the LSm domain of DCP5. This interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. The combination of in planta and in vitro analyses supports a model that explains how URT1 reduces the accumulation of oligo(A)-tailed mRNAs: first, by connecting decapping factors and second, because 3’ terminal uridines can intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs in Arabidopsis avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb plant growth and development.

2020-05-29 | PXD018672 | Pride

A combinatorial approach to uncover an additional Integrator subunit

Project description:RNA Polymerase II (RNAPII) controls expression of all protein coding genes and most noncoding loci in higher eukaryotes. Calibrating RNAPII activity requires an assortment of polymerase-associated factors that are recruited at sites of active transcription. The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Integrator is known to be composed of 14 subunits that assemble and operate in a modular fashion. We employed proteomics and machine-learning structure prediction (AlphaFold2) to identify an additional Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS13/INTS14/INTS10 module and interfaces with the Int-PP2A module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes. Our study shows that omics approaches combined with AlphaFold2-based predictions provide additional insights into the molecular architecture of large and dynamic multiprotein complexes.

2023-02-10 | PXD039995 | Pride

The protein-protein interaction network of the Drosophila Speciation Core Complex

Project description:In this study we described the protein-protein interaction network of the Drosophila Speciation Core Complex by analysing the interactome of its subunit: HMR, LHR, NLP, BOH1 (CG33213), BOH2 (CG4788) and HP1a. For this purpose we performed Affinity Purification coupled with Mass Spectrometry (AP-MS) in D. melanogaster SL2 cells using as bait the two hybrid incompatibility proteins HMR (n = 8) and LHR (n = 4), as well as NLP (n = 3), BOH1(CG33213, n = 4), BOH2 (CG4788, n = 5) and HP1a (n = 4). Each bait was targeted with at least one antibody (rat anti-LHR 12F4, mouse anti-HP1a 2C09, rabbit anti-Nlp, anti-FLAG-M2 for FLAG-CG33213 and FLAG-CG4788), while HMR was targeted with three different antibodies (rat anti-HMR 2C10 and 12F1, anti-FLAG-M2 for FLAG-HMR). Individual replicates and antibodies used are listed in samples_table.

2021-08-11 | PXD023188 | Pride

Reverse phase protein array based protein profiling of breast cancer specimens

Project description:Expression levels of proteins and phosphoproteins, covering major cancer signaling pathways with a special focus on breast cancer biology, were obtained for a series of 109 breast cancer tumor specimens with positive estrogen receptor status. Tumor specimens from patients diagnosed with primary invasive breast carcinoma were collected at the time of surgery between 2008 and 2010 at the Department of Gynecology and Obstetrics / National Center for Tumor Diseases Heidelberg. None of the patients had received neoadjuvant therapy. Institutional Review Board approval was received as ethics vote no. S039/2008 and informed consent was obtained from all patients. Tumor specimens were processed within 20 min after surgery. Samples were stored snap frozen at -80M-BM-0C until further use. Only tumor samples with > 70% tumour cells and positive estrogen receptor status (immunoreactive score M-bM-^IM-% 3) as assessed by routine immunohistochemistry were selected for this study (n = 109). Tumor lysates were printed on a series of nitrocellulose coated glass slides and probed with 128 different primary antibodies directed against proteins and phosphoproteins of interest. Primary antibodies were selected to recognize proteins involved in major cancer signaling pathways with a special focus on breast cancer biology.

2014-02-18 | E-GEOD-47066 | biostudies-arrayexpress

Reverse phase protein array based protein profiling of breast cancer specimens (2)

Project description:Expression levels of proteins and phosphoproteins, covering major cancer signaling pathways with a special focus on breast cancer biology, were obtained for a series of 164 breast cancer tumor specimens with positive estrogen receptor status. Tumor specimens from patients diagnosed with primary invasive breast carcinoma were collected at the time of surgery between 2005 and 2011 and provided by the NCT Tissue Bank Heidelberg as well as by the Department of Gynecology and Obstetrics / National Center for Tumor Diseases Heidelberg. None of the patients had received neoadjuvant therapy.Tumor specimens were processed within 20 min after surgery. Samples were stored snap frozen at -80M-BM-0C until further use. Only tumor samples with > 70% tumour cells and positive estrogen receptor status (immunoreactive score M-bM-^IM-% 3) as assessed by routine immunohistochemistry were selected for this study (n = 164). Tumor lysates were printed on a series of nitrocellulose coated glass slides and probed with with differnt primary antibodies directed against proteins and phosphoproteins of interest. Primary antibodies were selected to recognize proteins involved in major cancer signaling pathways with a special focus on breast cancer biology.

2014-02-18 | E-GEOD-50861 | biostudies-arrayexpress

ADAR1 interactome analysis, LC-MS/MS

Project description:Adenosine-to-inosine (A-to-I) RNA editing catalyzed by ADAR1 plays essential roles in the regulation of diverse molecular and cellular processes both under physiological conditions and in the disease states, including cancer. However, mechanisms governing the protein abundance and activity of ADAR1, particularly of its constitutively-expressed and ubiquitous form, ADAR1p110, are largely unknown. Here, we report the E3 ubiquitin ligase SMURF2 as an essential positive regulator of ADAR1p110. We show that SMURF2 directly interacts with ADAR1p110, oligo-ubiquitinates it in an E3 ubiquitin ligase-dependent manner, and stabilizes ADAR1p110 expression by reducing its proteolysis through the proteasomal and lysosomal degradation pathways. The ability of SMURF2 to positively regulate ADAR1p110 was observed in different types of human cells and tissues, and in mouse tissues derived from Smurf2 knock-out and wild-type animals. Further investigations uncovered that SMURF2 regulates ADAR1p110 through its ubiquitination on K744 residue. Mutation of K744 to arginine (K744R), which is also linked to several human disorders, abolished SMURF2-mediated protection of ADAR1p110 from the proteolysis and inactivated its RNA editing activity. Additionally, SMURF2 showed a significant impact on ADAR1p110 protein-protein interactions, influencing its stability and functions. Altogether, these findings reveal SMURF2 as a multilayer regulator of ADAR1p110, governing its protein expression, interactions and functions.

2022-05-29 | PXD025338 | Pride

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