Ubiquitin C-terminal Hydrolase L1 Protein Research Articles

UCHL1 acts as a potential oncogene and affects sensitivity of common anti-tumor drugs in lung adenocarcinoma

BackgroundLung adenocarcinoma is the leading cause of cancer death worldwide. Recently, ubiquitin C-terminal hydrolase L1 (UCHL1) has been demonstrated to be highly expressed in many tumors and plays the role of an oncogene. However, the functional mechanism of UCHL1 is unclear in lung adenocarcinoma progression.MethodsWe analyzed the differential expression of the UCHL1 gene in lung adenocarcinoma and normal lung tissues, and the correlation between the UCHL1 gene and prognosis was also analyzed by the bioinformatics database TCGA. Meanwhile, we detected and analyzed the expression of UCHL1 and Ki-67 protein in a tissue microarray (TMA) containing 150 patients with lung adenocarcinoma by immunohistochemistry (IHC) and clinicopathological characteristics by TCGA database. In vitro experiments, we knocked down the UCHL1 gene of A549 cells and detected the changes in cell migration, invasion, and apoptosis. At the same time, we analyzed the effect of UCHL1 on anti-tumor drug sensitivity of lung adenocarcinoma by a bioinformatics database. In terms of the detection rate of lung adenocarcinoma indicators, we analyzed the impact of UCHL1 combined with common clinical indicators on the detection rate of lung adenocarcinoma through a bioinformatics database.ResultsIn this study, the analysis of UCHL1 protein expression in lung adenocarcinoma proved that obviously higher UCHL1 protein level was discovered in lung adenocarcinoma tissues. The expression of UCHL1 was closely related to poor clinical outcomes. Interestingly, a significantly positive correlation between the expression of UCHL1 and Ki-67-indicated UCHL1 was associated with tumor migration and invasion. Through executing loss of function tests, we affirmed that silencing of UCHL1 expression significantly inhibited migration and invasion of lung adenocarcinoma cells in vitro. Furthermore, lung adenocarcinoma cells with silenced UCHL1 showed a higher probability of apoptosis. In terms of the detection rate of lung adenocarcinoma indicators, we discovered UCHL1 could improve the detection rate of clinical lung adenocarcinoma and affect drug sensitivity.ConclusionIn lung adenocarcinoma, UCHL1 promotes tumor migration, invasion, and metastasis by inhibiting apoptosis and has an important impact on the clinical drug treatment of lung adenocarcinoma. In addition, UCHL1 can improve the detection rate of clinical lung adenocarcinoma. Above all, UCHL1 may be a new marker for the diagnosis of lung adenocarcinoma and provide a new target for the treatment of clinical diseases.

The requirement of ubiquitin C-terminal hydrolase L1 in mouse ovarian development and fertility†.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health.

UCHL1 Regulates Lipid and Perilipin 2 Level in Skeletal Muscle.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally found in neurons. We found that UCHL1 is highly expressed in slow oxidative skeletal muscles, but its functions remain to be fully understood. In this study, we observed that UCHL1 protein levels in skeletal muscle and C2C12 myotubes were downregulated by fasting or glucose starvation respectively. Skeletal muscle selective knockout (smKO) of UCHL1 resulted in a significant reduction of lipid content in skeletal muscle and improved glucose tolerance. UCHL1 smKO did not significantly change the levels of key proteins involved in oxidative metabolism such as SDHA, Akt, or PDH. Interestingly, while the levels of the major lipases and lipid transporters were unchanged, perilipin 2 was significantly downregulated in UCHL1 smKO muscle. Consistently, in C2C12 myotubes, UCHL1 siRNA knockdown also reduced perilipin 2 protein level. This data suggests that UCHL1 may stabilize perilipin 2 and thus lipid storage in skeletal muscle.

UCHL1 regulates oxidative activity in skeletal muscle.

Ubiquitin C-terminal Hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally identified in neurons. Our recent study showed that UCHL1 was expressed in C2C12 myoblast cells and mouse skeletal muscle. Here we report that in mouse skeletal muscle, UCHL1 is primarily expressed in oxidative muscle fibers. Skeletal muscle specific gene knockout (smKO) of UCHL1 in mice reduced oxidative activity in skeletal muscle measured by SDH staining. The in situ muscle contraction test revealed that gastrocnemius muscle from UCHL1 smKO mice was more prone to fatigue in response to the repetitive stimulation. This data suggests that UCHL1 plays a role in maintenance of muscle oxidative metabolism. Moreover, UCHL1 smKO caused a significant reduction in key proteins that are involved in mitochondrial oxidative phosphorylation in soleus muscles, suggesting that UCHL1 may be involved in regulation of mitochondrial content and function. Immunostaining showed the co-localization of UCHL1 and mitochondrial marker VDAC in skeletal muscle. Mitochondrial fractionation assay revealed that, although UCHL1 was primarily present in the cytosolic fraction, a low level of UCHL1 protein was present in mitochondrial fraction. The level of phosphorylation of AMPKα, a master regulator of mitochondrial biogenesis, were unchanged in UCHL1 smKO muscle. On the other hand, immunoprecipitation from soleus muscle sample indicated the interaction between UCHL1 and HSP60, a chaperon protein that is involved in mitochondrial protein transport. There was a trend of downregulation of HSP60 in UCHL1 smKO muscle. Overall, our data suggests UCHL1 is a novel regulator of mitochondrial function and oxidative activity in skeletal muscle.

Metals and oxidative potential in urban particulate matter influence systemic inflammatory and neural biomarkers: A controlled exposure study

BackgroundOxidative stress and inflammation are considered to be important pathways leading to particulate matter (PM)-associated disease. In this exploratory study, we examined the effects of metals and oxidative potential (OP) in urban PM on biomarkers of systemic inflammation, oxidative stress and neural function. MethodsFifty-three healthy non-smoking volunteers (mean age 28 years, twenty-eight females) were exposed to coarse (2.5–10 μm, mean 213 μg/m3), fine (0.15–2.5 μm, 238 μg/m3), and/or ultrafine concentrated ambient PM (<0.3 μm, 136 μg/m3). Exposures lasted 130 min, separated by ≥2 weeks. Metal concentrations and OP (measured by ascorbate and glutathione depletion in synthetic airway fluid) in PM were analyzed. Blood and urine samples were collected pre-exposure, and 1-h and 21-h post exposure for assessment of biomarkers. We used mixed-regression models to analyze associations adjusting for PM size and mass concentration. ResultsResults for metals were expressed as change (%) from daily pre-exposure biomarker levels after exposure to a metal at a level equivalent to the mean concentration. Exposure to various metals (silver, aluminum, barium, copper, iron, potassium, lithium, nickel, tin, and/or vanadium) was significantly associated with increased levels of various blood or urinary biomarkers. For example, the blood inflammatory marker vascular endothelia growth factor (VEGF) increased 5.3% (95% confidence interval: 0.3%, 10.2%) 1-h post exposure to nickel; the traumatic brain injury marker ubiquitin C-terminal hydrolase L1 (UCHL1) increased 11% (1.2%, 21%) and 14% (0.3%, 29%) 1-h and 21-h post exposure to barium, respectively; and the systemic stress marker cortisol increased 1.5% (0%, 2.9%) and 1.5% (0.5%, 2.8%) 1-h and 21-h post exposure to silver, respectively. Urinary DNA oxidation marker 8‑hydroxy‑deoxy‑guanosine increased 14% (6.4%, 21%) 1-h post exposure to copper; urinary neural marker vanillylmandelic acid increased 29% (3%, 54%) 1-h post exposure to aluminum; and urinary cortisol increased 88% (0.9%, 176%) 1-h post exposure to vanadium. Results for OP were expressed as change (%) from daily pre-exposure biomarker levels after exposure to ascorbate-related OP at a level equivalent to the mean concentration, or for exposure to glutathione-related OP at a level above the limit of detection. Exposure to ascorbate- or glutathione-related OP was significantly associated with increased inflammatory and neural biomarkers including interleukin-6, VEGF, UCHL1, and S100 calcium-binding protein B in blood, and malondialdehyde and 8-hydroxy-deoxy-guanosine in urine. For example, UCHL1 increased 9.4% (1.8%, 17%) in blood 21-h post exposure to ascorbate-related OP, while urinary malondialdehyde increased 19% (3.6%, 35%) and 8-hydroxy-deoxy-guanosine increased 24% (2.9%, 48%) 21-h post exposure to ascorbate- and glutathione-related OP, respectively. ConclusionOur results from this exploratory study suggest that metal constituents and OP in ambient PM may influence biomarker levels associated with systemic inflammation, oxidative stress, perturbations of neural function, and systemic physiological stress.

High Expression of Ubiquitin C-terminal Hydrolase L1 Is Associated With Poor Prognosis in Endometrial Cancer Patients.

The ubiquitin C-terminal hydrolase L1 (UCHL1) plays a key role in tumor invasion and metastasis. Ubiquitin C-terminal hydrolase L1 is overexpressed in various cancers and reported to be correlated with a poor prognosis. The objective of this study was to determine the prognostic significance of UCHL1 in endometrial cancer. The expression of UCHL1 in endometrial cancer was assessed using quantitative reverse transcription polymerase chain reaction and immunohistochemistry in 56 and 215 resected tumor specimens, respectively. The 4-year survival rates of the high UCHL1 messenger RNA expression group and high UCHL1 protein expression group were 78% and 71%, respectively, compared with 96% and 95% for the low UCHL1 messenger RNA expression group and low UCHL1 protein expression group, respectively. Kaplan-Meier and log-rank tests indicated a significant correlation between expression of UCHL1 and disease-free survival and overall survival. Moreover, multivariate stepwise Cox proportional hazard regression model analysis showed that UCHL1 was a significant independent marker for predicting a poor disease-free survival and overall survival. In 43 patients with metastatic lesions, immunohistochemical analysis of metastatic lesions revealed that the recurrence rate and mortality rate were 62% and 41%, respectively, in 29 UCHL1-positive patients and 36% and 29%, respectively, in 14 UCHL1-negative patients. The results of this study suggest that high UCHL1 expression is a strong marker of poor prognosis of endometrial cancer. Furthermore, we suggest that UCHL1 may be involved in the development of distant metastasis in endometrial cancer.

Deubiquitinase UCHL1 Regulates Myogenesis in Skeletal Muscles

Myogenesis is crucial for skeletal muscle regeneration in injury and disease. This multi‐step process is closely regulated by several myogenic factors. Timely activation and inactivation of these factors require ubiquitin‐proteasome system (UPS)‐mediated protein degradation. In this aspect, deubiquitinases (DUBs) could also play a role in myogenesis. Ubiquitin C‐terminal hydrolase L1 (UCHL1) is a DUB that is highly expressed in the brain. Though undetectable in adult skeletal muscles, UCHL1 protein level is significantly increased in injured muscles. However, the functional significance of UCHL1 in skeletal muscles is unknown. Using in vitro and in vivo approaches, this study tested a hypothesis that UCHL1 can play a role in skeletal muscle myogenesis and regeneration.Our data show that UCHL1 protein level was gradually downregulated during myoblast C2C12 differentiation. Knockdown of UCHL1 expression significantly reduced C2C12 cell proliferation. In contrast, UCHL1 knockdown markedly accelerated cell differentiation, evidenced by more myotubes in UCHL1 deficient cells, which was accompanied by significant upregulation of myogenic factors.In mouse muscles, UCHL1 was expressed by type I and IIa muscle fibers. Denervation increased UCHL1 protein levels in muscle fibers, which was completely abolished in muscles from the mice with UCHL1 skeletal muscle specific knockout (smKO). smKO mice showed reduced satellite cells in soleus and declined type I fiber number. Moreover, smKO muscles exhibited right shift of fiber distribution and a remarkable loss of type I muscle fiber upon denervation as compared with wildtype ones. These observations further suggest the potential involvement of UCHL1 in myogenesis for the regeneration of type I fibers.Support or Funding InformationNIH grants No. 1R15HL118696 and 1R03AG051926 and USD SSOM BBS bridge grant, CBBRe pilot grant, PQCD pilot grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ubiquitin C-Terminal Hydrolase L1 regulates myoblast proliferation and differentiation

Skeletal muscles are dynamic tissues that possess regenerative abilities, which require multiple processes and regulatory factors. Ubiquitin C-Terminal Hydrolase L1 (UCHL1), which is primarily expressed in neuronal tissues, was upregulated in skeletal muscles in disease conditions but its functional role in skeletal muscles is unknown. Using mouse myoblast cells C2C12 as an in vitro model, this study reported that UCHL1 elicits different regulation in myoblast cell proliferation and differentiation. We first observed that UCHL1 protein level was continuously declined during cell differentiation. Gene knockdown of UCHL1 by siRNA resulted in a significant decrease in cell proliferation but marked acceleration of cell differentiation and myotube formation. Meanwhile, UCHL1 gene knockdown upregulated myogenic factors myoD and Myogenin (MyoG). In mice, UCHL1 was significantly upregulated in denervated skeletal muscle. Overall, these novel data suggest that UCHL1 may play a role in myogenesis by promoting myoblast proliferation and inhibiting differentiation.

CPKCγ-mediated down-regulation of UCHL1 alleviates ischaemic neuronal injuries by decreasing autophagy via ERK-mTOR pathway.

Stroke is one of the leading causes of death in the world, but its underlying mechanisms remain unclear. Both conventional protein kinase C (cPKC)γ and ubiquitin C‐terminal hydrolase L1 (UCHL1) are neuron‐specific proteins. In the models of 1‐hr middle cerebral artery occlusion (MCAO)/24‐hr reperfusion in mice and 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation in cortical neurons, we found that cPKCγ gene knockout remarkably aggravated ischaemic injuries and simultaneously increased the levels of cleaved (Cl)‐caspase‐3 and LC3‐I proteolysis product LC3‐II, and the ratio of TUNEL‐positive cells to total neurons. Moreover, cPKCγ gene knockout could increase UCHL1 protein expression via elevating its mRNA level regulated by the nuclear factor κB inhibitor alpha (IκB‐α)/nuclear factor κB (NF‐κB) pathway in cortical neurons. Both inhibitor and shRNA of UCHL1 significantly reduced the ratio of LC3‐II/total LC3, which contributed to neuronal survival after ischaemic stroke, but did not alter the level of Cl‐caspase‐3. In addition, UCHL1 shRNA reversed the effect of cPKCγ on the phosphorylation levels of mTOR and ERK rather than that of AMPK and GSK‐3β. In conclusion, our results suggest that cPKCγ activation alleviates ischaemic injuries of mice and cortical neurons through inhibiting UCHL1 expression, which may negatively regulate autophagy through ERK‐mTOR pathway.

Modification of ubiquitin C-terminal hydrolase L1 by reactive lipid species: role in neural regeneration and diseases of aging.

Modification of ubiquitin C-terminal hydrolase L1 by reactive lipid species: role in neural regeneration and diseases of aging.

The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.

Abstract 3559: Ubiquitin C-terminal hydrolase L1 (UCHL1) modulates uterine papillary serous cancer progression through interaction with cyclin B1

Abstract Background: Uterine papillary serous carcinoma (UPSC) comprises less than 10% of all endometrial cancers but accounts for a disproportionately high number of deaths. Few prospective studies focus solely on this aggressive subtype, making it difficult to determine optimal treatment strategies. The purpose of this study was therefore to identify novel therapeutic targets that could aid in the management of UPSC. Methods: Clinical and RNA sequencing data from The Cancer Genome Atlas (TCGA) was used to identify genes that were differentially expressed between endometrioid endometrial carcinoma (EEC) and UPSC, which also showed a significant association with overall patient survival duration in UPSC patients. Ubiquitin C-terminal hydrolase L1 (UCHL1) upregulation and correlation with survival was validated by immunohistochemical staining of an independent cohort of paraffin samples. UCHL1 was silenced by siRNA transfection and lentiviral shRNA transduction in UPSC cell lines ARK1, ARK2 and HEC50 to evaluate the role of UCHL1 on cell proliferation, migration and cell cycle progression. RPPA data from TCGA was used to identify proteins whose expression correlated with UCHL1 expression. Interaction between UCHL1 and cyclin B1 was determined by co-immunoprecipitation. The effect of UCHL1 silencing on cyclin B1 levels was evaluated by Western blot analysis, and the effect on cell cycle progression was determined by flow cytometry. Results: UCHL1 mRNA and protein expression levels were significantly higher in in UPSC compared to EEC. Kaplan-Meier analysis and cox regression of the TCGA data set and an independent cohort of patient samples revealed that UCHL1 expression correlated significantly with poorer overall survival in UPSC, especially in late-stage patients. siRNA-mediated silencing did not affect cell migration, but reduced the proliferation rates of ARK1, ARK2 and HEC50 in vitro. UCHL1 RNA expression was positively correlated with cyclin B1 protein levels in the TCGA data set. Accordingly, UCHL1 silencing reduced cyclin B1 protein but not mRNA expression levels in ARK1, ARK2 and HEC50. Cell cycle analysis of ARK1, ARK2 and HEC50 after UCHL1 silencing showed a reduction in the percentage of cells in the S and G2/M phases, with or without cell synchronization. Conclusion: These findings indicate that UCHL1 contributes to the aggressiveness of UPSC by stabilizing cyclin B1 and increasing cell proliferation, suggesting that it may be a therapeutic target for UPSC. Citation Format: Suet Ying Kwan, Samuel C. Mok, Kwong-Kwok Wong, Rosemarie E. Schmandt, Karen H. Lu. Ubiquitin C-terminal hydrolase L1 (UCHL1) modulates uterine papillary serous cancer progression through interaction with cyclin B1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2015-3559

Serum Glial Fibrillary Acidic Protein Predicts Tissue Glial Fibrillary Acidic Protein Break-Down Products and Therapeutic Efficacy after Penetrating Ballistic-Like Brain Injury.

Acute traumatic brain injury (TBI) is associated with neurological dysfunction, changes in brain proteins, and increased serum biomarkers. However, the relationship between these brain proteins and serum biomarkers, and the ability of these serum biomarkers to indicate a neuroprotective/therapeutic response, remains elusive. Penetrating ballistic-like brain injury (PBBI) was used to systematically analyze several key TBI biomarkers, glial fibrillary acidic protein (GFAP) and its break-down products (BDPs)-ubiquitin C-terminal hydrolase-L1 (UCH-L1), α-II spectrin, and α-II spectrin BDPs (SBDPs)-in brain tissues and serum during an extended acute-subacute time-frame. In addition, neurological improvement and serum GFAP theranostic value was evaluated after neuroprotective treatment. In brain tissues, total GFAP increased more than three-fold 2 to 7 d after PBBI. However, this change was primarily due to GFAP-BDPs which increased to 2.7-4.8 arbitrary units (AU). Alpha-II spectrin was nearly ablated 3 d after PBBI, but somewhat recovered after 7 d. In conjunction with α-II spectrin loss, SBDP-145/150 increased approximately three-fold 2 to 7 d after PBBI (vs. sham, p<0.05). UCH-L1 protein levels were slightly decreased 7 d after PBBI but otherwise were unaffected. Serum GFAP was elevated by 3.2- to 8.8-fold at 2 to 4 h (vs. sham; p<0.05) and the 4 h increase was strongly correlated to 3 d GFAP-BDP abundance (r=0.66; p<0.05). Serum GFAP showed such a strong injury effect that it also was evaluated after therapeutic intervention with cyclosporin A (CsA). Administration of 2.5 mg/kg CsA significantly reduced serum GFAP elevation by 22.4-fold 2 h after PBBI (vs. PBBI+vehicle; p<0.05) and improved neurological function 1 d post-injury. Serum biomarkers, particularly GFAP, may be correlative tools of brain protein changes and feasible theranostic markers of TBI progression and recovery.

Parkin-mediated K63-polyubiquitination targets ubiquitin C-terminal hydrolase L1 for degradation by the autophagy-lysosome system.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease.

Ubiquitin C-terminal hydrolase L1 deletion ameliorates glomerular injury in mice with ACTN4-associated focal segmental glomerulosclerosis

Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4pod+/UCHL1+/+ mice. UCHL1+/− mice were intercrossed with K256E-ACTN4pod+ mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4pod+/UCHL1−/− mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.

Ubiquitin C-terminal hydrolase l1 is expressed in mouse pituitary gonadotropes in vivo and gonadotrope cell lines in vitro.

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.

Prognostic relevance of ubiquitin C-terminal hydrolase L1 (UCH-L1) mRNA and protein expression in breast cancer patients

The ubiquitin C-terminal hydrolase L1 (UCH-L1) belongs to the family of deubiquitinating enzymes. It is overexpressed in various tumour entities and associated with metastases formation in some solid tumours. However, only limited information about its role in breast cancer is available. The aim of this study was to examine the UCH-L1 expression in primary breast cancer and to determine its relevance as a potential prognostic marker. We investigated both UCH-L1 mRNA expression in microarray data from 182 primary mammary carcinomas and UCH-L1 protein expression using a tissue microarray containing samples from 1,622 breast cancer patients. With both methods, high UCH-L1 expression correlated significantly with negative oestrogen receptor and progesterone receptor status and advanced tumour stage. Moreover by Kaplan-Meier analysis, high UCH-L1 mRNA and protein expression correlated with a significantly shorter overall survival. The data of our study suggest that high levels of UCH-L1 expression indicate a more aggressive tumour behaviour and might represent a potential target in breast cancer treatment.

Ubiquitin C-terminal hydrolase L1 is required for pancreatic beta cell survival and function in lipotoxic conditions

Ubiquitin C-terminal hydrolase L1 (UCHL1) is associated with neurodegenerative diseases and has been suggested to have roles in pancreatic beta cells. Our proteomic analysis revealed that UCHL1 was the most increased protein in MIN6 cells exposed to palmitate. The present study used a genetic loss-of-function model to test the hypothesis that UCHL1 is required for normal beta cell function and fate under lipotoxic conditions. Human islets, mouse islets and MIN6 cells were used to analyse UCHL1 protein levels and regulation of UCHL1 by palmitate. The levels of free mono-ubiquitin and poly-ubiquitinated proteins were assessed. Gracile axonal dystrophy (GAD) mutant mice lacking UCHL1 were fed a normal or lipotoxic high-fat diet. Glucose tolerance, insulin tolerance and insulin secretion were assessed in vivo. Beta cell death and proliferation were assessed by TUNEL and proliferating cell nuclear antigen (PCNA) staining. Insulin secretion, calcium signalling, endoplasmic reticulum (ER) stress, apoptosis and SNARE protein levels were assessed in vitro. UCHL1 protein, which was highly specific to beta cells, was increased by palmitate at basal glucose, but not in the context of hyperglycaemia associated with frank diabetes. Although islet development and function were initially normal in Uchl1 (-/-) mice, a 4-week high-fat diet caused glucose intolerance and impaired insulin secretion. Uchl1 (-/-) mice had increased ER stress and beta cell apoptosis. The levels of SNARE proteins were dysregulated in Uchl1 (-/-) islets. Palmitate-stimulated vesicle-associated membrane protein 2 (VAMP2) ubiquitination was modulated by a chemical UCHL1 inhibitor. Together, these data suggest that UCHL1 has essential functional and anti-apoptotic roles in beta cells under stress conditions associated with lipotoxicity.

Effect of liposomal transfection of UCH-L1 siRNA on proliferation and apoptosis of lung cancer cell line H157

背景与目的泛素羧基末端水解酶-1（ubiquitin C terminal hydrolase-L1, UCH-L1）是近期研究较多的一类癌基因，在多种恶性肿瘤如食管癌、肺癌、乳腺癌等中均有异常表达和活性增强。本研究旨在探讨脂质体转染UCH-L1 siRNA对人肺腺癌H157细胞增殖、凋亡的影响。方法人工合成抑制UCH-L1基因的siRNA片段，通过脂质体转染到H157细胞内，显微镜下观察其形态学变化；流式细胞术检测各组细胞周期变化和凋亡指数；RT-PCR检测UCH-L1 mRNA的表达；Western blot检测UCH-L1蛋白表达。结果转染siRNA后的H157细胞，细胞增殖受到显著抑制，细胞出现明显凋亡，UCH-L1 mRNA表达显著下降，UCH-L1蛋白水平显著下降。结论 应用RNA干扰靶向抑制UCH-L1基因可以显著抑制H157细胞增殖，促进调亡。UCH-L1可能成为肺癌基因治疗的一个新靶点。

Ubiquitin C‐terminal hydrolase‐L1 as a biomarker for ischemic and traumatic brain injury in rats

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).