Dithiocarbamates (DTCs) are simple organic compounds with many applications in industry and medicine. They are potent metal chelators forming complexes with various metal ions, including copper. Recently, bis(diethyldithiocarbamate)-copper complex (CuET) has been identified as a metabolic product of the anti-alcoholic drug Antabuse (disulfiram, DSF), standing behind DSF's reported anticancer activity. Mechanistically, CuET in cells causes aggregation of NPL4 protein, an essential cofactor of the p97 segregase, an integral part of the ubiquitin-proteasome system. The malfunction of p97/NPL4 caused by CuET leads to proteotoxic stress accompanied by heat shock and unfolded protein responses and cancer cell death. However, it is not known whether the NPL4 inhibition is unique for CuET or whether it is shared with other dithiocarbamate-copper complexes. Thus, we tested 20 DTCs-copper complexes in this work for their ability to target and aggregate NPL4 protein. Surprisingly, we have found that certain potency against NPL4 is relatively common for structurally different DTCs-copper complexes, as thirteen compounds scored in the cellular NPL4 aggregation assay. These compounds also shared typical cellular phenotypes reported previously for CuET, including the NPL4/p97 proteins immobilization, accumulation of polyubiquitinated proteins, the unfolded protein, and the heat shock responses. Moreover, the active complexes were also toxic to cancer cells (the most potent in the nanomolar range), and we have found a strong positive correlation between NPL4 aggregation and cytotoxicity, confirming NPL4 as a relevant target. These results show the widespread potency of DTCs-copper complexes to target NPL4 with subsequent induction of lethal proteotoxic stress in cancer cells with implications for drug development.

• Klíčová slova
• Cancer, Copper complex, CuET, Dithiocarbamates, NPL4 protein, Proteotoxic stress,
• MeSH
• disulfiram farmakologie   MeSH
• měď * farmakologie   MeSH
• nádory * farmakoterapie   MeSH
• proteasomový endopeptidasový komplex MeSH
• proteiny metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• disulfiram MeSH
• měď * MeSH
• proteasomový endopeptidasový komplex MeSH
• proteiny MeSH
• ubikvitin MeSH

Endocytosis controls the perception of stimuli by modulating protein abundance at the plasma membrane. In plants, clathrin-mediated endocytosis is the most prominent internalization pathway and relies on two multimeric adaptor complexes, the AP-2 and the TPLATE complex (TPC). Ubiquitination is a well-established modification triggering endocytosis of cargo proteins, but how this modification is recognized to initiate the endocytic event remains elusive. Here we show that TASH3, one of the large subunits of TPC, recognizes ubiquitinated cargo at the plasma membrane via its SH3 domain-containing appendage. TASH3 lacking this evolutionary specific appendage modification allows TPC formation but the plants show severely reduced endocytic densities, which correlates with reduced endocytic flux. Moreover, comparative plasma membrane proteomics identified differential accumulation of multiple ubiquitinated cargo proteins for which we confirm altered trafficking. Our findings position TPC as a key player for ubiquitinated cargo internalization, allowing future identification of target proteins under specific stress conditions.

• MeSH
• buněčná membrána metabolismus   MeSH
• endocytóza * MeSH
• klathrin * genetika   metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• klathrin * MeSH
• ubikvitin MeSH

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

• MeSH
• deubikvitinasy metabolismus   MeSH
• elektroforéza kapilární MeSH
• elektroforéza mikročipová * MeSH
• lidé MeSH
• proteom MeSH
• ubikvitin * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• deubikvitinasy MeSH
• proteom MeSH
• ubikvitin * MeSH

BACKGROUND: Ubiquitin ligases (Ub-ligases) are essential intracellular enzymes responsible for the regulation of proteome homeostasis, signaling pathway crosstalk, cell differentiation and stress responses. Individual Ub-ligases exhibit their unique functions based on the nature of their substrates. They create a complex regulatory network with alternative and feedback pathways to maintain cell homeostasis, being thus important players in many physiological and pathological conditions. However, the functional classification of Ub-ligases needs to be revised and extended. METHODS: In the current study, we used a novel semantic biclustering technique for expression profiling of Ub-ligases and ubiquitination-related genes in the murine gastrointestinal tract (GIT). We accommodated a general framework of the algorithm for finding tissue-specific gene expression clusters in GIT. In order to test identified clusters in a biological system, we used a model of epithelial regeneration. For this purpose, a dextran sulfate sodium (DSS) mouse model, following with in situ hybridization, was used to expose genes with possible compensatory features. To determine cell-type specific distribution of Ub-ligases and ubiquitination-related genes, principal component analysis (PCA) and Uniform Manifold Approximation and Projection technique (UMAP) were used to analyze the Tabula Muris scRNA-seq data of murine colon followed by comparison with our clustering results. RESULTS: Our established clustering protocol, that incorporates the semantic biclustering algorithm, demonstrated the potential to reveal interesting expression patterns. In this manner, we statistically defined gene clusters consisting of the same genes involved in distinct regulatory pathways vs distinct genes playing roles in functionally similar signaling pathways. This allowed us to uncover the potentially redundant features of GIT-specific Ub-ligases and ubiquitination-related genes. Testing the statistically obtained results on the mouse model showed that genes clustered to the same ontology group simultaneously alter their expression pattern after induced epithelial damage, illustrating their complementary role during tissue regeneration. CONCLUSIONS: An optimized semantic clustering protocol demonstrates the potential to reveal a readable and unique pattern in the expression profiling of GIT-specific Ub-ligases, exposing ontologically relevant gene clusters with potentially redundant features. This extends our knowledge of ontological relationships among Ub-ligases and ubiquitination-related genes, providing an alternative and more functional gene classification. In a similar way, semantic cluster analysis could be used for studding of other enzyme families, tissues and systems.

• Klíčová slova
• Cluster analysis, GIT, Gene redundancy, Regeneration, Semantic biclustering, Ub-ligase,
• MeSH
• gastrointestinální trakt metabolismus   MeSH
• lidé MeSH
• myši MeSH
• sémantika * MeSH
• shluková analýza MeSH
• ubikvitin genetika   metabolismus   MeSH
• ubikvitinligasy * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ubikvitin MeSH
• ubikvitinligasy * MeSH

BACKGROUND: Many regulatory circuits in plants contain steps of targeted proteolysis, with the ubiquitin proteasome system (UPS) as the mediator of these proteolytic events. In order to decrease ubiquitin-dependent proteolysis, we inducibly expressed a ubiquitin variant with Arg at position 48 instead of Lys (ubK48R). This variant acts as an inhibitor of proteolysis via the UPS, and allowed us to uncover processes that are particularly sensitive to UPS perturbation. RESULTS: Expression of ubK48R during germination leads to seedling death. We analyzed the seedling transcriptome, proteome and metabolome 24 h post ubK48R induction and confirmed defects in chloroplast development. We found that mutations in single genes can suppress seedling lethality, indicating that a single process in seedlings is critically sensitive to decreased performance of the UPS. Suppressor mutations in phototropin 2 (PHOT2) suggest that a contribution of PHOT2 to chloroplast protection is compromised by proteolysis inhibition. CONCLUSIONS: Overall, the results reveal protein turnover as an integral part of a signal transduction chain that protects chloroplasts during development.

• Klíčová slova
• Chlorophagy, Chloroplast development, Light signal transduction, Light stress, Photomorphogenesis, Phototropin, Ubiquitin K48 chains,
• MeSH
• chloroplasty genetika   metabolismus   MeSH
• metabolom MeSH
• proteasomový endopeptidasový komplex * genetika   metabolismus   MeSH
• proteolýza MeSH
• semenáček genetika   metabolismus   MeSH
• transkriptom MeSH
• ubikvitin * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteasomový endopeptidasový komplex * MeSH
• ubikvitin * MeSH

Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.

• Klíčová slova
• Nse1, Nse4 kleisin, SMC5/6, Ubc13/Mms2, ubiquitin ligase, ubiquitination,
• MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• lidé MeSH
• ligasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinace imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• ligasy MeSH
• NSMCE1 protein, human MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• SMC5 protein, human MeSH Prohlížeč
• transportní proteiny MeSH
• ubikvitin MeSH

Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.

• Klíčová slova
• C-terminal degron, DCAF12, MOV10, T cell activation, WDR40A, spermatogenesis,
• MeSH
• aktivace lymfocytů fyziologie   MeSH
• antigeny nádorové metabolismus   MeSH
• buněčné linie MeSH
• CD4-pozitivní T-lymfocyty metabolismus   MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• NKT buňky metabolismus   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• RNA-helikasy metabolismus   MeSH
• spermatogeneze fyziologie   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• DCAF12 protein, human MeSH Prohlížeč
• IL17RB protein, human MeSH Prohlížeč
• Mov10 protein, human MeSH Prohlížeč
• proteasomový endopeptidasový komplex MeSH
• RNA-helikasy MeSH
• ubikvitin MeSH
• ubikvitinligasy MeSH

Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• povrchová plasmonová rezonance MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• protein obsahující valosin genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP70 metabolismus   MeSH
• reakce na tepelný šok * MeSH
• stříbro chemie   MeSH
• ubikvitin metabolismus   MeSH
• vysoká teplota škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteasomový endopeptidasový komplex MeSH
• protein obsahující valosin MeSH
• proteiny tepelného šoku HSP70 MeSH
• stříbro MeSH
• ubikvitin MeSH
• VCP protein, human MeSH Prohlížeč

Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.

• MeSH
• kmenové buňky metabolismus   patologie   MeSH
• lymfopoéza * MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• T-lymfocyty metabolismus   patologie   MeSH
• ubikvitin genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ubikvitin MeSH
• zelené fluorescenční proteiny MeSH

The Epstein-Barr virus (EBV) immediate early transactivator Zta plays a key role in regulating the transition from latency to the lytic replication stages of EBV infection. Regulation of Zta is known to be controlled through a number of transcriptional and posttranscriptional events. Here, we show that Zta is targeted for ubiquitin modification and that this can occur in EBV-negative and in EBV-infected cells. Genetic studies show critical roles for both an amino-terminal region of Zta and the basic DNA binding domain of Zta in regulating Zta ubiquitination. Pulse-chase experiments demonstrate that the bulk population of Zta is relatively stable but that at least a subset of ubiquitinated Zta molecules are targeted for degradation in the cell. Mutation of four out of a total of nine lysine residues in Zta largely abrogates its ubiquitination, indicating that these are primary ubiquitination target sites. A Zta mutant carrying mutations at these four lysine residues (lysine 12, lysine 188, lysine 207, and lysine 219) cannot induce latently infected cells to produce and/or release infectious virions. Nevertheless, this mutant can induce early gene expression, suggesting a possible defect at the level of viral replication or later in the lytic cascade. As far as we know, this is the first study that has investigated the targeting of Zta by ubiquitination or its role in Zta function.IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen and associated with various human diseases. EBV undergoes latency and lytic replication stages in its life cycle. The transition into the lytic replication stage, at which virus is produced, is mainly regulated by the viral gene product, Zta. Therefore, the regulation of Zta function becomes a central issue regarding viral biology and pathogenesis. Known modifications of Zta include phosphorylation and sumoylation. Here, we report the role of ubiquitination in regulating Zta function. We found that Zta is subjected to ubiquitination in both EBV-infected and EBV-negative cells. The ubiquitin modification targets 4 lysine residues on Zta, leading to both mono- and polyubiquitination of Zta. Ubiquitination of Zta affects the protein's stability and likely contributes to the progression of viral lytic replication. The function and fate of Zta may be determined by the specific lysine residue being modified.

• Klíčová slova
• Epstein-Barr virus, Zta, lytic replication, reactivation, ubiquitination,
• MeSH
• buněčné linie MeSH
• infekce virem Epsteina-Barrové virologie   MeSH
• lidé MeSH
• mutace MeSH
• promotorové oblasti (genetika) MeSH
• proteinové domény MeSH
• regulace exprese virových genů MeSH
• replikace viru MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• vazba proteinů MeSH
• virové proteiny genetika   metabolismus   MeSH
• virus Epsteinův-Barrové genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• BZLF1 protein, Herpesvirus 4, Human MeSH Prohlížeč
• trans-aktivátory MeSH
• ubikvitin MeSH
• virové proteiny MeSH