Ubiquitin Carboxyl-terminal Hydrolase Isozyme L1 Research Articles

Data-independent acquisition proteomics of cerebrospinal fluid implicates endoplasmic reticulum and inflammatory mechanisms in amyotrophic lateral sclerosis.

While unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of amyotrophic lateral sclerosis (ALS), high-abundance proteins mask the presence of lower abundance proteins that may have diagnostic and prognostic value. However, developments in mass spectrometry (MS) proteomic data acquisition methods offer improved protein depth. In this study, MS with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of people with ALS (n = 40), healthy (n = 15) and disease (n = 8) controls. Quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins, all significantly upregulated in ALS versus healthy controls, and 9 with altered abundance in ALS versus disease controls (FDR < 0.1). Elevated chitotriosidase-1 (CHIT1) was common to both comparisons and was proportional to ALS disability progression rate (Pearson r = 0.41, FDR-adjusted p = 0.035) but not overall survival. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS versus healthy controls) was proportional to disability progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and survival (Kaplan Meier log-rank p = 0.013) but not independently in multivariate proportional hazards models. Weighted correlation network analysis was used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was associated with age at symptom onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio = 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, was negatively correlated with disability progression rate (r = -0.42, FDR-adjusted p = 0.109). DIA acquisition methodology therefore strengthened the biomarker candidacy of CHIT1 and UCHL1 in ALS, while additionally highlighted inflammatory and endoplasmic reticulum proteins as novel sources of prognostic biomarkers.

Neurobiomarker and body temperature responses to recreational marathon running

ObjectivesTo assess how biomarkers indicating central nervous system insult (neurobiomarkers) vary in peripheral blood with exertional-heat stress from prolonged endurance exercise. DesignObservational study of changes in neuron specific enolase (NSE), S100 calcium-binding protein B (S100β), Glial Fibrillary Acid Protein (GFAP) and Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) at Brighton Marathon 2022. MethodsIn 38 marathoners with in-race core temperature (Tc) monitoring, exposure (High, Intermediate or Low) was classified by cumulative hyperthermia – calculated as area under curve of Time × Tc > 38 °C – and also by running duration (finishing time). Blood was sampled for neurobiomarkers, cortisol and fluid-regulatory stress surrogates, including copeptin and creatinine (at rested baseline; within 30 min of finishing; and at 24 h). ResultsFinishing in 236 ± 40 min, runners showed stable GFAP and UCH-L1 across the marathon and next-day. Significant (P < 0.05) increases from baseline were shown post-marathon and at 24 h for S100β (8.52 [3.65, 22.95] vs 39.0 [26.48, 52.33] vs 80.3 [49.1, 99.7] ng·L−1) and post-marathon only for NSE (3.73 [3.30, 4.32] vs 4.85 [4.45, 5.80] μg·L−1, P < 0.0001). Whilst differential response to hyperthermia was observed for cortisol, copeptin and creatinine, neurobiomarker responses did not vary. Post-marathon, only NSE differed by exercise duration (High vs Low, 5.81 ± 1.77 vs. 4.69 ± 0.73 μg·L−1, adjusted P = 0.0358). ConclusionsSuccessful marathon performance did not associate with evidence for substantial neuronal insult. To account for variation in neurobiomarkers with prolonged endurance exercise, factors additional to hyperthermia, such as exercise duration and intensity, should be further investigated.

Use of iTRAQ-based quantitative proteomic identification of CHGA and UCHL1 correlated with lymph node metastasis in colorectal carcinoma.

Metastatic dissemination of colorectal cancer (CRC), the third most common cancer type, is responsible for CRC deaths. Understanding the transition of lymph node metastasis (LNM) from Stage II to Stage III is beneficial in the prognosis and intervention of CRC. In this study, a quantitative proteomic survey was conducted to investigate the LNM-associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC. By using the LC-MS/MS iTRAQ technology, we analysed the proteomic changes between LMN II and LMN III. Fresh tumours from the CRC specimens consisting of 12 node-negative (Stage II) and 12 node-positive (Stage III) cases were analysed by LC-MS/MS iTRAQ proteome analysis. Subsequently, tissue microarray with immunohistochemistry staining was conducted to access the clinicopathological characteristics of these proteins in 116 paraffin-embedded CRC samples, each for non-LNM and LNM CRC. To study the effects of the differentially expressed proteins on the potential mechanism, Boyden chamber assay, flow cytometry and shRNA-based assessments were conducted to examine the role of the epithelial-mesenchymal transition (EMT) and the invasiveness of CRC cells and others in vivo xenograft mouse model experiments. Forty-eight proteins were found differentially expressed between non-LNM and LNM CRC tissues. Protein abundances of chromogranin-A (CHGA) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) were observed in node-positive CRC (p < 0.05). Knockdown of CHGA and UCHL1 significantly regulate cancer behaviours of HCT-116, including inhibition of cell migration, invasiveness, cell cycle G1/S arrest and reactive oxygen species (ROS) generation. Mechanistically, the CHGA and UCHL1 inactivation displayed decreased levels of UCH-L1, chromogranin A, β-catenin, cyclin E, twist-1/2, vimentin, MMP-9, N-cadherin and PCNA through the activation of the Rho-GTPase/AKT/NFκB pathways. Histone modification of H3K4 trimethylation of CHGA and UCHL1 promoter were increased to activate their transcription through the signalling transduction such as Rho-GTPase, AKT and NFκB pathways. Our results indicated that UCHL1 and chromogranin A are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as biomarkers for CRC diagnosis at the metastatic stage.

Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant public health burden with limited treatment options. Many β-coronaviruses, including SARS-CoV-2, gain entry to host cells through the interaction of SARS-CoV-2 spike protein with membrane-bound ACE2 (angiotensin-converting enzyme 2). Given its necessity for SARS-CoV-2 infection, ACE2 represents a potential therapeutic target in COVID-19. However, early attempts focusing on ACE2 in COVID-19 have not validated it as a druggable target nor identified other ACE2-related novel proteins for therapeutic intervention. Here, we identify a mechanism for ACE2 protein modulation by the deubiquitinase (DUB) enzyme UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1). ACE2 is constitutively ubiquitinated and degraded by the proteasome in lung epithelia. SARS-CoV-2 spike protein cellular internalization increased ACE2 protein abundance by decreasing its degradation. Using an siRNA library targeting 96 human DUBs, we identified UCHL1 as a putative regulator of ACE2 function as a viral receptor. Overexpressed UCHL1 preserved ACE2 protein abundance, whereas silencing of the DUB in cells destabilized ACE2 through increased polyubiquitination. A commercially available small molecule inhibitor of UCHL1 DUB activity decreased ACE2 protein concentrations coupled with inhibition of SARS-CoV-2 infection in epithelial cells. These findings describe a unique pathway of ACE2 regulation uncovering UCHL1 as a potential therapeutic target to modulate COVID-19 viral entry as a platform for future small molecule design and testing.

Raw Inonotus obliquus polysaccharide counteracts Alzheimer's disease in a transgenic mouse model by activating the ubiquitin-proteosome system.

Inonotus obliquus has been used as antidiabetic herb around the world, especially in the Russian and Scandinavian countries. Diabetes is widely believed to be a key factor in Alzheimer's disease (AD), which is widely considered to be type III diabetes. To investigate whether I. obliquus can also ameliorate AD, it would be interesting to identify new clues for AD treatment. We tested the anti-AD effects of raw Inonotus obliquus polysaccharide (IOP) in a mouse model of AD (3×Tg-AD transgenic mice). SPF-grade 3×Tg-AD mice were randomly divided into three groups (Control, Metformin, and raw IOP groups, n = 5 per group). β-Amyloid deposition in the brain was analyzed using immunohistochemistry for AD characterization. Gene and protein expression of pertinent factors of the ubiquitin-proteasome system (UPS) was determined using real-time quantitative polymerase chain reaction and Western blotting. Raw IOP significantly reduced the accumulation of amyloid aggregates and facilitated UPS activity, resulting in a significant reduction in AD-related symptoms in an AD mouse model. The presence of raw IOP significantly enhanced the expression of ubiquitin, E1, and Parkin (E3) at both the mRNA and protein levels in the mouse hippocampus. The mRNA level of ubiquitin carboxyl-terminal hydrolase isozyme L1, a key factor involved in UPS activation, also increased by approximately 50%. Raw IOP could contribute to AD amelioration via the UPS pathway, which could be considered as a new potential strategy for AD treatment, although we could not exclude other mechanisms involved in counteracting AD processing.

Multi-Omics Analysis Revealed a Significant Alteration of Critical Metabolic Pathways Due to Sorafenib-Resistance in Hep3B Cell Lines.

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells’ survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.

Integrated evaluation of a panel of neurochemical biomarkers to optimize diagnosis and prognosis in amyotrophic lateral sclerosis.

Background and purposeThis study was undertaken to determine the diagnostic and prognostic value of a panel of serum biomarkers and to correlate their concentrations with several clinical parameters in a large cohort of patients with amyotrophic lateral sclerosis (ALS).MethodsOne hundred forty‐three consecutive patients with ALS and a control cohort consisting of 70 patients with other neurodegenerative disorders (DEG), 70 patients with ALS mimic disorders (ALSmd), and 45 healthy controls (HC) were included. Serum neurofilament light chain (NfL), ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1), glial fibrillary acidic protein (GFAP), and total tau protein levels were measured using ultrasensitive single molecule array.ResultsNfL correlated with disease progression rate (p < 0.001) and with the measures of upper motor neuron burden (p < 0.001). NfL was higher in the ALS patients with classic and pyramidal phenotype. GFAP was raised in ALS with cognitive–behavioral impairment compared with ALS with normal cognition. NfL displayed the best diagnostic performance in discriminating ALS from HC (area under the curve [AUC] = 0.990), DEG (AUC = 0.946), and ALSmd (AUC = 0.850). UCHL1 performed well in distinguishing ALS from HC (AUC = 0.761), whereas it was not helpful in differentiating ALS from DEG and ALSmd. In multivariate analysis, NfL (p < 0.001) and UCHL1 (p = 0.038) were independent prognostic factors. Survival analysis combining NfL and UCHL1 effectively stratified patients with lower NfL levels (p < 0.001).ConclusionsNfL is a useful biomarker for the diagnosis of ALS and the strongest predictor of survival. UCHL1 is an independent prognostic factor helpful in stratifying survival in patients with low NfL levels, likely to have slowly progressive disease. GFAP reflects extramotor involvement, namely cognitive impairment or frontotemporal dementia.

Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia.

Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.

Effects of intraoperative lung-protective ventilation on clinical outcomes in patients with traumatic brain injury: a randomized controlled trial

BackgroundSecondary lung injury is the most common non-neurological complication after traumatic brain injury (TBI). Lung-protective ventilation (LPV) has been proven to improve perioperative oxygenation and lung compliance in some critical patients. This study aimed to investigate whether intraoperative LPV could improve respiratory function and prevent postoperative complications in emergency TBI patients.MethodsNinety TBI patients were randomly allocated to three groups (1:1:1): Group A, conventional mechanical ventilation [tidal volume (VT) 10 mL/kg only]; Group B, small VT (8 mL/kg) + positive end-expiratory pressure (PEEP) (5 cmH2O); and Group C, small VT (8 mL/kg) + PEEP (5 cmH2O) + recruitment maneuvers (RMs). The primary outcome was the incidence of total postoperative pulmonary complications; Secondary outcomes were intraoperative respiratory mechanics parameters and serum levels of brain injury markers, and the incidence of each postoperative pulmonary and neurological complication.ResultsSeventy-nine patients completed the final analysis. The intraoperative PaO2 and dynamic pulmonary compliance of Groups B and C were higher than those of Group A (P = 0.028; P = 0.005), while their airway peak pressure and plateau pressure were lower than those of group A (P = 0.004; P = 0.005). Compared to Group A, Groups B and C had decreased 30-day postoperative incidences of total pulmonary complications, hypoxemia, pulmonary infection, and atelectasis (84.0 % vs. 57.1 % vs. 53.8 %, P = 0.047; 52.0 % vs. 14.3 % vs. 19.2 %, P = 0.005; 84.0 % vs. 50.0 % vs. 42.3 %, P = 0.006; 24.0 % vs. 3.6 % vs. 0.0 %, P = 0.004). Moreover, intraoperative hypotension was more frequent in Group C than in Groups A and B (P = 0.007). At the end of surgery, the serum levels of glial fibrillary acidic protein and ubiquitin carboxyl-terminal hydrolase isozyme L1 in Group B were lower than those in Groups A and C (P = 0.002; P < 0.001). The postoperative incidences of neurological complications among the three groups were comparable.ConclusionsContinuous intraoperative administration of small VT + PEEP is beneficial to TBI patients. Additional RMs can be performed with caution to prevent disturbances in the stability of cerebral hemodynamics.Trial registrationChinese Clinical Trial Registry (ChiCTR2000038314), retrospectively registered on September 17, 2020.

Angiotensin-(1-7) Central Mechanisms After ICV Infusion in Hypertensive Transgenic (mRen2)27 Rats.

Previous data showed hypertensive rats subjected to chronic intracerebroventricular (ICV) infusion of angiotensin-(1-7) presented attenuation of arterial hypertension, improvement of baroreflex sensitivity, restoration of cardiac autonomic balance and a shift of cardiac renin-angiotensin system (RAS) balance toward Ang-(1-7)/Mas receptor. In the present study, we investigated putative central mechanisms related to the antihypertensive effect induced by ICV Ang-(1-7), including inflammatory mediators and the expression/activity of the RAS components in hypertensive rats. Furthermore, we performed a proteomic analysis to evaluate differentially regulated proteins in the hypothalamus of these animals. For this, Sprague Dawley (SD) and transgenic (mRen2)27 hypertensive rats (TG) were subjected to 14 days of ICV infusion with Ang-(1-7) (200 ng/h) or 0.9% sterile saline (0.5 μl/h) through osmotic mini-pumps. We observed that Ang-(1-7) treatment modulated inflammatory cytokines by decreasing TNF-α levels while increasing the anti-inflammatory IL-10. Moreover, we showed a reduction in ACE activity and gene expression of AT1 receptor and iNOS. Finally, our proteomic evaluation suggested an anti-inflammatory mechanism of Ang-(1-7) toward the ROS modulators Uchl1 and Prdx1.

Ubiquitin carboxyl-terminal hydrolase isozyme L1/UCHL1 suppresses epithelial-mesenchymal transition and is under-expressed in cadmium-transformed human bronchial epithelial cells.

Cadmium (Cd), a highly toxic heavy metal, is widespreadly distributed in the environment. Chronic exposure to Cd is associated with the development of several diseases including cancers. Over the decade, many researches have been carried on various models to examine the acute effects of Cd; yet, limited knowledge is known about the long-term Cd exposure, especially in the human lung cells. Previously, we showed that chronic Cd-exposed human bronchial epithelial BEAS-2B cells exhibited transformed cell properties, such as anchorage-independent growth, augmented cell migration, and epithelial-mesenchymal transition (EMT). To study these Cd-transformed cells more comprehensively, here, we further characterized their subproteomes. Overall, a total of 63 differentially expressed proteins between Cd-transformed and passage-matched control cells among the five subcellular fractions (cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal) were identified by mass spectrometric analysis and database searching. Interestingly, we found that the thiol protease ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is one of the severely downregulated proteins in the Cd-transformed cells. Notably, the EMT phenotype of Cd-transformed cells can be suppressed by forced ectopic expression of UCHL1, suggesting UCHL1 as a crucial modulator in the maintenance of the proper differentiation status in lung epithelial cells. Since EMT is considered as a critical step during malignant cell transformation, finding novel cellular targets that can antagonize this transition may lead to more efficient strategies to inhibit cancer development. Our data report for the first time that UCHL1 may play a function in the suppression of EMT in Cd-transformed human lung epithelial cells, indicating that UCHL1 might be a new therapeutic target for chronic Cd-induced carcinogenesis. Graphical abstract.

Different CSF protein profiles in amyotrophic lateral sclerosis and frontotemporal dementia with C9orf72 hexanucleotide repeat expansion

ObjectivesThe hexanucleotide repeat expansion in the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (C9-ALS) and frontotemporal dementia (C9-FTD). Until now, it is unknown which factors...

Intraoperative Oxidative Damage and Delirium after Cardiac Surgery.

Mechanisms of postoperative delirium remain poorly understood, limiting development of effective treatments. We tested the hypothesis that intraoperative oxidative damage is associated with delirium and neuronal injury and that disruption of the blood-brain barrier modifies these associations. In a prespecified cohort study of 400 cardiac surgery patients enrolled in a clinical trial of atorvastatin to reduce kidney injury and delirium, we measured plasma concentrations of F2-isoprostanes and isofurans using gas chromatography-mass spectrometry to quantify oxidative damage, ubiquitin carboxyl-terminal hydrolase isozyme L1 to quantify neuronal injury, and S100 calcium-binding protein B using enzyme-linked immunosorbent assays to quantify blood-brain barrier disruption before, during, and after surgery. We performed the Confusion Assessment Method for the Intensive Care Unit twice daily to diagnose delirium. We measured the independent associations between intraoperative F2-isoprostanes and isofurans and delirium (primary outcome) and postoperative ubiquitin carboxyl-terminal hydrolase isozyme L1 (secondary outcome), and we assessed if S100 calcium-binding protein B modified these associations. Delirium occurred in 109 of 400 (27.3%) patients for a median (10th, 90th percentile) of 1.0 (0.5, 3.0) days. In the total cohort, plasma ubiquitin carboxyl-terminal hydrolase isozyme L1 concentration was 6.3 ng/ml (2.7, 14.9) at baseline and 12.4 ng/ml (7.9, 31.2) on postoperative day 1. F2-isoprostanes and isofurans increased throughout surgery, and the log-transformed sum of intraoperative F2-isoprostanes and isofurans was independently associated with increased odds of postoperative delirium (odds ratio, 3.70 [95% CI, 1.41 to 9.70]; P = 0.008) and with increased postoperative ubiquitin carboxyl-terminal hydrolase isozyme L1 (ratio of geometric means, 1.42 [1.11 to 1.81]; P = 0.005). The association between increased intraoperative F2-isoprostanes and isofurans and increased postoperative ubiquitin carboxyl-terminal hydrolase isozyme L1 was amplified in patients with elevated S100 calcium-binding protein B (P = 0.049). Intraoperative oxidative damage was associated with increased postoperative delirium and neuronal injury, and the association between oxidative damage and neuronal injury was stronger among patients with increased blood-brain barrier disruption.

Proteomic and ultrastructural analysis of Clara cell and type II alveolar epithelial cell-type lung cancer cells.

BackgroundCurrently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling.MethodsSix human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed.ResultsNCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers.ConclusionsCytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.

Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys.

Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFRα-1) Fα and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFRα1 and PLZF were all positive. After 2.5 months’ culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3–6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.

Downregulated UCHL1 Accelerates Gentamicin-Induced Auditory Cell Death via Autophagy.

The clinical use of aminoglycoside antibiotics is partly limited by their ototoxicity. The pathogenesis of aminoglycoside-induced ototoxicity still remains unknown. Here, RNA-sequencing was conducted to identify differentially expressed genes in rat cochlear organotypic cultures treated with gentamicin (GM), and 232 and 43 genes were commonly up- and downregulated, respectively, at day 1 and 2 after exposure. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (Uchl1) was one of the downregulated genes whose expression was prominent in spiral ganglion cells (SGCs), lateral walls, as well as efferent nerve terminal and nerve fibers. We further investigated if a deficit of Uchl1 in organotypic cochlea and the House Ear Institute-Organ of Corti 1 (HEI-OC1) cells accelerates ototoxicity. We found that a deficit in Uchl1 accelerated GM-induced ototoxicity by showing a decreased number of SGCs and nerve fibers in organotypic cochlear cultures and HEI-OC1 cells. Furthermore, Uchl1-depleted HEI-OC1 cells revealed an increased number of autophagosomes accompanied by decreased lysosomal fusion. These data indicate that the downregulation of Uchl1 following GM treatment is deleterious to auditory cell survival, which results from the impaired autophagic flux. Our results provide evidence that UCHL1-dependent autophagic flux may have a potential as an otoprotective target for the treatment of GM-induced auditory cell death.

Fatigue in Sjögren's Syndrome: A Search for Biomarkers and Treatment Targets.

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, where patients often suffer from fatigue. Biological pathways underlying fatigue are unknown. In this study aptamer-based SOMAscan technology is used to identify potential biomarkers and treatment targets for fatigue in pSS.Methods: SOMAscan® Assay 1.3k was performed on serum samples of healthy controls (HCs) and pSS patients characterized for interferon upregulation and fatigue. Differentially expressed proteins (DEPs) between pSS patients and HC or fatigued and non-fatigued pSS patients were validated and discriminatory capacity of markers was tested using independent technology.Results: Serum concentrations of over 1,300 proteins were compared between 63 pSS patients and 20 HCs resulting in 58 upregulated and 46 downregulated proteins. Additionally, serum concentrations of 30 interferon positive (IFNpos) and 30 interferon negative (IFNneg) pSS patients were compared resulting in 25 upregulated and 13 downregulated proteins. ELISAs were performed for several DEPs between pSS patients and HCs or IFNpos and IFNneg all showing a good correlation between protein levels measured by ELISA and relative fluorescence units (RFU) measured by the SOMAscan. Comparing 22 fatigued and 23 non-fatigued pSS patients, 16 serum proteins were differentially expressed, of which 14 were upregulated and 2 were downregulated. Top upregulated DEPs included neuroactive synaptosomal-associated protein 25 (SNAP-25), alpha-enolase (ENO1) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Furthermore, the proinflammatory mediator IL36a and several complement factors were upregulated in fatigued compared to non-fatigued pSS patients. ROC analysis indicated that DEPs showed good capacity to discriminate fatigued and non-fatigued pSS patients.Conclusion: In this study we validated the use of aptamer-based proteomics and identified a novel set of proteins which were able to distinguish fatigued from non-fatigued pSS patients and identified a so-called “fatigue signature.”

Proteomic Profile of Carbonylated Proteins Screen Regulation of Apoptosis via CaMK Signaling in Response to Regular Aerobic Exercise.

To research carbonylated proteins and screen molecular targets in the rat striatum on regular aerobic exercise, male Sprague-Dawley rats (13 months old, n = 24) were randomly divided into middle-aged sedentary control (M-SED) and aerobic exercise (M-EX) groups (n = 12 each). Maximum oxygen consumption (VO2max) gradually increased from 50%–55% to 65%–70% for a total of 10 weeks. A total of 36 carbonylated proteins with modified oxidative sites were identified by Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometer (ESI-Q-TOF-MS), including 17 carbonylated proteins unique to the M-SED group, calcium/calmodulin-dependent protein kinase type II subunit beta (CaMKIIβ), and heterogeneous nuclear ribonucleoprotein A2/B1 (Hnrnpa2b1), among others, and 19 specific to the M-EX group, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), and malic enzyme, among others. Regular aerobic exercise improved behavioral and stereological indicators, promoted normal apoptosis (P < 0.01), alleviated carbonylation of the CaMKIIβ and Hnrnpa2b1, but induced carbonylation of the UCH-L1, and significantly upregulated the expression levels of CaMKIIβ, CaMKIIα, and Vdac1 (p < 0.01) and Hnrnpa2b1 and UCH-L1 (p < 0.01), as well as the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways (PI3K/Akt/mTOR) pathway-related genes Akt and mTOR. Regular aerobic exercise for 10 weeks (incremental for the first 6 weeks followed by constant loading for 4 weeks) enhanced carbonylation of CaMKIIβ, Hnrnpa2b1, and modulated apoptosis via activation of CaMK and phosphoinositide 3-kinase/protein kinase B/mTOR signaling. It also promoted normal apoptosis in the rat striatum, which may have protective effects in neurons.

Proteomic Analysis After Status Epilepticus Identifies UCHL1 as Protective Against Hippocampal Injury.

Brief, non-harmful seizures (preconditioning) can temporarily protect the brain against prolonged, otherwise injurious seizures. Following focal-onset status epilepticus (SE) in preconditioned (tolerance) and sham-preconditioned (injury) mice, we screened for protein changes using a proteomic approach and identified several putative candidates of epileptic tolerance. Among SE-induced changes to both proteomic screens, proteins clustered in key regulatory pathways, including protein trafficking and cytoskeletal regulation. Downregulation of one such protein, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), was unique to injury and not evident in tolerance. UCHL1 inhibition decreased hippocampal ubiquitin, disrupted UPS function, interfered with seizure termination and exacerbated seizure-induced cell death. Though UCHL1 transcription was maintained after SE, we observed downregulation of the pro-translational antisense Uchl1 (AsUchl1) and confirmed that both AsUchl1 and rapamycin can increase UCHL1 expression in vivo. These data indicate that the post-transcriptional loss of UCHL1 following SE is deleterious to neuronal survival and may contribute to hyperexcitability, and are suggestive of a novel modality of rapamycin therapy.

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy.

BackgroundX-linked muscular dystrophy is a primary disease of the neuromuscular system. Primary abnormalities in the Dmd gene result in the absence of the full-length isoform of the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting and cardio-respiratory complications, developmental cognitive deficits and behavioural abnormalities are clinical features of Duchenne muscular dystrophy. In order to better understand the mechanisms that underlie impaired brain functions in Duchenne patients, we have carried out a proteomic analysis of total brain extracts from the mdx-4cv mouse model of dystrophinopathy.ResultsThe comparative proteomic profiling of the mdx-4cv brain revealed a significant increase in 39 proteins and a decrease in 7 proteins. Interesting brain tissue-associated proteins with an increased concentration in the mdx-4cv animal model were represented by the glial fibrillary acidic protein GFAP, the neuronal Ca2+-binding protein calretinin, annexin AnxA5, vimentin, the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97. Decreased proteins were identified as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1, as well as the plasma membrane Ca2+-transporting ATPase PMCA2 that is mostly found in the brain cortex. The differential expression patterns of GFAP, vimentin, PMCA2 and AnxA5 were confirmed by immunoblotting. Increased GFAP levels were also verified by immunofluorescence microscopy.ConclusionsThe large number of mass spectrometrically identified proteins with an altered abundance suggests complex changes in the mdx-4cv brain proteome. Increased levels of the glial fibrillary acidic protein, an intermediate filament component that is uniquely associated with astrocytes in the central nervous system, imply neurodegeneration-associated astrogliosis. The up-regulation of annexin and vimentin probably represent compensatory mechanisms involved in membrane repair and cytoskeletal stabilization in the absence of brain dystrophin. Differential alterations in the Ca2+-binding protein calretinin and the Ca2+-pumping protein PMCA2 suggest altered Ca2+-handling mechanisms in the Dp427-deficient brain. In addition, the proteomic findings demonstrated metabolic adaptations and functional changes in the central nervous system from the dystrophic phenotype. Candidate proteins can now be evaluated for their suitability as proteomic biomarkers and their potential in predictive, diagnostic, prognostic and/or therapy-monitoring approaches to treat brain abnormalities in dystrophinopathies.