UCH-L1 regulates eye differentiation-related genes and modulates EGFR signalling in Drosophila melanogaster

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), which is a member of the ubiquitin carboxyl-terminal hydrolase (UCH) family, is highly expressed in neurons. In vitro, UCH- L1 exhibits both ubiquitin hydrolase and ligase activity. Many studies have suggested that UCH-L1 is involved in the pathogenesis of Parkinson's disease and some different human cancer diseases, but its role in a living system is still unclear. Recently, Drosophila melanogaster has been shown to be a compatible model for studying human diseases. To investigate the role of UCH-L1 in a living system, the UCH-L1 homologous protein in Drosophila melanogaster (dUCH) is used for analyzing the role of the protein's function in transgenic Drosophila. Here, we used DNA molecular techniques to clone, express, and purify dUCH protein from Escherichia coli. The purified dUCH protein was injected into a rabbit to produce an anti-dUCH antibody, which was shown to have high specificity and sensitivity to the dUCH protein. The affinity of the antibody is 1:320,000 at 7.81 ng/μL antigen concentration. The 1:40,000 dilution-produced antibodies can detect antigen at a low concentration of 0.98 ng/μL. Success in producing this antibody provides good material for further experiments in the study of the role of UCH-L1 by a Drosophila model.

Hypoxia-inducible factor 1 (HIF-1) is a key transcription factor for tumor malignancy. Recently, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme, has been reported to stabilize HIF-1α by releasing the pVHL complex conjugated Ub moieties. In this study, we investigated whether inhibition of UCHL1 can be an effective strategy for HIF-1-related tumor chemotherapy. First, we found that UCHL1 inhibition by siRNA or LDN57444, a well-known UCHL1 inhibitor, drastically lowered HIF-1α protein levels in UCHL1-expressing MDA-MB-436 breast cancer cells, by Western blotting analysis and immunostaining experiments. In UCHL1 non-expressing MDA-MB-231 cells, the ectopic expression of UCHL1 significantly increased HIF-1α protein expression levels, which was canceled by the treatment of LDN57444. We further analyzed whether the UCHL1 inhibitor could affect the HIF-dependent transactivation using 5HRE-luciferase reporter assay and RT-qPCR. LDN57444 dose-dependently inhibited the HIF activity and decreased the transcription of HIF downstream genes in MDA-MB-436 cells. Next we tested the effects of the UCHL1 inhibitor on tumor cell migration. LDN57444 significantly blocked cell migration in MDA-MB-436 cells in wound healing and transwell migration assays, suggesting a crucial role of the enzyme in tumor migration. We finally investigated potential roles of UCHL1 in tumor malignancy by using a 3D spheroid culture model, which has been more widely applied in the preclinical studies to provide more physiologically relevant information. Image analysis using open source image processing suites, AnaSP and ReViSP revealed that ectopic expression of UCHL1 in MDA-MB-231 cells significantly upregulated malignancy representing factors such as integrity and volume. Whole cell number of UCHL1 transfected MDA-MB-231 spheroids was also much higher than that of the mock treated spheroids when measured by 3D cell viability assay. Depletion of UCHL1 or inhibition of UCHL1 enzyme activity in MDA-MB-436 cells, on the contrary, downregulated these malignant factors. Besides, in spheroid viability staining and spheroid invasion assay, LDN57444 abolished the UCHL1 mediated cell proliferation and invasiveness as well. Moreover, the ectopic expression of UCHL1 in MDA-MB-231 cells stabilized HIF-1α protein level and promoted the expression of HIF-1 target genes in the 3D model as in the case of 2D monolayer culture. In conclusion, we clearly proved the involvement of UCHL1 in the HIF-1 induced tumor malignancy employing the 3D spheroid culture model. Our research indicates that the UHCL1-HIF-1 pathway is a promising therapeutic target for cancer chemotherapy. Citation Format: Xuebing Li, Akira Hattori, Senye Takahashi, Yoko Goto, Hiroshi Harada, Hideaki Kakeya. Inhibition of UCHL1 blocks proliferation and metastasis in HIF-1 dependent tumor malignancy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B119. doi:10.1158/1535-7163.TARG-19-B119

Ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) is one of the deubiquitinating enzymes in the ubiquitin-proteasome system. It has been shown that UCH-L1 could markedly decrease neointima formation through suppressing vascular smooth muscle cell (VSMC) proliferation in the balloon-injured rat carotid. However, whether UCH-L1 plays roles in VSMC migration remains to be determined. In this study, the primary VSMCs were isolated from aortic media of rats and TNF-α to was used to induce VSMC migration. Using a modified Boyden chamber and wound healing assay, it was found that TNF-α can dose and time-dependently induce VSMC migration with a maximal effect at 10 ng/mL. Moreover, UCH-L1 expression increased gradually with the prolonged induction time at 10 ng/mL of TNF-α. UCH-L1 content in VSMC was then modulated by recombinant adenoviruses expressing UCH-L1 or RNA interference to evaluate its roles in cell migration. The results showed that over-expression of UCH-L1 attenuated VSMC migration, while knockdown of it enhanced cell migration significantly no matter whether TNF-α treatment or not. Finally, the effect of UCH-L1 on NF-κB activation was demonstrated by NF-κB nuclear translocation and DNA binding activity, and the levels of IL-6 and IL-8 in cell culture media were examined by ELISA. It was showed that UCH-L1 over-expression inhibited NF-κB activation and decrease IL-6 and IL-8 levels, while knockdown of it enhanced NF-κB activation and increase IL-6 and IL-8 levels during TNF-α treatment. These data suggest that UCH-L1 can inhibit TNF-α-induced VSMCs migration, and this kind of effect may partially due to its suppression role in NF-κB activation.

UCHL1 Regulates Melanogenesis through Controlling MITF Stability in Human Melanocytes

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease.

Age-related hearing loss (ARHL) is the most common cause of hearing loss in the elderly. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme involved in several types of human disease. The present study aimed to investigate the effect of UCHL1 on a hydrogen peroxide (H2O2)-induced ARHL model in cochlear hair cells and uncover its underlying mechanism. Reverse transcription-quantitative (RT-q)PCR and western blot analysis were used to assess UCHL1 expression in HEI-OC1 cells exposed to H2O2. Following UCHL1 overexpression in H2O2-induced HEI-OC1 cells, cell activity was assessed by Cell Counting Kit-8 assay. The content of oxidative stress-associated markers including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and reactive oxygen species (ROS ) was measured using corresponding commercial kits. Cell apoptosis was evaluated by TUNEL assay and western blot analysis. Cell senescence was assessed by senescence-associated β-galactosidase staining and western blot analysis. RT-qPCR and western blot analysis were applied to measure mRNA and protein expression levels, respectively, of specificity protein 1 (Sp1) in H2O2-treated HEI-OC1 cells. In addition, the association between UCHL1 and Sp1 was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The mRNA and protein expression levels of UCHL1 were also determined in Sp1-overexpressing cells by RT-qPCR and western blot analysis, respectively. Following Sp1 overexpression in UCHL1-overexpressing H2O2-treated HEI-OC1 cells, cell activity, oxidative stress, apoptosis and senescence were assessed. Finally, the expression levels of NF-κB signaling-related proteins p-NF-κB p65 and NF-κB p65 were detected using western blot analysis. The results showed that UCHL1 was downregulated in H2O2-treated HEI-OC1 cells. In addition, UCHL1 overexpression enhanced cell viability and promoted oxidative damage, apoptosis and senescence in H2O2-induced HEI-OC1 cells. Furthermore, Sp1 was upregulated in H2O2-treated HEI-OC1 cells. Additionally, luciferase reporter and ChIP assays demonstrated that Sp1 interacted with the UCHL1 promoter to inhibit UCHL1 transcription. Sp1 overexpression reversed the effect of UCHL1 overexpression on cell viability, oxidative stress, apoptosis, senescence and activation of the NF-κB signaling pathway in H2O2-exposed HEI-OC1 cells. Collectively, the results suggested that UCHL1 transcriptional suppression by Sp1 protected cochlear hair cells from H2O2-triggered senescence and oxidative damage.

Contents to volume 21

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a critical deubiquitinating enzyme that is highly expressed in the central nervous system, where it participates in protein degradation and turnover as part of the ubiquitin–proteasome system (UPS). Convincing evidence supports the role of UCH-L1 dysfunction in several neurodegenerative disorders, given its unique position at the crossroad of several aetiopathogenic pathways, including those implicated in Alzheimer’s disease (AD) onset. Indeed, UCH-L1 depletion correlates with decreased levels of triggering receptor expressed on myeloid cells 2 (TREM2), with consequent effects on neuroinflammation. Notably, UCH-L1 can affect the level of phosphorylated tau protein, thus contributing to the formation of neurofibrillary tangles (NFTs). In addition, UCH-L1 influences β-Secretase 1 (BACE1) expression, resulting in the abnormal accumulation of amyloid-β plaques in brain parenchyma. These findings underline UCH-L1’s centrality in maintaining the homeostasis of protein folding and aggregation, which are significantly impaired in AD and AD-related dementias. Given these assumptions, UCH-L1 is recognized as a potential biomarker for AD, highlighting its relevance in governing the fate of crucial pathological mediators of cognitive impairment and neurodegeneration. Herein, we contextualize the involvement of UCH-L1 in different dementia-associated pathways and summarize the state of the art of UCH-L1 as a biomarker for AD diagnosis.

A pivotal strategy in immuno-oncology is the initiation and modulation of adaptive immune responses. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), known for its role in protein homeostasis and functionality, is implicated in tumorigenesis. However, its part in the antitumor immunity mediated by CD8+ T cells in lung adenocarcinoma (LUAD) is not yet clear. We harnessed bioinformatics to evaluate the clinical relevance of UCHL1 in LUAD, performed IHC to detect the expression of UCHL1 in LUAD tissue, and utilised qPCR to assess UCHL1 levels in LUAD cells, exploring its correlation with the presence of CD8+ T cells. The effects of UCHL1 on CD8+ T cell vigour have been investigated using lactate dehydrogenase and enzyme-linked immunosorbent assay kits, as well as flow cytometry. The contribution of UCHL1 to ferroptosis was examined with ferrous ion and manganese dioxide assay kits, alongside western blot. Furthermore, we utilised bioinformatics software UbiBrowser and Hdock, in conjunction with co-immunoprecipitation (Co-IP), immunofluorescence, and IP methods, to dissect the interaction between UCHL1 and FHL2. Rescue experiments further clarified the mechanism by which UCHL1 modulates FHL2 in tumour immunity. Invivo experiments confirmed the promoting effect of UCHL1 on tumour growth. Elevated UCHL1 levels in LUAD tissues and cells were observed. Dampening UCHL1 triggered ferroptosis in LUAD cells, which in turn ramped up CD8+ T cell activity and enhanced their tumour-killing potential. Mechanistically, UCHL1 was shown to deubiquitinate the downstream factor FHL2, and knocking down FHL2 could counteract the immunosuppressive effects induced by high UCHL1 levels on CD8+ T cells. UCHL1 inhibitor LDN57444 significantly inhibited tumour growth in mice. Therapies aimed at the UCHL1/FHL2 axis could be effectively paired with immunotherapies, opening new avenues in cancer treatment strategies.

α-Synuclein (α-syn) positive glial cytoplasmic inclusions (GCI) originating in oligodendrocytes (ODC) are a characteristic hallmark in multiple system atrophy (MSA). Their occurrence may be linked to a failure of the ubiquitin proteasome system (UPS) or the autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin, and deubiquitinated by deubiquitinating enzymes (DUBs) before proteolytic degradation is performed. The DUB ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the UPS, it is abundantly expressed in neuronal brain cells and has been connected to Parkinson’s disease (PD). It interacts with α-syn and tubulin. The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC, the myelin forming cells of the CNS, and is associated with GCIs in MSA. Furthermore, LDN-57444 (LDN), a specific UCH-L1 inhibitor, was used to analyze its effects on cell morphology, microtubule (MT) organization and the proteolytic degradation system. Towards this an oligodendroglial cell line (OLN cells), stably transfected with α-syn or with α-syn and GFP-LC3, to monitor the autophagic flux, was used. The data show that UCH-L1 is expressed in ODC derived from the brains of newborn rats and colocalizes with α-syn in GCIs of MSA brain sections. LDN treatment had a direct impact on the MT network by affecting tubulin posttranslational modifications, i.e., acetylation and tyrosination. An increase in α-tubulin detyrosination was observed and detyrosinated MT were abundantly recruited to the cellular extensions. Furthermore, small α-syn aggregates, which are constitutively expressed in OLN cells overexpressing α-syn, were abolished, and LDN caused the upregulation of the autophagic pathway. Our data add to the knowledge that the UPS and the autophagy-lysosomal pathway are tightly balanced, and that UCH-L1 and its regulation may play a role in neurodegenerative diseases with oligodendroglia pathology.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitinproteasome system and distributes mostly in the brain. Previous studies showed that the mutation form or reduction of UCH-L1 related to neurodegenerative disorders such as Parkinson disease, Alzheimer disease and also involved to the oxidative stress. However the specific role of UCH-L1 on the activities of the nervous system has not yet well been understood. Among several models to study the role of UCH-L1, Drosophila melanogaster one showed several advantages particularly in the study of the neurodegeneration. To clarify the effects of UCH-L1 deficiency in neurons, we used Drosophila with motor and sensory neuron-specific knockdown of dUCH (Drosophila Ubiquitin carboxyl-terminal hydrolase) which is a homolog gene of human UCH-L1. The obtained results showed that the knockdown dUCH decreased the locomotive function in both larval and adult stages of Drosophila, and also gave negative effects to their learning and memorying abilities. These results emphasized the roles of UCH-L1 in the neuronal health and suggested the possibility of using D. melanogaster as model for further studies of disease mechanisms or drug screening.

Breast cancer has the highest incidence and death rate among cancers in women worldwide. In particular, metastatic estrogen receptor negative (ER–) breast cancer and triple-negative breast cancer (TNBC) subtypes have very limited treatment options, with low survival rates. Ubiquitin carboxyl terminal hydrolase L1 (UCHL1), a ubiquitin C-terminal hydrolase belonging to the deubiquitinase (DUB) family of enzymes, is highly expressed in these cancer types, and several key reports have revealed emerging and important roles for UCHL1 in breast cancer. However, selective and potent small-molecule UCHL1 inhibitors have been disclosed only very recently, alongside chemical biology approaches to detect regulated UHCL1 activity in cancer cells. These tools will enable novel insights into oncogenic mechanisms driven by UCHL1, and identification of substrate proteins deubiquitinated by UCHL1, with the ultimate goal of realising the potential of UCHL1 as a drug target in breast cancer.

Aims: Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitin-proteasome system and is distributed mostly in the brain. Previous studies have shown that mutated forms or reduction of UCH-L1 are related to neurodegenerative disorders, but the mechanisms of pathogenesis are still not well understood. To study its roles in motor neuronal health, we utilized the Drosophila model in which dUCH, a homolog of human UCH-L1, was specifically knocked down in motor neurons. Results: The reduction of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) in motor neurons induced excessive reactive oxygen species production and multiple aging-like phenotypes, including locomotive defects, muscle degeneration, enhanced apoptosis, and shortened longevity. In addition, there is a decrease in the density of the synaptic active zone and glutamate receptor area at the neuromuscular junction. Interestingly, all these defects were rescued by vitamin C treatment, suggesting a close association with oxidative stress. Strikingly, the knockdown of dUCH at motor neurons exhibited aberrant morphology and function of mitochondria, such as mitochondrial DNA (mtDNA) depletion, an increase in mitochondrial size, and overexpression of antioxidant enzymes. Innovation: This research indicates a new, possible pathogenesis of dUCH deficiency in the ventral nerve cord and peripheral nervous systems, which starts with abnormal mitochondria, leading to oxidative stress and accumulation aging-like defects in general. Conclusion: Taken together, by using the Drosophila model, our findings strongly emphasize how the UCH-L1 shortage affects motor neurons and further demonstrate the crucial roles of UCH-L1 in neuronal health. Antioxid. Redox Signal. 37, 257-273.

Nasopharyngeal carcinoma is prevalent in southern China and Southeast Asia, with distinct geographic and ethnic distribution. One candidate susceptibility locus has been identified at 4p11-14, with the associated candidate gene(s) not identified yet. This study investigated the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in nasopharyngeal carcinoma pathogenesis. UCHL1 expression and methylation were examined in nasopharyngeal carcinoma. Furthermore, the mechanism of its tumor-suppressive function was elucidated in nasopharyngeal carcinoma cells. Through genomewide expression profiling, we identified UCHL1, a 4p14 gene normally expressed in normal upper respiratory tract tissues, being silenced in all nasopharyngeal carcinoma cell lines. Its silencing is mediated by CpG methylation because UCHL1 promoter methylation was detected in all silenced cell lines, and pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. UCHL1 methylation was also frequently detected in primary tumors but only weakly detected in few normal nasopharyngeal tissues, indicating that the methylation-mediated silencing of UCHL1 is important in nasopharyngeal carcinoma pathogenesis. Ectopic UCHL1 expression dramatically inhibited the growth of nasopharyngeal carcinoma cells through promoting tumor cell apoptosis. We further found that UCHL1 formed a complex with p53/p14(ARF)/Mdm2 p53 binding protein homolog (mouse), MDM2 and activated the p53 signaling pathway. UCHL1 expression extended p53 and p14(ARF) protein half-life and shortened MDM2 protein half-life. These results indicate that UCHL1 could deubiquitinate p53 and p14(ARF) and ubiquitinate MDM2 for p53 stabilization to promote p53 signaling, thus involved in nasopharyngeal carcinoma pathogenesis, whereas it is frequently silenced in this tumor.

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function.