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.

• 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
• 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
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The monitoring of intracellular cholesterol homeostasis and trafficking is of great importance because their imbalance leads to many pathologies. Reliable tools for cholesterol detection are in demand. This study presents the design and synthesis of fluorescent probes for cholesterol recognition and demonstrates their selectivity by a variety of methods. The construction of dedicated library of 14 probes was based on heterocyclic (pyridine)-sterol derivatives with various attached fluorophores. The most promising probe, a P1-BODIPY conjugate FP-5, was analysed in detail and showed an intensive labelling of cellular membranes followed by intracellular redistribution into various cholesterol rich organelles and vesicles. FP-5 displayed a stronger signal, with faster kinetics, than the commercial TF-Chol probe. In addition, cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, exhibited strong and fast FP-5 signal in the endo/lysosomal compartment, co-localizing with filipin staining of cholesterol. Hence, FP-5 has high potential as a new probe for monitoring cholesterol trafficking and its disorders.

• MeSH
• biologický transport MeSH
• buněčná membrána chemie   metabolismus   MeSH
• buněčné linie MeSH
• cholesterol analýza   metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• lyzozomální nemoci z ukládání diagnóza   metabolismus   MeSH
• pyridiny chemie   MeSH
• sloučeniny boru chemie   MeSH
• steroly chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH