Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

Role of E3 ubiquitin ligases in gastric cancer

E3 ubiquitin ligases have an important role in carcinogenesis and include a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.So far,E3 ubiquitin ligases have been reported to have a role in a variety of biological processes including cell cycle regulation,cell proliferation,and apoptosis.Recently,several kinds of E3 ubiquitin ligases were demonstrated to be generally highly expressed in gastric cancer(GC) tissues and to contribute to carcinogenesis.In this review,we summarize thecurrent knowledge and information about the clinical significance of E3 ubiquitin ligases in GC.Bortezomib,a proteasome inhibitor,encouraged the evaluation of other components of the ubiquitin proteasome system for pharmaceutical intervention.The clinical value of novel treatment strategies targeting aberrant E3 ubiquitin ligases for GC are discussed in the review.

Role of E3 ubiquitin ligases in lung cancer

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome.Therefore,E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation,proliferation and apoptosis.E3 ubiquitin ligases are often found overexpressed in human cancers,including lung cancer,and their deregulation has been shown to contribute to cancer development.However,the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting.In this review,we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer.Furthermore,we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets.By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis,we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.

Research Progress in Function and Regulation of E3 Ubiquitin Ligase SMURF1

Smad ubiquitylation regulatory factor 1(Smurf1)is an important homologous member of E6-AP C-terminus type E3 ubiquitin ligase.Initially,Smurf1 was reportedly involved in the negative regulation of the bone morphogenesis protein(BMP)pathway.After further research,several studies have confirmed that Smurf1 is widely involved in various biological processes,such as bone homeostasis regulation,cell migration,apoptosis,and planar cell polarity.At the same time,recent studies have provided a deeper understanding of the regulatory mechanisms of Smurf1’s expression,activity,and substrate selectivity.In our review,a brief summary of recent important biological functions and regulatory mechanisms of E3 ubiquitin ligase Smurf1 is proposed.

Changes and significance of serum ubiquitin carboxyl-terminal hydrolase L1 and glial fibrillary acidic protein in patients with glioma

BACKGROUND Brain gliomas are malignant tumors with high postoperative recurrence rates.Early prediction of prognosis using specific indicators is of great significance.AIM To assess changes in ubiquitin carboxy-terminal hydrolase L1(UCH-L1)and glial fibrillary acidic protein(GFAP)levels in patients with glioma pre-and postoperatively.METHODS Between June 2018 and June 2021,91 patients with gliomas who underwent surgery at our hospital were enrolled in the glioma group.Sixty healthy volunteers were included in the control group.Serum UCH-L1 and GFAP levels were measured in peripheral blood collected from patients with glioma before and 3 d after surgery.UCH-L1 and GFAP levels in patients with glioma with different clinicopathological characteristics were compared before and after surgery.The patients were followed-up until February 2022.Postoperative glioma recurrence was recorded to determine the serum UCH-L1 and GFAP levels,which could assist in predicting postoperative glioma recurrence.RESULTS UCH-L1 and GFAP levels in patients with glioma decreased significantly 3 d after surgery compared to those before therapy(P<0.05).However,UCH-L1 and GFAP levels in the glioma group were significantly higher than those in the control group before and after surgery(P<0.05).There were no statistically significant differences in preoperative serum UCH-L1 and GFAP levels among patients with glioma according to sex,age,pathological type,tumor location,or number of lesions(P>0.05).Serum UCH-L1 and GFAP levels were significantly lower in the patients with WHO grade I-II tumors than in those with gradeⅢ-IV tumors(P<0.05).Serum UCH-L1 and GFAP levels were lower in the patients with tumor diameter≤5 cm than in those with diameter>5 cm,in which the differences were statistically significant(P<0.05).Glioma recurred in 22 patients.The preoperative and 3-d postoperative serum UCH-L1 and GFAP levels were significantly higher in the recurrence group than these in the non-recurrence group(P<0.05).Receiver operating characteristic curves were plotted.The areas under the curves of preoperative serum UCH-L1 and GFAP levels for predicting postoperative glioma recurrence were 0.785 and 0.775,respectively.However,the efficacy of serum UCH-L1 and GFAP levels 3 d after surgery in predicting postoperative glioma recurrence was slightly lower compared with their preoperative levels.CONCLUSION UCH-L1 and GFAP efficiently reflected the development and recurrence of gliomas and could be used as potential indicators for the recurrence and prognosis of glioma.

The Cbl family of ubiquitin ligases regulates FcεRI expression and mast cell activation

Antigen interaction with specific IgE bound to the high-affinity Fc receptor for IgE, constitutively expressed on the cell-surface of mast cells, generates signals that cause a shift in the resting state equilibrium of phosphorylation and dephosphorylation events that serves to maintain homeostasis. The outcome of this activated state is the release of a wide array of preformed and newly synthesized pro-inflammatory mediators. During the past few years, the existence of a negative feedback loop initiated upon FcεRI engagement has also been envisaged. This negative signal involves the coordinated action of adaptors, phosphatases and ubiquitin ligases that limits the intensity and duration of positive signals, thus modulating mast cell functions. Relevant to this, others and we have demonstrated that Cbl family proteins control the amplitude of FcεRI-generated signals by specific ubiquitin modification of activated receptor subunits and associated protein tyrosine kinases. In this article, we review advances in our understanding of the molecular mechanisms through which Cbl proteins regulate FcεRI expression and signaling.

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

Molecular cloning and functional characterization of apple U-box E3 ubiquitin ligase gene MdPUB29 reveals its involvement in salt tolerance

An E3 ubiquitin ligase gene(Genbank accession no.:MD01 G1010900) was cloned from the Royal Gala apple genome(Malus×domestica Borkh.).Sequence analysis showed that the length of the MdPUB29 gene was 1 275 bp,encoding 424 amino acids.Phylogenetic tree analysis indicated that the apple E3 ubiquitin ligase exhibited the greatest sequence similarity to Pyrus×bretschneideri.The predicted protein structural domain of MdPUB29 showed that it contained a U-box domain.qRT-PCR analysis showed that Md PUB29 was expressed widely in different tissues of the Royal Gala apple species,and was highly expressed in the root,while the expression of MdPUB29 was significantly inhibited by exogenous NaCl.Immunoblotting assays revealed that MdPUB29 protein abundance in tissue cultures of the Royal Gala apple accumulated under NaC l stress conditions.Three-dimensional protein structure prediction indicated that MdPUB29 was highly homologous with AtPUB29.The growing potential of MdPUB29-expressing apple calli and Arabidopsis were much stronger than that of the control under salt stress conditions,suggesting that MdPUB29 may positively regulate salt tolerance.

Ubiquitination-mediated protein degradation and modification:an emerging theme in plant-microbe interactions

Post-translational modification is central to protein stability and to the modulation of protein activity. Various types of protein modification, such as phosphorylation, methylation, acetylation, myristoylation, glycosylation, and ubiquitination, have been reported. Among them, ubiquitination distinguishes itself from others in that most of the ubiquitinated proteins are targeted to the 26S proteasome for degradation. The ubiquitin/26S proteasome system constitutes the major protein degradation pathway in the cell. In recent years, the importance of the ubiquitination machinery in the control of numerous eukaryotic cellular functions has been increasingly appreciated. Increasing number of E3 ubiquitin ligases and their substrates, including a variety of essential cellular regulators have been identified. Studies in the past several years have revealed that the ubiquitination system is important for a broad range of plant developmental processes and responses to abiotic and biotic stresses. This review discusses recent advances in the functional analysis of ubiquitination-associated proteins from plants and pathogens that play important roles in plant-microbe interactions.

RNF13： a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin ligase activity. Immunohistochemical analysis of pancreatic ductal adenocarcinoma （PDAC） and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples （41.7%, 30/72）, and its expression was significantly associated with histological grading （P= 0.024）. In addition, RNF13 was detected in precancerous lesions： tubular complexes in chronic pancreatitis （CP） and pancreatic intraepithelial neoplasia （PanIN） （79.3%, 23/29 and 62.8%, 22/35, respectively）. Moreover, RNF13 staining was significantly correlated with Tenascin-C expression （P = 0.004） in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

Identification of Ubiquitinated Proteins from Human Multiple Myeloma U266 Cells by Proteomics

Objective To identify ubiquitinated proteins from complex human multiple myeloma （MM） U266 cells,a malignant disorder of differentiated human B cells.Methods Employing a globally proteomic strategy combining of immunoprecipitation,LC-MS/MS and SCX-LC-MS analysis to identified ubiquitination sites,which were identified by detecting signature peptides containing a GG-tag （114.1 Da） and an LRGG-tag （383.2 Da）.Results In total,52 ubiquitinated proteins containing 73 ubiquitination sites of which 14 and 59 sites contained LRGG-tag and GG-tag were identified,respectively.Conclusion Classification analysis by of the proteins identified in the study based on the PANTHER showed that they were associated with multiple functional groups.This suggested the involvement of many endogenous proteins in the ubiquitination in MM.

Characterization of a Novel RING-Type Ubiquitin E3 Ligase GhRING2 Differentially Expressed in Cotton Fiber

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is differentially expressed between two Gossypium hirsutum lines-Texas Marker-1 (TM-1) and Chromosome Substitution Line CS-B25. The CS-B25 line has chromosome 25 from G. barbadense substituted into TM-1. The complete nucleotide sequences of GhRING2 along with its 5’-flanking region were obtained by genomic walking. GhRING2 was highly expressed in elongating fiber, and GUS expression directed by the GhRING2 promoter was found in hypocotyls and young stems of transgenic Arabidopsis plants. Using a yeast two-hybrid assay GhRING2 was found to interact with a PROTODERMAL FACTOR1 (GhPDF1) protein. GhPDF1 was expressed preferentially in immature ovules and fiber initials, and the GhPDF1 gene had been suggested to play a role in cell fate determination and fiber development. Pull down and plasmid swap assays further confirmed the interaction between GhRING2 and GhPDF1. The expression and protein interaction data indicate that GhRING2 may be involved in the turnover of GhPDF1 and participation in the transition from initiation to elongation stages during fiber development. Our data strongly suggest that the ubiquitin-proteasome pathway may regulate cotton fiber growth and development. The nucleotide sequence data of GhRING2 in this article have been submitted to the Gen Bank Nucleotide Sequence Data Bases under the accession number BankIt 1,742,008 SeqKM 108,000.

Effects of Nephrolithiasis on Serum DNase(Deoxyribonuclease Ⅰ and Ⅱ) Activity and E3 SUMO-Protein Ligase NSE2(NSMCE2) in Malaysian Individuals

Objective Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase Ⅰ/Ⅱ activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Methods Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase Ⅰ/Ⅱ activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. Results The result indicated that mean levels of sera NSMCE2 have a significantly increase（P〈0.01） in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase Ⅰ and Ⅱ were significantly elevated in nephrolithiasis patients（P〈0.01）. Conclusion This study suggests that an increase in serum concentrations of DNase Ⅰ/Ⅱ and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

F-box only protein 2 exacerbates non-alcoholic fatty liver disease by targeting the hydroxyl CoA dehydrogenase alpha subunit

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a major health burden with an increasing global incidence.Unfortunately,the unavailability of knowledge underlying NAFLD pathogenesis inhibits effective preventive and therapeutic measures.AIM To explore the molecular mechanism of NAFLD.METHODS Whole genome sequencing(WGS)analysis was performed on liver tissues from patients with NAFLD(n=6)and patients with normal metabolic conditions(n=6)to identify the target genes.A NAFLD C57BL6/J mouse model induced by 16 wk of high-fat diet feeding and a hepatocyte-specific F-box only protein 2(FBXO2)overexpression mouse model were used for in vivo studies.Plasmid transfection,co-immunoprecipitation-based mass spectrometry assays,and ubiquitination in HepG2 cells and HEK293T cells were used for in vitro studies.RESULTS A total of 30982 genes were detected in WGS analysis,with 649 up-regulated and 178 down-regulated.Expression of FBXO2,an E3 ligase,was upregulated in the liver tissues of patients with NAFLD.Hepatocyte-specific FBXO2 overexpression facilitated NAFLD-associated phenotypes in mice.Overexpression of FBXO2 aggravated odium oleate(OA)-induced lipid accumulation in HepG2 cells,resulting in an abnormal expression of genes related to lipid metabolism,such as fatty acid synthase,peroxisome proliferator-activated receptor alpha,and so on.In contrast,knocking down FBXO2 in HepG2 cells significantly alleviated the OA-induced lipid accumulation and aberrant expression of lipid metabolism genes.The hydroxyl CoA dehydrogenase alpha subunit(HADHA),a protein involved in oxidative stress,was a target of FBXO2-mediated ubiquitination.FBXO2 directly bound to HADHA and facilitated its proteasomal degradation in HepG2 and HEK293T cells.Supplementation with HADHA alleviated lipid accumulation caused by FBXO2 overexpression in HepG2 cells.CONCLUSION FBXO2 exacerbates lipid accumulation by targeting HADHA and is a potential therapeutic target for NAFLD。

The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease

In this study, we investigated the effect of an antibody against E3 ubiquitin ligase seven in absentia homolog 1（SIAH-1） in PC12 cells. 1-Methyl-4-phenylpyridinium（MPP＋） treatment increased α-synuclein, E1 and SIAH-1 protein levels in PC12 cells, and it reduced cell viability; however, there was no significant change in light chain 3 expression. Treatment with an SIAH-1 antibody decreased m RNA expression levels of α-synuclein, light chain 3 and SIAH-1, but increased E1 m RNA expression. It also increased cell viability. Combined treatment with MPP＋ and rapamycin reduced SIAH-1 and α-synuclein levels. Treatment with SIAH-1 antibody alone diminished α-synuclein immunoreactivity in PC12 cells, and reduced the colocalization of α-synuclein and light chain 3. These findings suggest that the SIAH-1 antibody reduces the monoubiquitination and aggregation of α-synuclein, promoting its degradation by the ubiquitin-proteasome pathway. Consequently, SIAH-1 may be a potential new therapeutic target for Parkinson’s disease.

Research status of E3 ubiquitin ligase Smurf2 and related signaling pathways in glioma

Glioma is the tumor with the highest incidence in the brain,and it is eager to seek new and efiective treatment.The interaction of ubiquitination and deubiquitination regulates many cell activities in organisms,and participates in tumor occurrence,development,migration,invasion and other processes.This article summarized the progress of E3 ubiquitination ligase smad ubiquitination regulatory factor 2(Smurf2)and glioma-related signaling pathways to assist clinical diagnosis and treatment of glioma.

Study the effect of kidney stones on serum xanthine oxidase, ecto-5'-nucleotidase activity and E3 SUMO-protein ligase NSE2(NSMCE2) in Malaysian individuals

Objective: To verify possible relations between 5'-nucleotidase, xanthine oxidase to E3 small ubiquitin-like modifier-protein ligase non structural maintenance of chromosomes elements 2 in sera patients with kidney stones and to evaluate the possibility of a new biomarker for the evaluation of kidney damage. Methods: A sixty patients with known kidney stones who appeared the government health clinics in Kuantan–Pahang and fifty apparently healthy were taken as control group. The 5'-nucleotidase,xanthine oxidase and other biochemical parameters were measured by colorimetric tests. The serum NSMCE2 were measured by enzyme linked immunosorbent assay.Results: The mean serum xanthine oxidase [(39.98±19.70) IU/L] and ecto-5'-nucleotidase activity(40.03±9.53 IU/L) were significantly higher than the controls' levels of(18.04 ±6.26) and(16.06 ±4.61) IU/L respectively. There were 85.00% and 83.33%, of patients with kidney stones who had abnormal ecto-5'-nucleotidase activity and uric acid respectively while xanthine oxidase activity was less sensitive 58.33%.Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5'-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone,also in developments of change DNA damage and inflammation disorders in these patients.

Serum E3 SUMO-protein ligase NSE2 level and peroxynitrite related to oxidative stress in nephrolithiasis patients

Objective: To prove probable relations between serum E3 SUMO-protein ligase NSE2(NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients.Methods: A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA.Results: A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased.Conclusions: The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

Isolation and characterization ofβ-transducin repeat-containing protein ligands screened using a high-throughput screening system

β-transducin repeat-containing protein(β-TrCP)is an F-box protein subunit of the E3 Skp1-Cullin-F box(SCF)type ubiquitin-ligase complex,and provides the substrate specificity for the ligase.To find potent ligands ofβ-TrCP useful for the proteolysis targeting chimera(PROTAC)system usingβ-TrCP in the future,we developed a high-throughput screening system for small moleculeβ-TrCP ligands.We screened the chemical library utilizing the system and obtained several hit compounds.The effects of the hit compounds on in vitro ubiquitination activity of SCFβ-TrCP1 and on downstream signaling pathways were examined.Hit compounds NPD5943,NPL62020-01,and NPL42040-01 inhibited the TNFα-induced degradation of IκBαand its phosphorylated form.Hence,they inhibited the activation of the transcription activity of NF-κB,indicating the effective inhibition ofβ-TrCP by the hit compounds in cells.Next,we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds.Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds withβ-TrCP similar to those created by intrinsic target phosphopeptides ofβ-TrCP.Our findings enhance our knowledge of useful small molecule ligands ofβ-TrCP and the importance of residues that can be ligands ofβ-TrCP.