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Yulan Huang,* Shitong He,* Yitian Chen, Junli Sheng, Yuling Fu, Xialin Du, Yalong Yang, Honglin Liu, Zhenyu Han, Yingqi Huang, Qian Wen, Chaoying Zhou, Xinying Zhou, Shengfeng Hu, Li Ma Institute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li Ma; Shengfeng HuInstitute of Molecular Immunology, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, 510515, People’s Republic of China, Email mali_61648322@smu.edu.cn; hushengfeng@smu.edu.cnBackground: As deubiquitinases (DUBs), ubiquitin C-terminal hydrolase (UCH)-L1 has been shown to play a crucial role in regulating diverse biological processes. However, its function in macrophage polarization remains unclear.Methods: We performed in vivo and in vitro experiments to investigate the role of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a kind of DUBs, in macrophage differentiation by using UCHL1-deficiency mice.Results: We demonstrated that LPS stimulation induced UCHL1 expression in macrophages. The deficiency of UCHL1 expression decreased the expression of CD80 and CD86 but increased the expression of CD206. The expression of TNF-α, IL-6, iNOS, and IL-10 was downregulated, while that of Arg1, Ym1, and Fizz1 was upregulated in UCHL1 deficient macrophages. Moreover, we observed that UCHL1 promoted the degradation of p110α through autophagy, but paradoxically increased the activity of AKT, thereby promoting polarization of macrophages into pro-inflammatory states.Conclusion: In this study, we identified UCHL1 as a positive regulator of M1 macrophage polarization. Our findings may help in developing therapeutic interventions for the treatment of inflammatory diseases and pathogenic infections.Keywords: UCHL1, macrophages, AKT, p110α, autophagy

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-β1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

Radiation-induced lung injury (RILI) is a potential complication of thoracic radiotherapy that can result in pneumonitis or pulmonary fibrosis and is associated with significant morbidity and mortality. The pathobiology of RILI is complex and includes the generation of free radicals and DNA damage that precipitate oxidative stress, endothelial cell (EC), and epithelial cell injury and inflammation. While the cellular events involved continue to be elucidated and characterized, targeted and effective therapies for RILI remain elusive. Sphingolipids are known to mediate EC function including many of the cell signaling events associated with the elaboration of RILI. Sphingosine-1-phosphate (S1P) and S1P analogs enhance EC barrier function in vitro and have demonstrated significant protective effects in vivo in a variety of acute lung injury models including RILI. Similarly, statin drugs that have pleiotropic effects that include upregulation of EC S1P receptor 1 (S1PR1) have been found to be strongly protective in a small animal RILI model. Thus, targeting of EC sphingosine signaling, either directly or indirectly, to augment EC function and thereby attenuate EC permeability and inflammatory responses, represents a novel and promising therapeutic strategy for the prevention or treatment of RILI.

The mainstay of Amyotrophic lateral sclerosis (ALS) diagnosis is the clinical evaluation, and it can be challenging when its first manifestations overlap with those of ALS mimic disorders (ALSmd). The lack of specific diagnostic test prevents an early diagnosis. The definition of prognosis in ALS is hampered by the heterogeneity of its clinical features, with variability in survival being the most salient feature. Therefore, wet biomarkers are needed to aid clinical decision, track disease progression, and better define disease trajectories. Recent advances highlighted neurofilaments as the most promising biomarkers for ALS. In the first part of our results, we assessed serum phosphorylated neurofilament heavy chain (pNfH) in a large ALS cohort (n=219). pNfH was an independent predictor of survival for ALS and its concentration was heterogenous across the ALS phenotypes, patients with fast disease progression and a predominant upper motor neuron burden showed the highest serum concentration. Subsequently, we performed gene expression and pathways analyses, on 8 motor nerve biopsies of patients with ALS and compared with 7 motor neuropathies as controls, identifying Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) as a potential candidate biomarker for ALS. Therefore, we tested UCHL1 in an independent large ALS and control cohorts to assess its diagnostic and prognostic performances. At the same time, we also tested serum Neurofilament light chain (NfL) and Glial fibrillary acidic protein (GFAP). In our analysis, UCHL1 resulted not promising as diagnostic biomarkers, conversely, it was an independent prognostic factor, proving itself helpful in the stratification of survival for patients with lower NfL levels. NfL consistently reached the best diagnostic performance for ALS showing almost optimal performance in discriminating ALS from healthy controls and other neurodegenerative diseases. Conversely, the diagnostic yield in distinguishing ALS from ALSmd was lower with specificity decreasing to 78.0%. As similarly observed for the pNfH, NfL concentrations were heterogenous across the ALS phenotypes and higher concentrations were detected in patients with a fast disease progression and a predominant upper motor neuron burden. Serum GFAP, a known marker of astrogliosis, differs among cognitive phenotypes, namely ALS with concomitant cognitive impairment or FTD had higher levels compared with ALS with normal cognition. Therefore, GFAP might be instrumental in tracking the occurrence of cognitive decline in ALS. La diagnosi della Sclerosi Laterale Amiotrofica (SLA) è principalmente basata sulla valutazione clinica e può essere talvolta problematica soprattutto quando le manifestazioni cliniche sono sovrapponibili a quelle di malattie neurologiche che possono mimare la SLA. L’assenza di test diagnostici specifici limitano la possibilità di formulare la diagnosi in fase precoce di malattia. La definizione della prognosi dei malati di SLA è limitata dall’eterogeneità fenotipica della malattia e dalla sua intrinseca variabilità inter-individuale. Alla luce di tali considerazioni, eventuali biomarcatori potrebbero essere utili nel guidare decisioni diagnostiche, terapeutiche e di valutazione prognostica. Recenti evidenze di letteratura hanno individuato i neurofilamenti a catena leggera e pesante (NfL e pNfH) come i due più promettenti biomarcatori per la SLA. Nella prima parte del nostro studio, abbiamo valutato la concentrazione sierica dei pNfH in una numerosa cohorte di pazienti affetti da SLA (n=219). I nostri risultati evidenziano che in analisi multivariata i pNfH sono risultati fattori indipendenti nel predizione della sopravvivenza dei pazienti. Inoltre, la concentrazione dei pNfH è risultata eterogenea nella popolazione SLA; i valori più elevati sono stati riscontrati nei pazienti con prevalente interessamento del I neurone di moto e concomitante elevata velocità di progressione di malattia. In seguito, abbiamo effettuato un’analisi di espressione genica e sequenziale analisi di pathways su 8 nervi motori ottenuti da pazienti SLA e 7 da pazienti con neuropatia motoria considerati come controlli. Da tale analisi abbiamo indentificato UCHL1 come una proteina potenzialmente promettente come biomarcatore per la SLA. Abbiamo analizzato i valori plasmatici di tale proteina esplorandone le potenzialità diagnostiche e prognostiche, in parallelo abbiamo misurato anche i valori plasmatici di NfL, GFAP e tTAU in coorte di pazienti SLA (n=143) e di controllo. Nel nostro studio UCHL1 non si è confermato un promettente biomarcatore per la diagnosti di SLA, tuttavia è risultato essere un fattore determinante per la definizione prognostica dei pazienti stratificando ulteriormente la prognosi dei pazienti SLA con bassi valori plasmatici di NfL. Dalle nostre analisi si conferma che NfL come i migliori biomarcatori diagnostici sia per discriminare i pazienti SLA dai controlli sani (HC) e da pazienti affetti da altre malattie neurodegenerative (DEG). Tuttavia, la performance diagnostica degli NfL nel distinguere le SLA dalle patologie SLA mimic è sensibilmente ridotta con una sensibilità del 78%. Come osservato per i pNfH, anche i livelli di NfL sono più elevati in pazienti con prevalente interessamento del I neurone di moto e concomitante elevata velocità di progressione di malattia. I livelli plasmatici di GFAP riflettono i diversi gradi di interessamento cognitivo nei pazienti affetti da SLA, infatti pazienti SLA con concomitante demenza fronto-temporale o interessamento cognitivo presentavano più elevati valori di GFAP rispetto a pazienti SLA con selettivo interessamento motorio. Alla luce di questi dati GFAP potrebbe essere un biomarcatore utile per tracciare il declino cognitivo dei pazienti con SLA.

Programmed cell death‐ligand 1 (PD‐L1) expressed on cancer cells can cause immune escape of non‐small‐cell lung cancer (NSCLC). Elucidation of the regulatory mechanisms of the PD‐L1 expression is a prerequisite for establishing new tumor immunotherapy strategies. Ubiquitin C‐terminal hydrolase L1 (UCHL1) is a regulator of cellular signaling transduction that is aberrantly expressed in NSCLC. However, it is not known whether UCHL1 regulates the expression of PD‐L1 in NSCLC cells. In the present study, we found that UCHL1 promotes the expression of PD‐L1 in NSCLC cell lines. In addition, UCHL1 expressed in NSCLC cells inhibited activation of Jurkat cells through upregulation of PD‐L1 expression in in vitro experiments. Moreover, UCHL1 upregulates PD‐L1 expression through facilitating activation of the AKT‐P65 signaling pathway. In conclusion, these results indicated that UCHL1 promoted PD‐L1 expression in NSCLC cells. This finding implied that inhibition of UCHL1 might suppress immune escape of NSCLC through downregulation of PD‐L1 expression in NSCLC cells., Deubiquitinase ubiquitin C‐terminal hydrolase L1 (UCHL1) is a regulator of cellular signaling transduction that is aberrantly expressed in non‐small‐cell lung cancer (NSCLC). We analyzed the relationship between deubiquitinase UCHL1 and immune checkpoint molecule programmed cell death‐ligand 1 (PD‐L1) in NSCLC cells. Our results showed that UCHL1 promoted expression of PD‐L1 through the Akt‐P65 signaling axis.

Background Despite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F‐box and WD repeat domain containing 7 (FBXW7). Methods To identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)‐edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry‐based proteomic profiles of CRISPR‐edited ARK1 and ARK4 serous EC cells to matched parental cells. Results Among distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7‐mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR‐edited FBXW7‐mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM‐1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors. Conclusion Our findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors., Mass spectrometry‐based profiling identified proteomic changes associated with recurrent somatic FBXW7 mutations and shared in two biologically distinct serous endometrial cancer cell lines. Most notably, a correlation between PADI2 protein and FBXW7 mutation was demonstrated in serous endometrial cancer cells and confirmed in an endometrioid endometrial cancer cell line. PADI2 protein expression was further demonstrated in primary serous endometrial tumors.

High‐grade neuroendocrine lung cancer (HGNEC), which includes small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC) of the lung is a rapidly proliferating, aggressive form of lung cancer. The initial standard chemotherapeutic regimens of platinum doublets are recommended for SCLC and have been frequently used for LCNEC. However, there are currently no molecularly targeted agents with proven clinical benefit for this disease. The deubiquitinating enzyme ubiquitin C‐terminal hydrolase‐L1 (UCHL1) is a neuroendocrine cell‐specific product that is known as a potential oncogene in several types of cancer, but little is known about the biological function of UCHL1 and its therapeutic potential in HGNEC. In this study, we found that preclinical efficacy evoked by targeting UCHL1 was relevant to prognosis in HGNEC. UCHL1 was found to be expressed in HGNEC, particularly in cell lines and patient samples of SCLC, and the combined use of platinum doublets with selective UCHL1 inhibitors improved its therapeutic response in vitro. Immunohistochemical expression of UCHL1 was significantly associated with postoperative survival in patients with HGNEC and contributed towards distinguishing SCLC from LCNEC. Circulating extracellular vesicles (EV), including exosomes isolated from lung cancer cell lines and serum from early‐stage HGNEC, were verified by electron microscopy and nanoparticle tracking analysis. Higher levels of UCHL1 mRNA in EV were found in the samples of patients with early‐stage HGNEC than those with early‐stage NSCLC and healthy donors’ EV. Taken together, UCHL1 may be a potential prognostic marker and a promising druggable target for HGNEC., In this study, we found that preclinical efficacy evoked by targeting UCHL1 was relevant to prognosis in HGNEC. UCHL1 may be a potential prognostic marker and a promising druggable target for HGNEC.

Parkinson’s disease (PD) is characterized by Lewy bodies containing α-synuclein and ubiquitin aggregates, their co-occurrence possibly linked to a failure of the ubiquitin proteasome system. Ubiquitin C-terminal hydrolase L1 (UCHL1) plays an important role in maintenance of nervous system integrity, and overexpression of UCHL1 has been shown to increase ubiquitin levels within neurons. While cerebrospinal fluid ubiquitin levels were reported to be lower in PD vs controls, plasma UCHL1 levels and their relationship with clinical measures in PD has not been reported. We measured plasma UCHL1 levels using single molecule array (Simoa) in 291 subjects (242 PD and 49 healthy controls, HC). We found that UCHL1 levels were significantly higher in PD patients at moderate stages (Hoehn and Yahr, H&Y stage >2) vs milder PD (H&Y ≤2, p

Rationale: Resistance to pemetrexed (PEM)-based chemotherapy is a major cause of progression in non-small cell lung cancer (NSCLC) patients. The deubiquitinating enzyme UCHL1 was recently found to play important roles in chemoresistance and tumor progression. However, the potential roles and mechanisms of UCHL1 in PEM resistance remain unclear. Methods: Bioinformatics analyses and immunohistochemistry were used to evaluate UCHL1 expression in NSCLC specimens. Kaplan-Meier analysis with the log-rank test was used for survival analyses. We established PEM-resistant NSCLC cell lines by exposing them to step-wise increases in PEM concentrations, and in vitro and in vivo assays were used to explore the roles and mechanisms of UCHL1 in PEM resistance using the NSCLC cells. Results: In chemoresistant tumors from NSCLC patients, UCHL1 was highly expressed and elevated UCHL1 expression was strongly associated with poor outcomes. Furthermore, UCHL1 expression was significantly upregulated in PEM-resistant NSCLC cells, while genetic silencing or inhibiting UCHL1 suppressed resistance to PEM and other drugs in NSCLC cells. Mechanistically, UCHL1 promoted PEM resistance in NSCLC by upregulating the expression of thymidylate synthase (TS), based on reduced TS expression after UCHL1 inhibition and re-emergence of PEM resistance upon TS restoration. Furthermore, UCHL1 upregulated TS expression, which mitigated PEM-induced DNA damage and cell cycle arrest in NSCLC cells, and also conferred resistance to PEM and other drugs. Conclusions: It appears that UCHL1 promotes PEM resistance by upregulating TS in NSCLC cells, which mitigated DNA damage and cell cycle arrest. Thus, UCHL1 may be a therapeutic target for overcoming PEM resistance in NSCLC patients.

Alzheimer’s disease (AD) is an incurable neurodegenerative disease. It is the most common form of dementia among the elderly population. So far, no effective methods of its treatment have been found. Research to better understand the mechanism of pathology may provide new methods for early diagnosis. This, in turn, could enable early intervention that could slow or halt disease progression and improve patients’ quality of life. Therefore, minimally invasive markers, including serum-based markers, are being sought to improve the diagnosis of AD. One of the important markers may be the concentration of UCHL1 and the proteasome in the blood serum. Their concentration can be affected by many factors, including eating habits. This study was conducted in 110 patients with early or moderate AD, with a mean age of 78.0 ± 8.1 years. The patients were under the care of the Podlasie Center of Psychogeriatrics and the Department of Neurology (Medical University of Białystok, Poland). The control group consisted of 60 healthy volunteers, matched for gender and age. The concentration of UCHL1 and the 20S proteasome subunit were measured by surface plasmon resonance imaging (SPRI). In addition, a nutritional interview was conducted with patients with AD, which assessed the frequency of consumption of 36 groups of products. In the group of patients with AD, compared to the control group, we showed a significantly higher concentration of UCHL1 (56.05 vs. 7.98 ng/mL) and the proteasome (13.02 vs. 5.72 µg/mL). Moreover, we found a low negative correlation between UCHL1 and the proteasome in the control group, and positive in the AD group. The analysis of eating habits showed that the consumption of selected groups of products may affect the concentration of the tested components, and therefore may have a protective effect on AD.

Purpose Biallelic variants in UCHL1 have been associated with a progressive early-onset neurodegenerative disorder, autosomal recessive spastic paraplegia type 79. In this study, we investigated heterozygous UCHL1 variants on the basis of results from cohort-based burden analyses. Methods Gene-burden analyses were performed on exome and genome data of independent cohorts of patients with hereditary ataxia and spastic paraplegia from Germany and the United Kingdom in a total of 3169 patients and 33,141 controls. Clinical data of affected individuals and additional independent families were collected and evaluated. Patients’ fibroblasts were used to perform mass spectrometry-based proteomics. Results UCHL1 was prioritized in both independent cohorts as a candidate gene for an autosomal dominant disorder. We identified a total of 34 cases from 18 unrelated families, carrying 13 heterozygous loss-of-function variants (15 families) and an inframe insertion (3 families). Affected individuals mainly presented with spasticity (24/31), ataxia (28/31), neuropathy (11/21), and optic atrophy (9/17). The mass spectrometry-based proteomics showed approximately 50% reduction of UCHL1 expression in patients’ fibroblasts. Conclusion Our bioinformatic analysis, in-depth clinical and genetic workup, and functional studies established haploinsufficiency of UCHL1 as a novel disease mechanism in spastic ataxia.

Atrial fibrillation (AF) is the most common human arrhythmia in clinical practice and may be promoted by atrial inflammation and fibrosis. Ubiquitination is an important posttranslational modification process that is reversed by deubiquitinating enzymes (DUBs). DUBs play critical roles in modulating the degradation, activity, trafficking, and recycling of substrates. However, less research has focused on the role of DUBs in AF. Here, we investigated the effect of ubiquitin C-terminal hydrolase 1 (UCHL1), an important DUB, on the development of AF induced by angiotensin II (Ang II). Male wild-type mice were treated with the UCHL1 inhibitor LDN57444 (LDN) at a dose of 40 μg/kg and infused with Ang II (2000 ng/kg/min) for 3 weeks. Our results showed that Ang II-infused wild-type (WT) mice had higher systolic blood pressure and an increased incidence and duration of AF. Conversely, this effect was attenuated in LDN-treated mice. Moreover, the administration of LDN significantly reduced Ang II-induced left atrial dilation, fibrosis, inflammatory cell infiltration, and reactive oxygen species (ROS) production. Mechanistically, LDN treatment inhibited the activation of multiple signaling pathways (the AKT, ERK1/2, HIF-1α, and TGF-β/smad2/3 pathways) and the expression of CX43 protein in atrial tissues compared with that in vehicle-treated control mice. Overall, our study identified UCHL1 as a novel regulator that contributes to Ang II-induced AF and suggests that the administration of LDN may represent a potential therapeutic approach for treating hypertensive AF.

AIM:To explore the possible effects of genetic variant S18Y of ubiquitin carboxyl-terminal esterase L1(UCHL1)gene on age-related cataract formation.METHODS:We have investigated the frequency of this variant among the Han-Chinese population in a case-control study. A total of 242 cortical cataract patients and 144 controls were genotyped using polymerase chain reaction(PCR)and genomic sequencing.RESULTS:There is no statistical difference between cataract patients and healthy controls for the frequency of alleles(P=0.746)and genotypes(P=0.813). Since there was in our sample a significant difference between the groups in mean age(POR and CI for UCHL1 allele A positivity were computed by a binary Logistic regression model, no association was obtained for a positive UCHL1 allele A carrier status(P=0.818).CONCLUSION:We did not find any difference between the cases and the controls at allelic and genotypic level. Our results do not support a role for this variant in cataracts.

Significance Many neuroprotective strategies have failed to translate to clinical trials, perhaps because of a failure to preserve white matter function. Ubiquitin C-terminal hydrolase L1 (UCHL1), a neuron-specific protein essential for axonal function, is deactivated by reactive lipids produced after cerebral ischemia. Mutation of the cysteine residue 152-reactive lipid-binding site of UCHL1 decreased axonal injury after hypoxia and ischemia in vitro and in vivo, preserved axonal conductance and synaptic function, and improved motor behavior after ischemia in mice. These results suggest that UCHL1 may play an important role in maintaining axonal function after cerebral ischemia. Restoration of UCHL1 activity or prevention of degradation of UCHL1 activity by preventing binding of substrates to cysteine residue 152 could be useful approaches for treatment of stroke., Ubiquitin C-terminal hydrolase L1 (UCHL1) is a unique brain-specific deubiquitinating enzyme. Mutations in and aberrant function of UCHL1 have been linked to many neurological disorders. UCHL1 activity protects neurons from hypoxic injury, and binding of stroke-induced reactive lipid species to the cysteine 152 (C152) of UCHL1 unfolds the protein and disrupts its function. To investigate the role of UCHL1 and its adduction by reactive lipids in inhibiting repair and recovery of function following ischemic injury, a knock-in (KI) mouse expressing the UCHL1 C152A mutation was generated. Neurons derived from KI mice had less cell death and neurite injury after hypoxia. UCHL1 C152A KI and WT mice underwent middle cerebral artery occlusion (MCAO) or sham surgery. White matter injury was significantly decreased in KI compared with WT mice 7 d after MCAO. Histological analysis revealed decreased tissue loss at 21 d after injury in KI mice. There was also significantly improved sensorimotor recovery in postischemic KI mice. K63- and K48-linked polyubiquitinated proteins were increased in penumbra of WT mouse brains but not in KI mouse brains at 24 h post MCAO. The UCHL1 C152A mutation preserved excitatory synaptic drive to pyramidal neurons and their excitability in the periinfarct zone; axonal conduction velocity recovered by 21 d post MCAO in KI mice in corpus callosum. These results demonstrate that UCHL1 activity is an important determinant of function after ischemia and further demonstrate that the C152 site of UCHL1 plays a significant role in functional recovery after stroke.

International audience; Rare mutations in genes originally discovered in multigenerational families have been associated with increased risk of Parkinson's disease (PD). The involvement of rare variants in DNAJC13, UCHL1, HTRA2, GIGYF2, and EIF4G1 loci has been poorly studied or has produced conflicting results across cohorts. However, they are still being often referred to as "PD genes" and used in different models. To further elucidate the role of these 5 genes in PD, we fully sequenced them using molecular inversion probes in 2408 patients with PD and 3444 controls from 3 different cohorts. A total of 788 rare variants were identified across the 5 genes and 3 cohorts. Burden analyses and optimized sequence Kernel association tests revealed no significant association between any of the genes and PD after correction for multiple comparisons. Our results do not support an association of the 5 tested genes with PD. Combined with previous studies, it is unlikely that any of these genes plays an important role in PD. Their designation as "PARK" genes should be reconsidered.

Background Biomarkers that can objectively guide the diagnosis of sports-related concussion, and consequent return-to-play decisions, are urgently needed. In this study, we aimed to determine the temporal profile and diagnostic ability of serum levels of neurofilament light (NfL), ubiquitin carboxy-terminal hydrolase L1 (UCHL1), glial fibrillary acidic protein (GFAP), and tau in concussed male and female Australian footballers. Methods Blood was collected from 28 Australian rules footballers (20 males, 8 females) at 2-, 6-, and 13-days after a diagnosed concussion for comparison to their levels at baseline (i.e. pre-season), and with 27 control players (19 males, 8 females) without a diagnosis of concussion. Serum concentrations of protein markers associated with damage to neurons (UCHL1), axons (NfL, tau), and astrocytes (GFAP) were quantified using a Simoa HD-X Analyzer. Biomarker levels for concussed players were compared over time and between sex using generalised linear mixed effect models, and diagnostic performance was assessed using area under the receiver operating characteristic curve (AUROC) analysis. Results Serum NfL was increased from baseline in male footballers at 6- and 13-days post-concussion. GFAP and tau were increased in male footballers with concussion at 2- and 13-days respectively. NfL concentrations discriminated between concussed and non-concussed male footballers at all time-points (AUROC: 2d = 0.73, 6d = 0.85, 13d = 0.79), with tau also demonstrating utility at 13d (AUROC = 0.72). No biomarker differences were observed in female footballers after concussion. Conclusions Serum NfL may be a useful biomarker for the acute and sub-acute diagnosis of concussion in males, and could inform neurobiological recovery and return-to-play decisions. Future adequately powered studies are still needed to investigate biomarker changes in concussed females.

Archives of Allied Health Sciences, 33, 2, 1-8

Glycosphingolipids (GSLs) are amphipathic lipid moieties that make up only 3% of the total lipid content of the cell and are almost exclusively expressed at the Plasma Membrane(PM). They are key drivers of signalling hotspots known as ‘lipid-rafts’ as well as acting as primary signalling molecules themselves. Pathologically, perturbations in their synthesis (Knock-Out mice models) and disorders in their metabolism(Lysosomal storage disorders), both converge on neurological symptoms. In fact, during neuronal development, stem cells have been known to completely remodel their glycosphingolipid profiles, so much so, that the developmental stages of the nervous system have been associated with the expression of different glycosphingolipids. This coordinated change in the expression of glycosphingolipids has been previously attributed to the transcriptional shift in the the expression of genes encoding specific Glycosphingolipid Synthesizing Enzymes (GSEs) observed over neuronal development and, more recently, shown to be internally regulated by the GSLs themselves whereby globosides negatively regulate the expression of the ganglioside synthesizing enzyme GM3S via a neuronal transcription factor AUTS2. In this thesis I show that there exists an extra layer of post-translational regulation that is active over neuronal development that destabilizes the Gb3 synthesizing enzyme, Gb3S, on one hand while stabilizing the GM3S on the other. The regulatory mechanism achieves this effect by doubling the rate of degradation of Gb3S in neuronal cells, probably via membrane trafficking events. The exact details of the stabilization effect of GM3S remains to be determined as the results could not ascertain if it was the same mechanism or a completely different one. My study goes on to show that this regulatory mechanism is activated early during development, preceding the changes in canonical stem and neuronal markers. Morphologically, the study points towards a post-compaction but pre-cavitation (E2.5-E3.5) point of activation for the mechanism of Gb3S destabalization, relative to early mouse embryological development. Furthermore, I found that the Gb3S interactor and AUTS2 transcriptional target UCHL1 is a key regulatory unit of the machinery such that inhibiting its activity results in a specific rescue of the Gb3S enzyme.

Ubiquitin C-terminal hydrolase L1 (UCHL1), which is a deubiquitinating enzyme, is known to play a role in chemoresistance in cancers. However, its potential roles and mechanisms in the chemoresistance of breast cancer (BC) remain unclear. In this study, we examined its expression in patients with BC and employed Kaplan–Meier analysis and the log-rank test for survival analyses. It was found that up-regulated UCHL1 expression was positively associated with both chemoresistance and poor prognosis, especially in patients with HER2+ BC. Moreover, UCHL1 expression was elevated in HER2+ BC cells (SK-BR-3 and BT474). Similarly, doxorubicin (DOX)-resistant BC cells (MCF-7/DOX) had higher UCHL1 levels than MCF-7 cells. CCK-8 assay showed that BC cells with higher UCHL1 levels were more resistant to DOX. Furthermore, by inhibiting UCHL1 in BC cells with elevated UCHL1 expression, we demonstrated that UCHL1 promoted DOX-resistance in BC. Mechanistically, UCHL1 probably promoted DOX-resistance of BC by up-regulating free fatty acid (FFA) synthesis, as exhibited by reduced FFA synthase expression and resurrected DOX-sensitivity upon UCHL1 inhibition. Overall, UCHL1 up-regulation is associated with DOX-resistance and poor prognosis in patients with HER2+ BC. UCHL1 induces DOX-resistance by up-regulating FFA synthesis in HER2+ BC cells. Thus, UCHL1 might be a potential clinical target for overcoming DOX resistance in patients with HER2+ BC.

Renal cell carcinoma is characterized by high immunogenicity and infiltration of immune cells. CD45RO+CD8+ T cells are well known as a critical role in host defense of the immune environment. However, their role in clear cell renal carcinoma (ccRCC) remains unknown. To elucidate the clinical importance of CD45RO+CD8+ T cells in ccRCC as well as its underlying mechanism, we analyzed several types of peripheral immune cells from 274 patients with ccRCC who have received radical or partial nephrectomy and 350 healthy people. Flow cytomety assays showed there was no significant difference in the proportions of CD8+ T cells and its subtypes other than CD45RO+/CD45RA+CD8+ cells. Both gene and protein expression levels of CD45RO in ccRCC tissues were decreased. CD45RO+CD8+ T cells showed increased proliferative abilities but decreased apoptotic abilities through MAPK signaling activation in ccRCC. High expression level of CD45RO+CD8+ T cells inhibited ccRCC progression, including proliferation, invasion, as well as autophagy of ccRCC through many signaling pathways. Bioinformatics and immunohistochemical chip analysis measured gene and protein levels of CD45RO and other related proteins. The combination of UCHL1, HMGB3, and CD36 has diagnostic value in ccRCC and is able to predict prognosis. Collectively, CD45RO+CD8+ T cells play a critical role in ccRCC progression and may be regarded as clinical indicators.

Background Parkinson’s disease (PD) is the second most common neurodegenerative disorder. PD associated human UCHL1 (Ubiquitin C-terminal hydrolase L1) gene belongs to the family of deubiquitinases and is known to be highly expressed in neurons (1–2% in soluble form). Several functions of UCHL1 have been proposed including ubiquitin hydrolyze activity, ubiquitin ligase activity and stabilization of the mono-ubiquitin. Mutations in human UCHL1 gene have been associated with PD and other neurodegenerative disorders. The present study aims to decipher the sequence evolutionary pattern and structural dynamics of UCHL1. Furthermore, structural and interactional analysis of UCHL1 was performed to help elucidate the pathogenesis of PD. Results The phylogenetic tree topology suggests that the UCHL1 gene had originated in early gnathostome evolutionary history. Evolutionary rate analysis of orthologous sequences reveals strong purifying selection on UCHL1. Comparative structural analysis of UCHL1 pinpoints an important protein segment spanning amino acid residues 32 to 39 within secretion site with crucial implications in evolution and PD pathogenesis through a well known phenomenon called intragenic epistasis. Identified critical protein segment appears to play an indispensable role in protein stability, proper protein conformation as well as harboring critical interaction sites. Conclusions Conclusively, the critical protein segment of UCHL1 identified in the present study not only demonstrates the relevant role of intraprotein conformational epistasis in the pathophysiology of PD but also offers a novel therapeutic target for the disease.

Multiple myeloma (MM) patients considered to be at high cytogenetic risk commonly fail to respond to standard treatment. A thorough understanding of the molecular mechanism of MM development is, therefore, needed. We endeavored to explore the transcriptional signature among different subgroups of newly diagnosed MM using gene chip-based expression microarray. Bone marrow samples of 15 newly diagnosed Thai MM patients were included. The chromosomal translocation t(4;14) was the most frequently identified genetic alteration in the high-risk subgroup. Cluster analysis from expression profiling demonstrated that high-risk MM have a distinctly different expression pattern compared to standard-risk patients. The most significant differentially expressed gene was UCHL1. Functional enrichment analysis by Gene Set Enrichment Analysis, FUNRICH, and Gene Ontology Panther pathway revealed the gene sets involved in cell cycle control to be enriched in the t(4;14) high-risk group. Interestingly, among the well-established downstream targets of UCHL1, only CCND2 was significantly expressed in the t(4;14) high-risk group. Suppression of UCHL1 protein level by LDN-5744 inhibitor could arrest the cell cycle in G1 phase in cell lines. These findings shed light on the molecular mechanism of UCHL1 in t(4;14) high-risk MM and support the evidence that alteration of the UCHL1 pathway may play a role in the pathogenesis of high-risk MM.

In summary, this thesis focused on the understanding the underlying mechanisms driving TNBC metastatic progression. We established DUB activity profiling methods and identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis. Importantly, we found UCHL1 activity inhibitor as a potential drug for TNBC therapy and developed UCHL1 activity-based probe. For vemurafenib-resistance melanoma, we provided insights that targeting TGFβ signaling may help to overcome drug resistant phenotype.

Breast cancer is a highly heterogeneous disease with dynamic changes in the tumor microenvironment. Precision medicine will in the future provide the possibility to treat each individual cancer patient with the right (combination) therapy specifically tailored to personal needs. However, in order to accomplish this, more accurate biomarkers for precise diagnosis, prognosis, therapy response, and target-specific drugs are required. Although an increasing number of (epi)genetic driving alterations have been reported in breast cancer, the major stumbling block for clinical application of many of them is that they are difficult to therapeutically target. Deubiquitinases (DUBs) are emerging drug targets that play important roles in cancer progression. Hence, we devoted our efforts to uncover the global DUB activity landscape of breast cancer in order to discover potential novel biomarkers or therapeutic targets. We developed a specific DUB activity-based inhibitor and probe and applied it to obtain new insights into breast cancer.

Chemoresistance represents one of the main obstacles in treating several types of cancer, including bladder and ovarian cancers, and it is characterized by an increase of cellular antioxidant potential. Nrf2 and YAP proteins play an important role in increasing chemoresistance and in inducing antioxidant enzymes. It has been reported that Ailanthone (Aila), a compound extracted from the Ailanthus Altissima, has an anticancer activity toward several cancer cell lines, including chemoresistant cell lines. We have examined the effect of Aila on proliferation, migration and expression of Nrf2 and YAP proteins in A2780 (CDDP-sensitive) and A2780/CP70 (CDDP-resistant) ovarian cancer cells. Furthermore, to clarify the mechanism of Aila action we extended our studies to sensitive and CDDP-resistant 253J-BV bladder cancer cells, which have been used in a previous study on the effect of Aila. Results demonstrated that Aila exerted an inhibitory effect on growth and colony formation of sensitive and CDDP-resistant ovarian cancer cells and reduced oriented cell migration with higher effectiveness in CDDP resistant cells. Moreover, Aila strongly reduced Nrf2 and YAP protein expression and reduced the expression of the Nrf2 target GSTA4, and the YAP/TEAD target survivin. In CDDP-resistant ovarian and bladder cancer cells the intracellular oxidative stress level was lower with respect to the sensitive cells. Moreover, Aila treatment further reduced the superoxide anion content of CDDP-resistant cells in correlation with the reduction of Nrf2 and YAP proteins. However, Aila treatment increased Nrf2 and YAP mRNA expression in all cancer cell lines. The inhibition of proteolysis by MG132, a proteasoma inhibitor, restored Nrf2 and YAP protein expressions, suggesting that the Aila effect was at post-translational level. In accordance with this observation, we found an increase of the Nrf2 inhibitor Keap1, a reduction of p62/SQSTM1, a Nrf2 target which leads Keap1 protein to the autophagic degradation, and a reduction of P-YAP. Moreover, UCHL1 deubiquitinase expression, which was increased in bladder and ovarian resistant cells, was down-regulated by Aila treatment. In conclusion we demonstrated that Aila can reduce proliferation and migration of cancer cells through a mechanism involving a post translational reduction of Nrf2 and YAP proteins which, in turn, entailed an increase of oxidative stress particularly in the chemoresistant lines.

Made available in DSpace on 2022-04-28T19:48:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Venlafaxine, a norepinephrine and serotonin reuptake inhibitor, impairs rat sperm parameters, spermatogenesis and causes high intratesticular estrogen and testosterone levels, indicating that Leydig cells (LCs) may be a venlafaxine target. We evaluated the effect of venlafaxine treatment on rat LCs, focusing on adrenergic signaling, EGF immunoexpression and steroidogenesis. Germ cells mitotic/meiotic activity and UCHL1 levels were also evaluated in the seminiferous epithelium. Eighteen adult male rats received 30 mg/kg of venlafaxine (n = 9) or distilled water (n = 9). The seminiferous tubules, epithelium and LCs nuclear areas were measured, and the immunoexpression of Ki-67, UCHL1, StAR, EGF, c-Kit and 17β-HSD was evaluated. UCHL1, StAR and EGF protein levels and Adra1a, Nur77 and Ndrg2 expression were analyzed. Malondialdehyde (MDA) and nitrite testicular levels, and serum estrogen and testosterone levels were measured. Venlafaxine induced LCs hypertrophy and Ndrg2 upregulation in parallel to increased number of Ki-67, c-Kit- and 17β-HSD-positive interstitial cells, indicating that this antidepressant stimulates LCs lineage proliferation and differentiation. Upregulation of Adra1a and Nur77 could explain the high levels of StAR and testosterone levels, as well as aromatization. Enhanced EGF immunoexpression in LCs suggests that this growth fact is involved in adrenergically-induced steroidogenesis, likely via upregulation of Nur77. Slight tubular atrophy and weak Ki-67 immunoexpression in germ cells, in association with high UCHL1 levels, indicate that spermatogenesis is likely impaired by this enzyme under supraphysiological estrogen levels. These data corroborate the unchanged MDA and nitrite levels. Therefore, venlafaxine stimulates LCs steroidogenesis via adrenergic signaling, and EGF may be involved in this process. Federal University of São Paulo Department of Morphology and Genetics São Paulo State University (Unesp) School of Dentistry Department of Morphology Genetics Orthodontics and Pediatric Dentistry São Paulo State University (Unesp) School of Veterinary Medicine Department of Production and Animal Health São Paulo State University (Unesp) School of Dentistry Department of Morphology Genetics Orthodontics and Pediatric Dentistry São Paulo State University (Unesp) School of Veterinary Medicine Department of Production and Animal Health FAPESP: 2017/19829-6 FAPESP: 2018/13590-4

Purpose: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. Experimental Design: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. Results: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFβ signaling–induced metastasis by protecting TGFβ type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell–conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. Conclusions: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFβ-induced breast cancer metastasis and may provide a potential target for TNBC treatment.

Dysregulation of the neddylation pathway is related to various cancers. However, the specific role of the neddylation pathway in human hepatocellular carcinoma (HCC) remains largely unclear. In this study, the neddylation pathway in HCC and adjacent noncancerous liver (ANL) tissues was evaluated by immunohistochemistry (IHC), Western blotting, and qRT‐PCR (quantitative real‐time polymerase chain reaction). The results showed that the entire neddylation pathway, including NEDD8 (the IHC staining of NEDD8 represents the global‐protein neddylation), E1 NEDD8‐activating enzymes (NAE1 and UBA3), E2 NEDD8‐conjugating enzymes (UBE2F and UBE2M), E3 NEDD8‐ligases (MDM2, RBX1 and RNF7), and deneddylation enzymes (COPS5, UCHL1 and USP21), was overactivated in HCC. Furthermore, the upregulation of NEDD8 in HCC was correlated with aggressive characteristics and was an independent risk factor for overall survival (OS) and recurrence‐free survival (RFS) in patients with HCC after hepatectomy. The upregulation of NAE1, UBE2M, and UCHL1 in HCC was associated with aggressive characteristics and poor OS and RFS in patients with HCC after hepatectomy. In conclusion, our research reveals that the entire neddylation pathway is overactivated in HCC and associated with clinical characteristics and prognosis of patients with HCC.

As a member of the ubiquitin-dependent proteasome degradation pathway, Ubiquitin carboxyl-terminal hydrolase-L1 (UCHL1) plays a key role in post-translational modification and protein degradation, and it is extensive and important for the regulation of various biological functions of the organism. However, its function remains unclear in goose growth performance. In this study, the full-length genomic DNA and coding region of UCHL1 gene was firstly cloned and characterized in Yangzhou goose. Tissue expression profile revealed that UCHL1 was exclusively expressed in brain and gonads. A novel single nucleotide polymorphisms c.-652C>T which is significantly related to 64-d body weight of Yangzhou goose was found in UCHL1 promoter region by comparative sequencing. Correlation analysis in a population of 405 geese showed that TT genotype individuals had higher body weight than CC individuals in male, but not in female geese. Dual-luciferase reporter assay indicated that the single nucleotide polymorphisms c.-652C>T is located at the core promoter region of UCHL1, and the promoter transcription activity was significantly increased (P < 0.01) when allele C changed to T. Geese with TT genotype had higher mRNA level of UCHL1 in brain tissue than those of CC genotype (P < 0.01). Compared with CC individuals, neuropeptide Y and AdipoR1 mRNA level was significantly higher in TT individuals (P < 0.05), while FAS mRNA level was lower in the TT individuals (P < 0.05). In summary, we identify a novel mutation in the promoter of UCHL1 gene, which can alter transcriptional activity of UCHL1 gene, and affect the growth performance of male goose.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which is extensively expressed in vertebrates, is a deubiquitinating enzymes that inhibits the degradation of proteins by reversing ubiquitination modification. Herein, a 1087-bp sequence encoding UCHL1 was identified from the Chinese giant salamander (CGS; Andrias davidianus). The coding sequences (CDS) of UCHL1 encoded a putative poly peptide of 222 amino acids. The CGS UCHL1 isoforms were more related to their human and mouse counterparts. The phylogenic tree of vertebrate UCHL1 indicated that CGS UCHL1 has the closest relationship with human UCHL1 (up to 73.99 %). Before the gonads of male CGSs matured, the peak level of UCHL1 expression in testes appeared in 3-year-old CGSs according to RT-qPCR and western blot. In adult testes, the level of UCHL1 protein was lower in the breeding period than in the post-breeding period, whereas the level of UCHL1 protein in interstitial fluid of adult CGS testes was higher during the breeding period than during the post-breeding period. In testicular seminiferous lobules in the developmental stage of CGSs, immunohistochemistry displayed three kinds of localizing patterns of UCHL1, including nuclear localization at half year old, cytoplasmic localization from one year to three years old, and extracellular localization in adult. In testicular seminiferous lobules of adult CGS, the different developmental germ cells were separated by cysts containing UCHL1 protein, but UCHL1 did not localize on the mature sperm. The results showed that extracellular UCHL1 loaded on exosomes, as a component of the homogeneous germ cell cysts, could regulate the synchronous development of sperm in testes of adult CGS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and the proteins and pathways involved in the pathophysiology are not fully understood. Even less is known about the preclinical disease phase. To uncover new ALS-related proteins and pathways, we performed a comparative proteomic analysis in cerebrospinal fluid (CSF) of asymptomatic (n = 14) and symptomatic (n = 14) ALS mutation carriers and sporadic ALS patients (n = 12) as well as post-mortem human spinal cord tissue (controls: n = 7, ALS, n = 8). Using a CSF-optimized proteomic workflow, we identified novel (e.g., UCHL1, MAP2, CAPG, GPNMB, HIST1H4A, HIST1H2B) and well-described (e.g., NEFL, NEFH, NEFM, CHIT1, CHI3L1) protein level changes in CSF of sporadic and genetic ALS patients with enrichment of proteins related to transcription, cell cycle and lipoprotein remodeling (total protein IDs: 2303). No significant alteration was observed in asymptomatic ALS mutation carriers representing the prodromal disease phase. We confirmed UCHL1, MAP2, CAPG and GPNMB as novel biomarker candidates for ALS in an independent validation cohort of patients (n = 117) using multiple reaction monitoring. In spinal cord tissue, 292 out of 6810 identified proteins were significantly changed in ALS with enrichment of proteins involved in mRNA splicing and of the neurofilament compartment. In conclusion, our proteomic data in asymptomatic ALS mutation carriers support the hypothesis of a sudden disease onset instead of a long preclinical phase. Both CSF and tissue proteomic data indicate transcriptional pathways to be amongst the most affected. UCHL1, MAP2 and GPNMB are promising ALS biomarker candidates which might provide additional value to the established neurofilaments in patient follow-up and clinical trials. ispartof: ACTA NEUROPATHOLOGICA vol:139 issue:1 pages:119-134 ispartof: location:Germany status: published

Epigenetic regulation plays an important role in the development and progression of nasopharyngeal carcinoma (NPC), but the epigenetic mechanisms underlying NPC metastasis remain poorly understood. Here, we demonstrate that hypermethylation of the UCHL1 promoter leads to its downregulation in NPC. Restoration of UCHL1 inhibited the migration and invasion of NPC cells in vitro and in vivo, and knockdown of UCHL1 promoted NPC cell migration and invasion in vitro and in vivo. Importantly, we found that UCHL1 interacts with CTTN, and may function as a ligase promoting CTTN degradation by increasing K48-linked ubiquitination of CTTN. Additionally, restoration of CTTN in NPC cells that overexpressed UCHL1 rescued UCHL1 suppressive effects on NPC cell migration and invasion, which indicated that CTTN is a functional target of UCHL1 in NPC. Our findings revealed that UCHL1 acts as a tumor suppressor gene in NPC and thus provided a novel therapeutic target for NPC treatment.

Uterine serous carcinoma (USC) is the most aggressive form of endometrial cancer, with poor survival rates and high recurrence risk. Therefore, the purpose of this study was to identify therapeutic targets that could aid in the management of USC. By analyzing endometrial cancer samples from The Cancer Genome Atlas (TCGA), we found Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) to be highly expressed in USC and to correlate with poorer overall survival. UCHL1 silencing reduced cell proliferation in vitro and in vivo, cyclin B1 protein levels and cell cycle progression. Further studies showed that UCHL1 interacts with cyclin B1 and increases cyclin B1 protein stability by deubiquitination. Treatment of USC-bearing mice with the UCHL1-specific inhibitor reduced tumor growth and improved overall survival. Our findings suggest that cyclin B1 is a novel target of UCHL1 and targeting UCHL1 is a potential therapeutic strategy for USC.

International audience; Objective: Salivary gland tumors (SGTs) are one of the most heterogeneous and challenging neoplasms affects human. Severalbiomarkers have been used to study proliferation, angiogenesis, prognosis, metastasis and recurrence of SGTs. The aim of this studywas to evaluate, compare and correlate the co-expression of Budding Uninhibited by Benz imidazole Related 1 (BubR1) andUbiquitin C-terminal hydrolase-L1 (UCHL1) immunomarkers in SGTs.Methods: The immunohistochemical expression of BubR1 and UCHL1 were performed with formalin fixed paraffin embeddedtissue sections of 35 retrieved blokes of SGTs. The expression, pattern of reactivity, intensity and subcellular localization of thesemarkers are studied. T-test was used to find statistical difference in expression immunomarkers.Results: All of the cases were positive for both BubR1 and UCHL1. The intensity of reaction differed between the tumor types. Asignificant difference was seen in the expression of BubR1 in benign versus malignant tumors (P=.002) and pleomorphic adenomaversus mucoepidermoid carcinoma (P=.001). While statistically significant difference was not seen in the expression of UCHL1between the tumors mentioned above (P=.81 and P=.83, respectively). Finally, there was a significant difference between theexpressions of BubR1 and UCHL1 in SGTs (P=.001), indicating a higher expression of UCHL1 in SGTs.Conclusions: UCHL1 has a higher percentage and intensity of reactivity in SGTs as compared to BubR1, While BubR1 is a betterimmunomarker for distinguishing between benign and malignant tumors.

Deubiquitinases (DUBs) play important roles and therefore are potential drug targets in various diseases including cancer and neurodegeneration. In this review, we recapitulate structure–function studies of the most studied DUBs including USP7, USP22, CYLD, UCHL1, BAP1, A20, as well as ataxin 3 and connect them to regulatory mechanisms and their growing protein interaction networks. We then describe DUBs that have been associated with endocrine carcinogenesis with a focus on prostate, ovarian, and thyroid cancer, pheochromocytoma, and adrenocortical carcinoma. The goal is enhancing our understanding of the connection between dysregulated DUBs and cancer to permit the design of therapeutics and to establish biomarkers that could be used in diagnosis and prognosis.

Background Ubiquitin C-terminal hydrolase isozyme L1 (UCHL1) is primarily expressed in neuronal cells and neuroendocrine cells and has been associated with various diseases, including many cancers. It is a multifunctional protein involved in deubiquitination, ubiquitination and ubiquitin homeostasis, but its specific roles are disputed and still generally undetermined. Results Herein, we demonstrate that UCHL1 is associated with genomic DNA in certain prostate cancer cell lines, including DU 145 cells derived from a brain metastatic site, and in HEK293T embryonic kidney cells with a neuronal lineage. Chromatin immunoprecipitation and sequencing revealed that UCHL1 localizes to TTAGGG repeats at telomeres and interstitial telomeric sequences, as do TRF1 and TRF2, components of the shelterin complex. A weak or transient interaction between UCHL1 and the shelterin complex was confirmed by immunoprecipitation and proximity ligation assays. UCHL1 and RAP1, also known as TERF2IP and a component of the shelterin complex, were bound to the nuclear scaffold. Conclusions We demonstrated a novel feature of UCHL1 in binding telomeres and interstitial telomeric sites. Electronic supplementary material The online version of this article (10.1186/s13072-017-0160-2) contains supplementary material, which is available to authorized users.

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.

This work was supported by The RJAH Institute of Orthopaedics, UK (H.F.), The SMA Trust, UK (H.F.), Cedars-Sinai Institutional startup funds (D.S), California Institute for Regenerative Medicine Grant RT-02040 (D.S.), National Center for Advancing Translational Sciences (NCATS), Grant UL1TR000124 (D.S.), and the Wellcome Trust [grant number 094476/Z/10/Z] which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews (S.S. and C.B.). D.S. is also supported by funds from National Institute of Health (NINDS) grant U54NS091046. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons, and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts, whereas 30 proteins were increased in SMA motor neurons versus their fibroblasts. Notably, UBA1 was significantly decreased in SMA motor neurons, supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons, including beta III-tubulin and UCHL1, were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts, highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons, and for the identification of novel biomarker and therapeutic targets for SMA. Publisher PDF

Years of research in the field of neurotrauma have led to the concept of applying systems biology as a tool for biomarker discovery in traumatic brain injury (TBI). Biomarkers may lead to understanding mechanisms of injury and recovery in TBI and can be potential targets for wound healing, recovery, and increased survival with enhanced quality of life. The literature available on neurotrauma studies from both animal and clinical studies has provided rich insight on the molecular pathways and complex networks of TBI, elucidating the proteomics of this disease for the discovery of biomarkers. With such a plethora of information available, the data from the studies require databases with tools to analyze and infer new patterns and associations. The role of different systems biology tools and their use in biomarker discovery in TBI are discussed in this chapter.

Objective and design To explore the role of mammalian target of rapamycin 2 (mTORC2) in the activation of inflammatory and oxidative responses in rodent models of acute injury and metabolic stress. Material The impact of nephrilin, an inhibitor of mTORC2 complex, was assessed in three CD-1 mouse models of acute xenobiotic stress and in a hypertensive Dahl rat model of metabolic stress. Methods Animals received daily subcutaneous bolus injections of saline or 4 mg/kg nephrilin. Tissues were assayed by ELISA, gene arrays and immunohistochemical staining. Results Nephrilin significantly inhibited elevations in plasma tumor necrosis factor-alpha, kidney substance P, and CX3CR1, and urinary lipocalin-2 [urinary neutrophil gelatinase-associated lipocalin (uNGAL)] in models of acute xenobiotic stress. UCHL1 gene expression levels dropped and plasma HMGB1 levels rose in the rhabdomyolysis model. Both effects were reversed by nephrilin. The inhibitor also blocked diet-induced elevations of uNGAL and albumin-creatinine ratio (UACR) as well as kidney tissue phosphorylation of PKC-beta-2-T641 and p66shc-S36, and reduced dark ring-like staining of nuclei by anti-phos-p66shc-S36 antibody in frozen sections of diseased kidneys from hypertensive Dahl rats fed an 8 % NaCl diet for 4 weeks. Conclusions Taken together, our results suggest a role for mTORC2 in the inflammatory-oxidative responses to stress.

The ubiquitin carboxyl-terminal esterase L1 gene, UCHL1, located on chromosome 4p14, has been studied as a potential candidate gene for Parkinson's disease risk. The authors conducted a Human Genome Epidemiology review and meta-analysis of published case-control studies of the UCHL1 S18Y variant and Parkinson's disease in Asian and Caucasian samples. The meta-analysis of studies in populations of Asian ancestry showed a statistically significant association between the Y allele and reduced risk of Parkinson's disease under a recessive model (odds ratio (OR) for YY vs. SY + SS = 0.79, 95% confidence interval (CI): 0.67, 0.94; P = 0.006). For a dominant model, the association was not significant in Asian populations (OR for YY + SY vs. SS = 0.88, 95% CI: 0.68, 1.14; P = 0.33). For populations of European ancestry, the meta-analysis showed a significant association between the Y allele and decreased risk of Parkinson's disease under a dominant model (OR = 0.89, 95% CI: 0.81, 0.98; P = 0.02) but not under a recessive model (OR = 0.92, 95% CI: 0.66, 1.30; P = 0.65). Using the Venice criteria, developed by the Human Genome Epidemiology Network Working Group on the assessment of cumulative evidence, the authors concluded that moderate evidence exists for an association between the S18Y variant and Parkinson's disease.

HPVs need to avoid immune responses of the host in order to establish persistent infection. HPVs achieve this by dampening innate immunity of keratinocytes, the major cell type targeted by HPV. As there is reduced production of danger signals including antimicrobial molecules, proinflammatory cytokines and chemokines by keratinocytes, HPV infection remains undetected by the immune system. However, our further data showed that PRR signaling is not completely blocked by hrHPV. Thus, the activation of innate and adaptive immunity at the site of HPV infection is slowed down but not prevented. In order for cancers to grow out they need to suppress the local effector cells. We focused on the role of the PD-1 receptor and its ligands PD-L1 and PD-L2, our data showed that the majority (81%) of the tumors from cervical cancer patients do not express PD-L1. Furthermore, PD-L1 expression was not associated with patient survival. Finally, we presented evidence that the chemokine receptor CXCR7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients.

AIM: Levels of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) are robustly increased in spinal muscular atrophy (SMA) patient fibroblasts and mouse models. We therefore wanted to establish whether changes in UCHL1 contribute directly to disease pathogenesis, and to assess whether pharmacological inhibition of UCHL1 represents a viable therapeutic option for SMA.METHODS: SMA mice and control littermates received a pharmacological UCHL1 inhibitor (LDN-57444) or DMSO vehicle. Survival and weight were monitored daily, a righting test of motor performance was performed, and motor neuron loss, muscle fibre atrophy and neuromuscular junction pathology were all quantified. Ubiquitin-like modifier activating enzyme 1 (Uba1) was then pharmacologically inhibited in neurons in vitro to examine the relationship between Uba1 levels and UCHL1 in SMA.RESULTS: Pharmacological inhibition of UCHL1 failed to improve survival, motor symptoms, or neuromuscular pathology in SMA mice and actually precipitated the onset of weight loss. LDN-57444 treatment significantly decreased spinal cord mono-ubiquitin levels, further exacerbating ubiquitination defects in SMA mice. Pharmacological inhibition of Uba1, levels of which are robustly reduced in SMA, was sufficient to induce accumulation of UCHL1 in primary neuronal cultures.CONCLUSION: Pharmacological inhibition of UCHL1 exacerbates rather than ameliorates disease symptoms in a mouse model of SMA. Thus, pharmacological inhibition of UCHL1 is not a viable therapeutic target for SMA. Moreover, increased levels of UCHL1 in SMA likely represent a downstream consequence of decreased Uba1 levels, indicative of an attempted supportive compensatory response to defects in ubiquitin homeostasis caused by low levels of SMN protein.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist., Author Summary A persistent infection with high-risk human papillomavirus (hrHPV) may cause cancer. Whereas keratinocytes – the cells infected by hrHPV – are equipped with different receptors allowing them to recognize invading pathogens and to activate the immune system, hrHPV has developed ways to evade the host's immune response for sustained periods of time. We showed that hrHPV accomplishes this by interfering with the signaling of the pathogen receptors, thereby hampering the production of cytokines that are known to attract and activate the immune system. HrHPV accomplishes this by upregulating the expression of a cellular protein called ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). This protein suppresses the activation of signals downstream of the pathogen receptor leading to reduced transcription factor activation and downstream gene expression, in particular that of type I interferon and pro-inflammatory cytokines. This lowers the attraction of immune cells and thereby the chance of hrHPV-infected cells to be recognized and eliminated and as such enables hrHPV to persist.

Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...

Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...

The human UCHL1 gene encodes the ubiquitin C-terminal hydrolase UCHL1, which comprises more than 2% of total brain protein. UCHL1 is a component of the ubiquitin–proteasome system, which degrades overexpressed and damaged proteins. Mutations in the UCHL1 gene are associated with susceptibility to and protection from Parkinson’s disease. Here we report cloning, characterization, expression analysis and mapping of porcine UCHL1. The UCHL1 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine cDNA codes for a protein of 223 amino acids which shows a very high similarity to human (98%) and to mouse (97%) UCHL1. In addition, the genomic organization of the porcine UCHL1 gene was determined. The porcine UCHL1 gene was mapped to chromosome 8(½p21)–p23. Three SNPs were found in the porcine UCHL1 sequence. Expression analysis by quantitative real time RT-PCR demonstrated that porcine UCHL1 mRNA is differentially expressed in various organs and tissues and similar to its human counterpart. UCHL1 transcript is most abundant in brain tissues and in the spinal cord. The UCHL1 mRNA expression was also investigated in developing porcine embryos. UCHL1 transcript was detected as early as 40 days of gestation. A significant decrease in UCHL1 transcript was detected in basal ganglia from day 60 to day 115 of gestation.