Culling protein is a member of Cullin-Ring-based E3-ligases ( CRLs family) , which belong to E3 ubiquitin ligases. Cullin plays diverse and essential roles in many biological processes through mediating the ubiquitination of target proteins. This article summarizes the potential functions of Culling proteins in gamete genesis and maturation, embryo development, and reproductive related disorders.
Cullin Proteins ; Humans ; Urogenital System

Cereblon（CRBN) is a brain-associated protein with ionic protease activity, which interacts with DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B), and regulator of Cullins 1 (RoC1) to form the functional E3 ubiquitin ligase complex(CRBN-CRL4) that performs proteolysis via the ubiquitin-proteasome pathway. And CRBN is a necessary target protein for the anti-myeloma effect of immunomodulators. The combination of lenalidomide and CRBN recruited a new substrate that binds to the CRBN-CRL4 complex, leading to increased ubiquitination and proteasome-dependent degradation, thus resulting in anti-myeloma activity. The substrates binding to this complex are IKZF1, IKZF3 proteins and GS, etc. The CRBN-dependent degradation of IKZF1 and IKZF3 after lenalidomide treatment is also the result of HO-mediated oxidative stress. In addition to ubiquitination, lenalidomide also mediates ubiquitin-independent pathways that prevent CRBN from binding to CD147-MCT1 in a competitive manner to regulate its antitumor activity. Lenalidomide can also play a role in multiple myeloma(MM) cells by modulating miRNA levels and CRBN binding to downstream protein AGO2 expression. Thus, there are many molecular mechanisms of lenalidomide anti-myeloma activity. This review summarizes the molecular mechanisms of CRBN in lenalidomide against myeloma activity in terms of ubiquitin-dependent and ubiquitin-independent pathways.
Cullin Proteins ; Humans ; Hydrogen Peroxide ; Multiple Myeloma ; Peptide Hydrolases ; Proteolysis ; Thalidomide ; Ubiquitination

Cullin Proteins ; genetics ; Humans ; Mutation ; Protein-Serine-Threonine Kinases ; Pseudohypoaldosteronism ; genetics ; Vision Disorders ; genetics

Cullin-RING E3 ubiquitin ligase (CRL)-4 is a member of the large CRL family in eukaryotes. It plays important roles in a wide range of cellular processes, organismal development, and physiological and pathological conditions. DDB1- and CUL4-associated factor 8 (DCAF8) is a WD40 repeat-containing protein, which serves as a substrate receptor for CRL4. The physiological role of DCAF8 is unknown. In this study, we constructed Dcaf8 knockout mice. Homozygous mice were viable with no noticeable abnormalities. However, the fertility of Dcaf8-deficient male mice was markedly impaired, consistent with the high expression of DCAF8 in adult mouse testis. Sperm movement characteristics, including progressive motility, path velocity, progressive velocity, and track speed, were significantly lower in Dcaf8 knockout mice than in wild-type (WT) mice. However, the total motility was similar between WT and Dcaf8 knockout sperm. More than 40% of spermatids in Dcaf8 knockout mice showed pronounced morphological abnormalities with typical bent head malformation. The acrosome and nucleus of Dcaf8 knockout sperm looked similar to those of WT sperm. In vitro tests showed that the fertilization rate of Dcaf8 knockout mice was significantly reduced. The results demonstrated that DCAF8 plays a critical role in spermatogenesis, and DCAF8 is a key component of CRL4 function in the reproductive system.
Animals ; Cullin Proteins/genetics* ; DNA-Binding Proteins/genetics* ; Factor VIII ; Male ; Mice ; Mice, Knockout ; Spermatogenesis/genetics* ; Ubiquitin-Protein Ligases

AIMTo identify genes related to the human testis development by substrate hybridization technique.

METHODSA human testis cDNA microarray was constructed and hybridized with probes prepared from human adult and fetal testes and spermatozoa mRNAs by reverse transcription reactions. The differentially expressed genes were sequenced. And a newly identified cullin-3 (CUL-3) transcript variant (designated cul-3b) was bio-informatically analyzed with an online GenBank database. Multi-tissue reverse transcription polymerase chain reaction (RT-PCR) was used to determine the tissue expression profile of cul-3b.

RESULTSCul-3b, a novel CUL-3 transcript variant, was identified. The expression level of cul-3b in adult testes was 3.79-fold higher than that in fetal ones. Cul-3b differed from cul-3 (including NM_003590 and AY337761) in the opening reading frame and had three internal ribosomal entry sites IRESes in the 5'-UTR. These led to a 24 amino acid (aa) truncation at N-terminus of CUL-3b as compared with CUL-3 and a more motivated expression pattern of cul-3b under some strict circumstances. Additionally, cul-3b expressed ubiquitously in human tissues according to multi-tissue RT-PCR.

CONCLUSIONCul-3b is a novel transcript variant of CUL-3, which may be important not only for the development of human testis but also for that of other organs.

Base Sequence ; Cell Cycle Proteins ; genetics ; Cullin Proteins ; genetics ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Cullin 4A (CUL4A) is an E3 ubiquitin ligase that directly affects DNA repair and cell cycle progression by targeting substrates including damage-specific DNA-binding protein 2 (DDB2), xeroderma pigmentosum complementation group C (XPC), chromatin licensing and DNA replication factor 1 (Cdt1), and p21. Recent work from our laboratory has shown that Cul4a-deficient mice have greatly reduced rates of ultraviolet-induced skin carcinomas. On a cellular level, Cul4a-deficient cells have great capacity for DNA repair and demonstrate a slow rate of proliferation due primarily to increased expression of DDB2 and p21, respectively. This suggests that CUL4A promotes tumorigenesis (as well as accumulation of skin damage and subsequent premature aging) by limiting DNA repair activity and expediting S phase entry. In addition, CUL4A has been found to be up-regulated via gene amplification or overexpression in breast cancers, hepatocellular carcinomas, squamous cell carcinomas, adrenocortical carcinomas, childhood medulloblastomas, and malignant pleural mesotheliomas. Because of its oncogenic activity in skin cancer and up-regulation in other malignancies, CUL4A has arisen as a potential candidate for targeted therapeutic approaches. In this review, we outline the established functions of CUL4A and discuss the E3 ligase's emergence as a potential driver of tumorigenesis.
Animals ; Carcinogenesis ; metabolism ; Cell Cycle ; Cell Proliferation ; Cullin Proteins ; genetics ; metabolism ; DNA Damage ; DNA Repair ; DNA-Binding Proteins ; metabolism ; Drug Delivery Systems ; Humans ; Neoplasms ; genetics ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Skin Neoplasms ; genetics ; metabolism ; pathology

OBJECTIVETo study muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1) mRNA expression and its relationship with muscular contraction following free muscle transfer.

METHODSThe gracilis muscle was orthotopic transferred in adult rat to establish the animal model. The muscle at the unoperated side was used as control. The expression of MAFbx and MuRF1 mRNA, the muscle contraction and muscle function were measured by real-time PCR and multiple function physiological device. The relationship among the expression of MAFbx and MuRF1 mRNA, the muscle contraction and muscle function was analyzed.

RESULTSAfter muscle free transfer, muscle wet weight reservation, the maximum contraction and tetanus strength reduce first and increased later, but still lower than those at control side. The expression of MAFbx and MuRF1 mRNA reached peak level 3 - 4 weeks after muscle transfer which was 7.1 and 4.1 times as that at control side. It decreased later, but still higher than that at control side, showing a significant difference between them (P< 0. 05).

CONCLUSIONSPersistent over-expression of MAFbx and MuRF1 mRNA after muscle transfer has a close relationship with muscle atrophy and muscle dysfunction. MAFbx and MuRF1 can be used as markers for early muscle atrophy, and also as potential target for drug treatment of muscle atrophy.

Animals ; Female ; Muscle Contraction ; Muscle Proteins ; genetics ; Muscle, Skeletal ; pathology ; Muscular Atrophy ; genetics ; metabolism ; pathology ; RING Finger Domains ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; SKP Cullin F-Box Protein Ligases ; genetics ; Tripartite Motif Proteins ; Ubiquitin-Protein Ligases ; genetics

BACKGROUND: Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer. METHODS: Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot. RESULTS: Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05). CONCLUSIONS LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.
Humans ; Lung Neoplasms/pathology* ; RNA, Long Noncoding/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Up-Regulation ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Methyltransferases/metabolism* ; Cullin Proteins/genetics*

OBJECTIVETo explore the interaction between SerpinB5 and MAFbx in gastric cancer cell and to identify the interaction sites.

METHODSThe interaction between SerpinB5 and MAFbx was screened and validated by yeast two-hybrid screening and co-immunoprecipitation. The expression of MAFbx was analyzed after SerpinB5 expression being modified by RNA interference and pGBKT7-SerpinB5 transfection. The impact of SerpinB5 on the expression of MAFbx was studied in gastric cancer cell line SUN-16. A model of MAFbx was constructed by homology modeling. The related residues for interaction were analyzed by Autodock4.0.

RESULTSThe interaction between SerpinB5 and MAFbx was validated. The expression of MAFbx changed along with SerpinB5 expression. Amino acids including PRO261, ASN361, and LYS362 were key residue in the interaction of SerpinB5 and MAFbx.

CONCLUSIONSerpinB5 interacts with MAFbx in gastric cancer cell. Amino acids including PRO261, ASN361, and LYS362 are potential binding sites.

Cell Line, Tumor ; Humans ; Immunoprecipitation ; Muscle Proteins ; genetics ; metabolism ; RNA Interference ; SKP Cullin F-Box Protein Ligases ; genetics ; metabolism ; Serpins ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo investigate the protective effect of Atrogin-1 gene silencing via RNA interference technique on a model of muscle cell malnutrition.

METHODSSequences of five target Atrogin-1 siRNA and the control were selected and synthesized and cloned to vector pBS-hU6-I and then to vector FG12. The length and rightness of the sequences were confirmed. The recombinant FG12 vectors were cotransfected along with pRSVREV, pMDLg/pRRE and pHCMV-G into 293T cells to package lentivirus particles, with which C2C12 cells were infected. The infected C2C12 cells were cultured and differentiated to form myotubes before TNF-alpha was added to induce malnutrition. Expressed products of Atrogin-1 of myotubes were identified by real time PCR and Western blot methods. Myotubes were observed and photographed directly in culture plate without fixation.

RESULTSThe length and sequences of inserted DNA were right. Compared with the RNA interferencing group, significant atrophy and upregulated expression of Atrogin-1 of myotubes treated by TNF-alpha was found in the control group.

CONCLUSIONAtrogin-1 gene silencing could be used to inhibit malnutrition of muscle cells caused by TNF-alpha. Atrogin-1 could be an ideal target in the treatment of cancer cachexia.

Animals ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Gene Silencing ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; Muscle Cells ; cytology ; drug effects ; metabolism ; Muscle Proteins ; genetics ; RNA, Small Interfering ; genetics ; SKP Cullin F-Box Protein Ligases ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology