Ubiquitin-specific Protease 26 Research Articles

Acetylation-dependent deubiquitinase USP26 stabilizes BAG3 to promote breast cancer progression

Deubiquitylases (DUBs) have emerged as promising targets for cancer therapy due to their role in stabilizing substrate proteins within the ubiquitin machinery. Here, we identified ubiquitin-specific protease 26 (USP26) as an oncogene via screening prognostic DUBs in breast cancer. Through in vitro and in vivo experiments, we found that depletion of USP26 inhibited breast cancer cell proliferation and invasion, and suppressed tumor growth and metastasis in nude mice. Further investigation identified co-chaperone Bcl-2-associated athanogene 3 (BAG3) as the direct substrate of USP26, and ectopic expression of BAG3 partially reversed antitumor effect induced by USP26 knockdown. Mechanistically, the lysine acetyltransferase Tip60 targeted USP26 at K134 for acetylation, which enhanced USP26 binding affinity to BAG3, leading to BAG3 deubiquitination and increased protein stability. Importantly, we employed a structure-based virtual screening and discovered a drug-like molecule called 5813669 that targets USP26, destabilizing BAG3 and effectively mitigating tumor growth and metastasis in vivo. Clinically, high expression levels of USP26 were correlated with elevated BAG3 levels and poor prognosis in breast cancer patients. Overall, our findings highlight the critical role of USP26 in BAG3 protein stabilization and provide a promising therapeutic target for breast cancer.

USP26 suppresses type I interferon signaling by targeting TRAF3 for deubiquitination.

Deubiquitinating enzymes (DUBs) play a pivotal role in regulating the antiviral immune response by targeting members of the RLR signaling pathway. As a pivotal member of the RLR pathway, TRAF3 is essential for activating the MAVS/TBK-1/IRF3 signaling pathway in response to viral infection. Despite its importance, the function of DUBs in the TRAF3-mediated antiviral response is poorly understood. Ubiquitin-specific protease 26 (USP26) regulates the RLR signaling pathway to modulate the antiviral immune response. The results demonstrate that EV71 infection upregulates the expression of USP26. Knockdown of USP26 significantly enhances EV71-induced expression of IFN-β and downstream interferon-stimulated genes (ISGs). Deficiency of USP26 not only inhibits EV71 replication but also weakens the host's resistance to EV71 infection. USP26 physically interacts with TRAF3 and reduces the K63-linked polyubiquitination of TRAF3, thereby promoting pIRF3-mediated antiviral signaling. USP26 physically interacts with TRAF3 and reduces the K63-linked polyubiquitination of TRAF3, thereby promoting pIRF3-mediated antiviral signaling. Conversely, knockdown of USP26 leads to an increase in the K63-linked polyubiquitination of TRAF3. These findings unequivocally establish the essential role of USP26 in RLR signaling and significantly contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses.

The association between mutations in ubiquitin-specific protease 26 (USP26) and male infertility: a systematic review and meta-analysis.

During recent decades, the association between mutations in ubiquitin-specific protease 26 (USP26) and male infertility remains doubtful. We conducted this meta-analysis to evaluate the association between mutations in USP26 and male infertility according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines. It was registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42021225251). PubMed, Web of Science, and Scopus were systematically searched for comparative clinical studies, which were written in English and provided eligible data. Studies were included when they compared USP26 mutations in azoospermic, oligozoospermic, and asthenozoospermic patients with controls with normal sperm parameter values or whose partners had experienced spontaneous pregnancy. Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated with random effect models. Overall, twelve studies with 3927 infertility patients and 4648 healthy controls were included. The association between overall USP26 mutations and infertility was not significant (OR = 1.60, 95% CI: 0.51–5.01). For specific mutations, the pooled ORs were 1.65 (95% CI: 1.02–2.69) for cluster mutation (including 370–371insACA, 494T>C, and 1423C>T), 1.80 (95% CI: 0.35–9.15) for c.576G>A, 1.43 (95% CI: 0.79–2.56) for c.1090C>T, and 3.59 (95% CI: 2.30–5.59) for c.1737G>A. Our results suggest that several mutations (cluster mutation, c.1737G>A) may play roles in male infertility, while others (c.576G>A and c.1090C>T) do not show notable associations with male infertility. More high-quality clinical researches are needed for validation.

Preclinical Study Using ABT263 to Increase Enzalutamide Sensitivity to Suppress Prostate Cancer Progression Via Targeting BCL2/ROS/USP26 Axis Through Altering ARv7 Protein Degradation.

Background: The recently developed antiandrogen, Enzalutamide (Enz), has reformed the standard of care for castration resistant prostate cancer (CRPC) patients. However, Enz-resistance inevitably emerges despite success of Enz in prolonging CRPC patients’ survival. Here we found that Enz-resistant prostate cancer (PCa) cells had higher BCL2 expression. We aimed to test whether targeting BCL2 would influence Enz sensitivity of prostate cancer (PCa) and identify the potential mechanism. Methods: The study was designed to target Enz-induced BCL2 with inhibitor ABT263 and test Enz sensitivity in Enz-resistant PCa cells by MTT assay. Cellular reactive oxygen species (ROS) levels were detected with dihydroethidium staining, and in vitro deubiquitinating enzyme activity assay was used to evaluate ubiquitin specific protease 26 (USP26) activity. Results: ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells via inducing ROS generation. Elevated cellular ROS levels might then inhibit USP26 activity to increase the ubiquitination of androgen receptor (AR) and AR splice variant 7 (ARv7) and their ubiquitin/proteasome-dependent degradation, which contributed to the increase of Enz sensitivity. In vivo mouse model also demonstrates that ABT263 will suppress the PCa progression. Conclusion: This study demonstrated that targeting Enz-induced BCL2 with inhibitor ABT263 could increase Enz sensitivity in both Enz-sensitive and Enz-resistant PCa cells through induction of cellular ROS levels and suppression of USP26 activity with a consequent increase of ubiquitin/proteasome-dependent degradation of AR and ARv7 protein expression.

Ubiquitin-specific protease 26 (USP26) is not essential for mouse gametogenesis and fertility.

Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.

The testis-specific USP26 is a deubiquitinating enzyme of the ubiquitin ligase Mdm2

Murine double minute-2 (Mdm2) is one of the E3-ligases of the androgen receptor besides being the major regulator of the p53 tumor suppressor. The testis-specific USP26 was demonstrated to regulate the androgen receptor. In the present study we examined possible association between the deubiquitinating enzyme - ubiquitin specific protease 26 (USP26), and the oncoprotein E3 ligase - (Mdm-2).We analyzed the half-life time of USP26 in HEK293 cells. In a cell-free system we asked whether USP26 can be ubiquitinated by HeLa extract. In co-transfection experiments we expressed Mdm2 along with different constructs of USP26 in order to examine possible regulation of Mdm2 by USP26. We found that USP26 binds to Mdm2 through its coiled-coiled C-terminal domain. USP26 deubiquitinates Mdm2 and stabilizes it.The physiological significance of the findings is still not fully understood.The interaction between USP26 and Mdm2, and the subsequent deubiquitination of Mdm2, serves, most probably to regulate Mdm2. Future therapeutic modalities that interfere with the association between USP26 and Mdm2 will be used to destabilize the ligase in malignancies where it is upregulated.

Single nucleotide polymorphisms of USP26 in azoospermic men

ABSTRACTSome studies have focused on the association between male infertility and single nucleotide polymorphisms (SNPs) in the ubiquitin-specific protease 26 (USP26) gene, but the results are controversial. In this case-control study including both normozoospermic men and patients with nonobstructive azoospermia, we analyzed both the entire coding region and 5’ and 3’ untranslated regions of USP26 in order to identify genetic variants in this gene to investigate the role of USP26 on spermatogenesis. We reported variations in the USP26 gene sequence in 82% of azoospermic and in 50% normospermic men. The synonymous variation c.576G>A has a frequency significantly different in the azoospermic (60.2%) and normozoospermic (23.6%) groups, while the frequencies in the two groups of both c.1090C>T and c.1737G>A missense mutations did not reach statistical significance. A cluster mutation (c.371insACA, c.494T>C) was detected in 2 normozoospermic men (2.7%). In the 5’UTR we identified the -33C>T variation both in azoospermic (3.8%) and in normozoospermic (2.7%) men. In a normozoospermic man we detected the nonsense mutation c.882C>A, never reported to date. According to our results, we suggest that only the variation c.576G>A has a frequency significantly different in azoospermic compared to normozoospermic men. Moreover, the identification in a normozoospermic man of a nonsense mutation (c.882C>A) which causes the production of a truncated protein, suggests a marginal role of USP26 in male spermatogenesis. Additional studies may be useful as we cannot exclude that the other SNPs may represent risk factors for male fertility acting by an oligogenic/polygenic mechanism.

Ubiquitin Specific Protease 26 (USP26) expression analysis in human testicular and extragonadal tissues indicates diverse action of USP26 in cell differentiation and tumorigenesis.

Ubiquitin specific protease 26 (USP26), a deubiquitinating enzyme, is highly expressed early during murine spermatogenesis, in round spermatids, and at the blood-testis barrier. USP26 has also been recognized as a regulator of androgen receptor (AR) hormone-induced action involved in spermatogenesis and steroid production in in vitro studies. Prior mutation screening of USP26 demonstrated an association with human male infertility and low testosterone production, but protein localization and expression in the human testis has not been characterized previously. USP26 expression analysis of mRNA and protein was completed using murine and human testis tissue and human tissue arrays. USP26 and AR mRNA levels in human testis were quantitated using multiplex qRT-PCR. Immunofluorescence colocalization studies were performed with formalin-fixed/paraffin-embedded and frozen tissues using primary and secondary antibodies to detect USP26 and AR protein expression. Human microarray dot blots were used to identify protein expression in extra-gonadal tissues. For the first time, expression of USP26 and colocalization of USP26 with androgen receptor in human testis has been confirmed predominantly in Leydig cell nuclei, with less in Leydig cell cytoplasm, spermatogonia, primary spermatocytes, round spermatids, and Sertoli cells. USP26 likely affects regulatory proteins of early spermatogenesis, including androgen receptor with additional activity in round spermatids. This X-linked gene is not testis-specific, with USP26 mRNA and protein expression identified in multiple other human organ tissues (benign and malignant) including androgen-dependent tissues such as breast (myoepithelial cells and secretory luminal cells) and thyroid tissue (follicular cells). USP26/AR expression and interaction in spermatogenesis and androgen-dependent cancer warrants additional study and may prove useful in diagnosis and management of male infertility.

Association of 370-371insACA, 494T>C, and 1423C>T haplotype in ubiquitin-specific protease 26 gene and male infertility: a meta-analysis.

Whether the 370-371insACA, 494T>C, and 1423C>T haplotype in ubiquitin-specific protease 26 (USP26) gene is associated with male infertility is controversial. To clarify this issue, we conducted a meta-analysis based on the most recent studies. Eligible studies were screened by using PubMed and Embase. Pooled odd ratio (OR) with 95% confidence interval (CI) was calculated with fixed effect models. Ten studies with 1603 patients and 2505 controls were included. Overall, the results indicated that there was an association between the haplotype and male infertile risk (OR = 1.74, 95% CI: 1.09–2.77). The OR calculated based on the five studies in Asia and three in Europe was 1.96 (95% CI: 1.05–3.67) and 1.54 (95% CI: 0.75–3.16) respectively, however, the OR was 0.86 (95% CI: 0.05–15.29) based on the two investigations in America. In addition, the data from the patients with azoospermia (AZO) showed an increased pooled OR of 2.35 (95% CI: 1.22–4.50). In contrast, the studies with oligoasthenoteratozoospermia (OAT) exhibited that the pooled OR was 0.97 (95% CI: 0.43–2.16). Our analyses indicate that there is an association of alteration in USP26 with male infertility, especially in AZO and Asian population.

Localization of ubiquitin specific protease 26 at blood-testis barrier and near Sertoli cell-germ cell interface in mouse testes

Ubiquitin Specific Protease 26 (USP26) is a little studied ubiquitin-specific protease that is expressed specifically in the testis. In humans, some USP26 polymorphisms have been reported to be associated with impaired male fertility. However, how USP26 affects male reproduction remains unclear. We generated an antibody that stained specifically cultured cells expressing an epitope-tagged USP26 and used it to elucidate the biological function of USP26. Immunostaining of mouse testis sections as well as dispersed germ cells showed the presence of USP26 at the blood-testis barrier, near the Sertoli cell-germ cell interface of post-step 7 spermatids, and coating the dorsal surface of sperm head. Further RT-PCR assays detected the expression of Usp26 in germ cells, but not in primary Sertoli cell lines. In addition, USP26 immunoprecipitated from testis lysates exhibited deubiquitinating activities. The localization of USP26 in the testis suggests a possible role in the movement of germ cells along the seminiferous epithelium.

Association between ubiquitin-specific protease USP26 polymorphism and male infertility in Chinese men

Background Increased sperm ubiquitin was inversely associated with sperm count and motility. Ubiquitin-specific protease 26 ( USP26), which is an X-linked gene, has been studied as a potential infertility gene. There are conflicting reports on whether variations in USP26 are associated with spermatogenesis. Methods In order to assess that USP26 polymorphisms contribute to male infertility, we screened 221 infertile men with azoospermia, oligozoospermia, asthenozoospermia, or oligoasthenozoospermia, and 101 control fertile men using DNA sequencing. Results There were six polymorphisms identified, including an unreported variation (508G>A, G170R). Only the allele frequency of 576G>A was significantly higher in fertile men than infertile patients ( p < 0.001), although this variant does not result in an amino acid change. The major haplotypes in fertile and infertile men were TGATC (76.2% vs 47.5% of the population, p < 0.001) and TGGTC (14.9% vs 39.4%, p < 0.001). The haplotype TGATC was under-transmitted, whereas the haplotype TGGTC was over-transmitted in infertile men with asthenozoospermia and oligoasthenozoospermia. Conclusions Our results indicated the variation of USP26 was not directly associated with human sperm count but suggested it might be a potential role in sperm motility. The 576G>A synonymous single nucleotide polymorphism (SNP) might have a role in improving the sperm motility.

The expression of Usp26 gene in mouse testis and brain

Deubiquitinating enzymes (DUBs) play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 (USP26) is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription (RT)-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids (steps 9-16), Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial cells, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

Histone H2B Deubiquitination Is Required for Transcriptional Activation of FLOWERING LOCUS C and for Proper Control of Flowering in Arabidopsis

The spectrum of histone modifications at a given locus is a critical determinant for the correct output of gene expression. In Arabidopsis (Arabidopsis thaliana), many studies have examined the relationship between histone methylation and gene expression, but few studies exist on the relationship between other covalent histone modifications and gene expression. In this work, we describe the role of histone H2B deubiquitination in the activation of gene expression and the consequence of a perturbation of histone H2B deubiquitination in the timing of the floral transition in Arabidopsis. A mutation in a H2B deubiquitinase, UBIQUITIN-SPECIFIC PROTEASE26 (UBP26), results in an early-flowering phenotype. In the ubp26 mutant, mRNA levels of the floral repressor FLOWERING LOCUS C (FLC) and other related family members is decreased. Furthermore, this mutant accumulates H2B monoubiquitination, and has decreased levels of H3K36 trimethylation and increased levels of H3K27 trimethylation at the FLC locus. Thus, UBP26 is required for transcriptional activation of FLC through H2B deubiquitination and is consistent with a model in which deubiquitination is necessary for the accumulation of H3K36 trimethylation and the proper level of transcriptional activation.

USP26 gene variations in fertile and infertile men

The human X chromosome is enriched with testis-specific genes that may be crucial for male fertility. One is the ubiquitin-specific protease 26 (USP26). Five frequent mutations have been identified: 1737G>A, 1090C > T, 370-371insACA, 494T > C and 1423C>T (with the latter three usually detected in a cluster). Their role in infertility is still controversial. This study assesses the association of the most frequent USP26 mutations with male infertility and male infertility etiology factors. The study included 300 infertile and 287 fertile men. Data were collected on ethnicity (according to maternal origin) and family history of reproduction. Clinical records from 235 infertile and 62 fertile (sperm bank donors) men were available and summarized. The five mutations were investigated by bioinformatic tools and their frequencies were assessed by restriction analysis. The results were correlated with clinical findings. Segregation of the mutations in four families was analyzed. The five analyzed mutations were detected in 44 men from both fertile and infertile groups. The cluster and the 1090C>T mutations showed the highest frequency among Arabs and Sephardic Jews of the infertile group, respectively. Inheritance studies showed that mutations were not always associated with the infertility trait. Mutations 1090C>T and 1737G>A were significantly associated with a history of inguinal hernia (P = 0.007 and P = 0.043, respectively). The prevalence of inguinal hernia among men with the 1090C > T mutation was 33.3% (5/15 men), higher than that reported in infertile men (6.7%). Mutation 1090C > T may be a new genetic risk factor for developing inguinal hernia which may be associated with impaired male fertility.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China. Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Of 41 infertile men, 9 (22.0%, P = 0.01) had changes in USP26 gene on the X chromosome. A compound mutation (364insACA; 460G right triple arrow A) was detected in 8 patients (19.5%, P = 0.01) and a 1044T right triple arrow A substitution was found in 1 patient (2.4%, P > 0.05). All three variations led to changes in the coding amino acids. Two substitutions predict some changes: 460G right triple arrow A changes a valine into an isoleucine, and 1044T right triple arrow A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

A Specific Mutation in the USP26 Gene is Associated With Poor Sperm Recovery in Men With Non-obstructive Azoospermia

We have previously shown that mutations in the ubiquitin specific protease 26 (USP26) gene occurred in 10.6 % (20/188) of men with non-obstructive azoospermia (NOA) and severe oligospermia. In the current study we evaluated whether specific mutations can predict histology, hormonal profile, success of testicular sperm extraction (TESE), and/or IVF outcomes in 126 men undergoing TESE.

Possible role of USP26 in patients with severely impaired spermatogenesis

The ubiquitin-specific protease 26 (USP26) gene is an X-linked gene specifically expressed in testis tissue. This gene is therefore a potential infertility gene. In order to analyse its possible involvement in spermatogenesis and infertility, 42 patients with Sertoli cell-only syndrome were analysed for mutations in this gene. We found four patients with exactly the same three changes of the nucleotide and therefore also amino acid sequence. These patients showed 370-371insACA, 494T>C and 1423C>T causing T123-124ins, L165S and H475Y, respectively. These changes were not found in 10 control samples. Furthermore, two polymorphisms were observed which do not alter the amino-acid sequence. A restriction analysis that can make a distinction between a T and a C, at position 494, was set up in order to examine more patient and control samples. Another 69 patients with Sertoli cell-only syndrome, 32 patients with maturation arrest and 142 control samples were analysed using this method. None of the control samples or patients with maturation arrest featured the change at position 494. However, four more patients with Sertoli cell-only syndrome were identified with the three alterations. The frequency of alterations in this group is therefore 7.2% (8/111). One of the patients had a deletion of the long arm of the Y chromosome, while another patient had a varicocoele. These results indicate that these alterations might be involved in male infertility or might increase the risk of male infertility.