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UbiquitinC-terminal hydrolase-L1 (UCH-L1) is a cysteine hydrolase. It functions as a ubiquitin hydrolase, stabilizes the ubiquitin monomer, and affects cell division through cell cycle protein deubiquitination. Abnormal UCH-L1 expression is closely related to the occurrence and development of several tumors. Although some in vitro studies have demonstrated the significance of UCH-L1 in non-small-cell lung cancer (NSCLC), only few clinical studies have focused on the UCH-L1 expression in NSCLC, and the results are controversial and non-uniform. We investigated the UCH-L1 expression in 401 cases of surgically resected NSCLC, including 286 cases of adenocarcinoma (ADC) and 65 cases of squamous cell carcinoma. The associations between the UCH-L1 expression and clinicopathological features, programmed cell death-ligand 1 (PD-L1) expression, and prognostic significance were analyzed. For NSCLC, the UCH-L1 expression is associated with sex, smoking history, tumor size (>3 cm), lymphocyte infiltration, advanced pathological stages, and shortened overall survival (OS; 89.72 vs. 114.55 months; P = 0.005), but not PD-L1 expression. The UCH-L1 expression in ADC is associated with advanced pathological stages, pleural invasion, and shortened OS (90.38 vs. 118.55 months; P = 0.010). Multivariate analysis confirmed that UCH-L1 expression was an independent poor prognostic factor for NSCLC (OS: hazard ratio [HR], 1.854; 95% confidence interval [CI], 1.132-3.038; P = 0.014). Our results suggest that the UCH-L1 expression differs across tumors with different clinicopathological features, and it is related to poor prognosis.

Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.

We prospectively evaluated serum concentrations of glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), total tau (T-Tau), and neurofilament light (NF-L) from collegiate athletes at baseline and acutely after sport-related concussion (SRC) using the Quanterix Neurology 4Plex "B" (N4PB) multiplex assay. Uninjured controls were matched on age, sex, race, sport, and concussion history. Clinical outcomes included acute symptom severity, balance, rapid automated naming, computerized cognitive testing, and recovery duration. Baseline (

Abnormal cardiac fibrosis indicates cardiac dysfunction and poor prognosis in myocardial infarction (MI) patients. Many studies have demonstrated that the ubiquitin proteasome system (UPS) plays a significant role in the pathogenesis of fibrosis. Ubiquitin C-terminal hydrolase L1 (UCHL1), a member of the UPS, is related to fibrosis in several heart diseases. However, whether UCHL1 regulates cardiac fibrosis following MI has yet to be determined. In the present study, we found that UCHL1 was dramatically increased in infarct hearts and TGF-β1-stimulated cardiac fibroblasts (CFs). Inhibition of UCHL1 with LDN57444 (LDN) reversed the myocardial fibrosis in post-MI heart and improved cardiac function. Treatment of LDN or UCHL1 siRNA abolished the TGF-β1-induced fibrotic response of CFs. We further identified GRP78 as an interactor of UCHL1 through screening using immunoprecipitation-mass spectrometer. We determined that UCHL1 interacted with glucose-regulated protein of 78 kDa (GRP78) and prompted GRP78 degradation via ubiquitination. Furthermore, we found that GRP78 was upregulated after UCHL1 knockdown and that the GRP78 inhibitor HA15 diminished the antifibrotic function exerted by UCHL1 knockdown in CFs stimulated with TGF-β1. This suggests that UCHL1 regulates cardiac fibrosis post MI through interactions with GRP78. This work identifies that the UCHL1-GRP78 axis is involved in cardiac fibrosis after MI.

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

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein–protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.

Rapid risk-stratification of patients with acute traumatic brain injury (TBI) would inform management decisions and prognostication. The objective of this serum biomarker study (Biomarkers of Injury and Outcome [BIO]-Progesterone for Traumatic Brain Injury, Experimental Clinical Treatment [ProTECT]) was to test the hypothesis that serum biomarkers of structural brain injury, measured at a single, very early time-point, add value beyond relevant clinical covariates when predicting unfavorable outcome 6 months after moderate-to-severe acute TBI. BIO-ProTECT utilized prospectively collected samples obtained from subjects with moderate-to-severe TBI enrolled in the ProTECT III clinical trial of progesterone. Serum samples were obtained within 4 h after injury. Glial fibrillary acidic protein (GFAP), S100B, αII-spectrin breakdown product of molecular weight 150 (SBDP150), and ubiquitin C-terminal hydrolase-L1 (UCH-L1) were measured. The association between log-transformed biomarker levels and poor outcome, defined by a Glasgow Outcome Scale-Extended (GOS-E) score of 1–4 at 6 months post-injury, were estimated via logistic regression. Prognostic models and a biomarker risk score were developed using bootstrapping techniques. Of 882 ProTECT III subjects, samples were available for 566. Each biomarker was associated with 6-month GOS-E (p

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1–deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1–deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1–deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1–deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1–dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multi-state conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to the protein yeast ubiquitin hydrolase 1 (YUH1), we find large dynamics of its N-terminus and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus gets into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.

A major obstacle for translational research in acute spinal cord injury (SCI) is the lack of biomarkers that can objectively stratify injury severity and predict outcome. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a neuron-specific enzyme that shows promise as a diagnostic biomarker in traumatic brain injury (TBI), but has not been studied in SCI. In this study, cerebrospinal fluid (CSF) and serum samples were collected over the first 72-96 h post-injury from 32 acute SCI patients who were followed prospectively to determine neurological outcomes at 6 months post-injury. UCH-L1 concentration was measured using the Quanterix Simoa platform (Quanterix, Billerica, MA) and correlated to injury severity, time, and neurological recovery. We found that CSF UCH-L1 was significantly elevated by 10- to 100-fold over laminectomy controls in an injury severity- and time-dependent manner. Twenty-four-hour post-injury CSF UCH-L1 concentrations distinguished between American Spinal Injury Association Impairment Scale (AIS) A and AIS B, and AIS A and AIS C patients in the acute setting, and predicted who would remain "motor complete" (AIS A/B) at 6 months with a sensitivity of 100% and a specificity of 86%. AIS A patients who did not improve their AIS grade at 6 months post-injury were characterized by sustained elevations in CSF UCH-L1 up to 96 h. Similarly, the failure to gain8 points on the total motor score at 6 months post-injury was associated with higher 24-h CSF UCH-L1. Unfortunately, serum UCH-L1 levels were not informative about injury severity or outcome. In conclusion, CSF UCH-L1 in acute SCI shows promise as a biomarker to reflect injury severity and predict outcome.

Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.

Redox-dependent inactivation of deubiquitinases (DUBs) is a critical factor for attenuating their DUB activity in response to cellular oxidative stress. Ubiquitin C-terminal hydrolase isoform (UCH-L1) is an important DUB that is highly expressed in human neuronal cells and is implicated in a myriad of human diseases such as neurodegenerative diseases and cancer. Increasing evidence suggests an important role of UCH-L1 in redox regulation and the protection of neuronal cells from oxidative stress. In this study, we examined the molecular basis of how UCH-L1 responds to oxidation in a reversible manner. Using H2O2 as a model oxidant, we showed by mass spectrometry that a subset of methionine and cysteine residues, namely (M1, M6, M12, C90, and C152) were more susceptible to oxidation. Spectroscopic analysis showed that oxidation of C90 can lead to profound structural changes in addition to the loss of function. Importantly, we further demonstrated that C152, which is located at the substrate recognition cross-over loop, serves as a reactive oxygen species (ROS) scavenger to protect catalytic C90 from oxidation under moderate oxidative conditions. Hydrogen-deuterium exchange mass spectrometry analysis provided detailed structural mapping of the destabilizing effect of H2O2-mediated oxidation, which resulted in global destabilization far beyond the oxidation sites. These perturbations may be responsible for irreversible aggregation when subject to prolonged oxidative stress.

Ubiquitin C-terminal hydrolase L1 (UCHL-1) and glial fibrillary acidic protein (GFAP) are two blood-based biomarkers which have been investigated as a combined assay to predict traumatic intracranial injuries on head CT. Ubiquitin is found in nearly all cells of the body and is a regulatory protein, whereas GFAP is a member of the intermediate filament family of cytoskeletal proteins that provide a structural support to the neuroglia. The ALERT-traumatic brain injury study investigated GFAP/UCHL1 and found a sensitivity of 0.973 for prediction of acute intracranial injury on CT for all patients suffering a minor traumatic brain injury (GCS14–15) and a specificity of 0.006 (1) for identifying neurosurgically manageable lesions. These biomarkers could be useful in ruling out intracranial hemorrhages but further research will be needed to look at the impact on current clinical care models.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Many reagents have been emerged to study the function of specific enzymesin vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinson’s and Alzheimer’s disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 bothin vitroand in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

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

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), which catalyzes the hydrolysis of ubiquitin esters and amides, is highly expressed in brain. Recently, UCH-L1 has been found to increase cancer cell migration and invasion by modulating hydrogen peroxide generated by NADPH oxidase 4 (NOX4). Because angiogenesis is also mediated by hydrogen peroxide, we explored the role of UCH-L1 in angiogenesis in human umbilical vein endothelial cells (HUVECs). Silencing UCH-L1 suppressed tubule formation in HUVECs, indicating that UCH-L1 promotes angiogenesis in vitro. This was confirmed using in vivo Matrigel plug studies of HUVECs, after overexpressing or silencing UCH-L1. Silencing UCH-L1 significantly suppressed VEGF-induced ROS levels as well as activation of VEGFR, both of which are required for angiogenesis. This study also showed that UCH-L1 promotes angiogenesis of HUVECs, as well as invasion in cancer cells, by up-regulating ROS by deubiquitination of NOX4, suggesting that UCH-L1 plays a key role in angiogenesis of HUVECS by regulating ROS levels by deubiquitination of NOX4.

Glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) have been studied as potential biomarkers of mild traumatic brain injury (mTBI). We report the levels of GFAP and UCH-L1 in patients with acute orthopedic injuries without central nervous system involvement, and relate them to the type of extracranial injury, head magnetic resonance imaging (MRI) findings, and levels of GFAP and UCH-L1 in patients with CT-negative mTBI. Serum UCH-L1 and GFAP were longitudinally measured from 73 patients with acute orthopedic injury on arrival and on days 1, 2, 3, 7 after admission, and on the follow-up visit 3-10 months after the injury. The injury types were recorded, and 71% patients underwent also head MRI. The results were compared with those found in patients with CT-negative mTBI (n = 93). The levels of GFAP were higher in patients with acute orthopedic trauma than in patients with CT-negative mTBI (p = 0.026) on arrival; however, no differences were found on the following days. The levels of UCH-L1 were not significantly different between these two groups at any measured point of time. Levels of GFAP and UCH-L1 were not able to distinguish patients with CT-negative mTBI from patients with orthopedic trauma. Patients with orthopedic trauma and high levels of UCH-L1 or GFAP values may be falsely diagnosed as having a concomitant mTBI, predisposing them to unwarranted diagnostics and unnecessary brain imaging. This casts a significant doubt on the diagnostic value of GFAP and UCH-L1 in cases with mTBI.

Objective Traumatic brain injuries (TBIs) are largely underdiagnosed and may have persistent refractory consequences. Current assessments for acute TBI are limited to physical examination and imaging. Biomarkers such as glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCH-L1), and S100 calcium-binding protein B (S100B) have shown predictive value as indicators of TBI and potential screening tools. Methods In total, 37 controls and 118 unique trauma subjects who received a clinically ordered head computed tomography (CT) in the emergency department of a level 1 trauma center were evaluated. Blood samples collected at 0–8 hours (initial) and 12–32 hours (delayed) postinjury were analyzed for GFAP, UCH-L1, and S100B concentrations. These were then compared in CT-negative and CT-positive subjects. Results Median GFAP, UCH-L1, and S100B concentrations were greater in CT-positive subjects at both timepoints compared with CT-negative subjects. In addition, median UCH-L1 and S100B concentrations were lower at the delayed timepoint, whereas median GFAP concentrations were increased. As predictors of a positive CT of the head, GFAP outperformed UCH-L1 and S100B at both timepoints (initial: 0.89 sensitivity, 0.62 specificity; delayed: 0.94 sensitivity, 0.67 specificity). GFAP alone also outperformed all possible combinations of biomarkers. Conclusions GFAP, UCH-L1, and S100B demonstrated utility for rapid prediction of a CT-positive TBI within 0–8 hours of injury. GFAP exhibited the greatest predictive power at 12–32 hours. Furthermore, these results suggest that GFAP alone has greater utility for predicting a positive CT of the head than UCH-L1, S100B, or any combination of the 3.

We investigated the role of serum Glial Fibrillary Acidic Protein (GFAP) and Ubiquitin C-Terminal Hydrolase-L1 (UCH-L1) in diagnosis of sepsis-associated encephalopathy(SAE), predicting prognosis and long-term quality of life with patients of sepsis.This is a prospective single center study entailed 105 patients whosuffered from sepsis from Jan 2015 to Aug 2016. Serum concentrations of GFAP and UCH-L1 for diagnosis of SAE and predicting prognosis and long-term quality of life with patients of sepsis were analyzed.The serum concentrations of GFAP and UCH-L1 were higher in SAE group than in no-SAE group (p .001). GFAP and UCH-L1 produced an AUC of 0.824 and 0.812 respectively for diagnosis of SAE with optimal cut-off values 0.532 ng/ml and 7.72 ng/ml respectively. The optimal cut-off values of GFAP and UCH-L1 to distinguish patients with survivors from non-survivors were 0.536 ng/ml and 8.06 ng/ml with an area under the curve of 0.773 and 0.746. Patients with a higher GFAP levels had worse long-term usual activities and patients with a higher UCH-L1 levels had more long-term pain (P = .026).Serum concentrations GFAP and UCH-L1 early elevated and associated with sepsis-associated encephalopathy, poor prognosis and quality of life.

INTRODUCTION Traumatic brain injury (TBI) is defined as a neurotrauma caused by a mechanical force applied to a head. An appropriate clinical therapy after TBI is crucial, or patients may suffer lasting effects in physical and mental abilities [1]. To find TBI, cranial computer tomography (CT) is commonly used, however, CT sometimes fails to find mild TBI (mTBI) even it accounts for majority of all cases of TBI. Recently, a blood-based biomarker, ubiquitin C-terminal hydrolase L1 (UCH-L1) has been regarded a promising marker for mTBI, as it becomes present in serum soon after TBI [2,3]. Actually, it was reported the elevated serum UCH-L1 level was observed with the soldiers exposed to low-level blast [4] and the athletes after sub-concussive dead hits [5]. Enzyme-linked immuno sorbent assay (ELISA) is currently used to detect the biomarker, however, since the ELISA is time-consuming procedure and requires specific laboratory equipment, development of a hand-held device to be used on-site in the field is expected. In this study, we propose and report a novel principle for a rapid, one-step electrochemical detection system for UCH-L1 to be dedicated for mTBI diagnosis. METHOD To detect UCH-L1, we chose an electrochemical method in terms of a rapid response time, miniaturization, and simple instrumentation. We employed a competitive assay combined with square wave voltammetry (SWV) on anti-UCH-L1 antibody immobilized electrode. In this system, a redox probe (phenazine ethosulfate: PES) modified UCH-L1 and the target biomarker is going to competitively bind to the antibody on the electrode, resulting electrochemical signal depend on the concentration of UCH-L1 in a sample. To construct the system, PES modified UCH-L1 was prepared using amine-reactive PES. This molecule has N-hydroxysuccinimide linked to PES, therefore, upon mixing amine-reactive PES with UCH-L1, it will react with amine group of lysine residue and make a covalent bond. After the modification, enzyme-linked immuno sorbent assay (ELISA) was performed to investigate the binding affinity of anti-UCH-L1 antibody against UCHH-L1 and PES modified UCH-L1. To detect UCH-L1, we added UCH-L1 together with PES-modified UCH-L1 on the antibody immobilized gold electrode, and performed SWV at a potential range of -0.5 to 0.1 V with different frequencies of square wave. RESUTLS The result of ELISA showed that the antibody had almost same affinity against non-labeled UCH-L1 and PES-modified UCH-L1. Therefore, the modification of PES did not affect the recognition of antibody. When we incubated the electrode with different concentration of PES-modified UCH-L1, a square wave voltammetry signal was increased in a concentration dependent manner, indicating the PES modified on the UCH-L1 was electrochemically active, and the amount of PES-modified UCH-L1 was detected by SWV when it was captured by anti-UCH-L1 antibody on the electrode. In the competitive assay, the incubation of intact UCH-L1 together with PES-modified UCH-L1 resulted in the SWV signal change depend on the UCH-L1 concentration. With this detection principle, we could detect UCH-L1 within 10 minutes without any washing procedure, which is ideal for the on-site detection of mTBI. CONCLUSION In this study, we developed a rapid electrochemical detection system of TBI biomarker, UCH-L1, based on the competitive of redox probe-modified UCH-L1 with the target. Using this detection system, we were able to observe SWV signal change within 10 minutes upon addition of UCH-L1. This study shows the proof-of-concept of this rapid competitive assay, and we hope it would help future development of TBI detection device. REFERENCES [1] S. W. Hoffman and C. Harrison, Clin. Neuropsychol., 23(8), 1400–1415 (2009). [2] R. Diaz-Arrastia, K. K. W. Wang, L. Papa, M.D. Sorani, J. K. Yue, A. M. Puccio, P. J. McMahon, T. Inoue, E. L. Yuh, H. F. Lingsma, A. I. R. Maas, A. B. Valadka, D. O. Okonkwo, G. T. Manley, TRACK-TBI Investigators, J. Neurotrauma, 31(1), 19–25 (2013). [3] L. Papa, G. M. Brophy, R. D. Welch, L. M. Lewis, C. F. Braga, C. N. Tan, N. J. Ameli, M. A. Lopez, C. A. Haeussler, D. I. M. Giordano, S. Silvestri, P. Giordano, K. D. Weber, C. Hill-Pryor, D. C. Hack, JAMA Neurol., 73(5), 551–560 (2016). [4] C. M. Tate, K. K. W. Wang, S. Eonta, Y. Zhang, W. Carr, F. C. Tortella, R. L. Hayes, G. H. Kamimori, J. Neurotrauma, 30(19), 1620–1630 (2013). [5] V. Puvenna, C. Brennan, G. Shaw, C. Yang, N. Marchi, J. J. Bazarian, K. Merchant-Borna, D. Janigro, PLoS ONE, 9(5), e96296–9 (2015).

Deubiquitylating enzymes or DUBs are a class of enzymes that selectively remove the polypeptide posttranslational modification ubiquitin from a number of substrates. Approximately 100 DUBs exist in human cells and are involved in key regulatory cellular processes, which drive many disease states, making them attractive therapeutic targets. Several aspects of DUB biology have been studied through genetic knock-out or knock-down, genomic, or proteomic studies. However, investigation of enzyme activation and regulation requires additional tools to monitor cellular and physiological dynamics. A comparison between genetic ablation and dominant-negative target validation with pharmacological inhibition often leads to striking discrepancies. Activity probes have been used to profile classes of enzymes, including DUBs, and allow functional and dynamic properties to be assigned to individual proteins. The ability to directly monitor DUB activity within a native biological system is essential for understanding the physiological and pathological role of individual DUBs. We will discuss the evolution of DUB activity probes, from in vitro assay development to their use in monitoring DUB activity in cells and in animal tissues, as well as recent progress and prospects for assessing DUB inhibition in vivo.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1–5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology.