Ubiquitin Carboxyl-terminal Hydrolase L1 Research Articles

Curative effect of ganglioside sodium for adjuvant therapy on acute severe craniocerebral injury

Objective: To study the effect of adjuvant therapy of ganglioside sodium on intracranial pressure (ICP), partial pressure of brain tissue oxygen (PbtO2), nerve injury molecules, nerve protection molecules and indexes of oxidative stress in patients with acute severe craniocerebral injury. Methods: Forty-seven patients with severe craniocerebral injury treated in the emergency department of our hospital during the period time from December 2012 to October 2015 were selected for retrospective analyses. They were divided into the ganglioside group and the normal treatment group according to the usage of ganglioside sodium in the process of the emergency treatment. At days 1, 3, 5 and 7 before and after treatment, the ICP and PbtO2 in patients of the two groups were measured. After 7 days of treatment, the nerve injury molecules, nerve protection molecules and the indexes of oxidative stress in serum of the patients of the two groups were determined. Results: At days 1, 3, 5 and 7 before and after treatment, the ICP in patients of the ganglioside group were all significantly lower than those of the normal treatment group, while the PbtO2 were all significantly higher than those of normal treatment group. After 7 days of treatment, the contents of serum methane dicarboxylic aldehyde, advanced oxidation protein products, 8-hydroxy-2'-deoxyguanosine urine, S100β, glial fibrillary acidic portein, neuron specific enolase, myelin basic protein, neuroglobin and ubiquitin carboxyl-terminal hydrolase L1 in patients of the ganglioside group were notably lower than those of the normal treatment group, while the contents of superoxidase dismutase, glutathione peroxidase, catalase, nerve growth factor and brain derived neurotrophic factor were significantly higher than those of the normal treatment group. Conclusions: The adjuvant therapy of ganglioside sodium in patients with severe craniocerebral injury can effectively reduce ICP, improve PbtO2 and alleviate the injuries of neurons and glial cells caused by oxidative stress.

Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5′ UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3′ end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.

Increased Cerebrospinal Fluid Levels of Ubiquitin Carboxyl-Terminal Hydrolase L1 in Patients with Alzheimer's Disease

Background: Dysfunctions of the ubiquitin proteasome system (UPS), including the highly abundant neuronal enzyme ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), and autophagy-related changes (lysosomal degradation) are implicated in several neurodegenerative disorders including Alzheimer's disease (AD). Method: This study evaluated cerebrospinal fluid (CSF) levels of UCH-L1, protein deglycase (DJ-1), neuron-specific enolase (NSE), and tau phosphorylated at threonine 231 (P-tau₂₃₁) in two independent patient and control cohorts. Cohort 1 included CSF samples from subjects having an AD biomarker profile (n = 10) or a control biomarker profile (n = 31), while cohort 2 was a monocenter clinical study including patients with AD (n = 32), mild cognitive impairment (n = 13), other dementias (n = 15), as well as cognitively healthy controls (n = 20). Results: UCH-L1 and P-tau₂₃₁ were elevated in AD patients compared to controls in both cohorts. CSF levels of DJ-1 and NSE were unchanged in the AD group, whereas they were decreased in the group of other dementia compared to controls in the clinical study. Conclusion: Our main findings support that the UPS pathway may be impaired in AD, and UCH-L1 may serve as an additional CSF biomarker for AD.

Are UCH-L1 and GFAP promising biomarkers for children with mild traumatic brain injury?

Objectives: To compare serum biomarker levels between children with mild traumatic brain injury (mTBI) and orthopaedic injury (OI), acutely following injury. Secondarily, to explore the association between biomarker levels and symptom burden over 1 month post-injury.Methods: This was a prospective cohort study of children aged 11–16 years who presented to the emergency department within 6 hours of sustaining mTBI or isolated extremity OI. Serum was drawn at the time of study enrollment and levels of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) and glial fibrillary acid protein (GFAP) were analysed. Symptom burden was assessed by the Post-Concussion Symptom Scale (PCSS) acutely following injury and at three subsequent time points over 1 month.Results: Twenty-five children with mTBI and 20 children with OI were enrolled. The average age for the overall cohort was 13 (± 1.6) years and the majority were male and injured playing sports. GFAP levels and PCSS scores were significantly higher acutely following mTBI vs OI (p < 0.01). There was not a significant group difference in UCH-L1 levels. Neither GFAP nor UCH-L1 were predictive of PCSS scores over the 1month post-injury.Conclusions: GFAP may be a promising diagnostic tool for children with mTBI. Additional approaches are needed to predict symptom severity and persistence.

Overexpression of ubiquitin carboxyl terminal hydrolase-L1 enhances multidrug resistance and invasion/metastasis in breast cancer by activating the MAPK/Erk signaling pathway.

Multidrug resistant (MDR) cancer cells overexpressing P-glycoprotein (P-gp) exhibit enhanced invasive/metastatic ability as compared with the sensitive cells. We aimed to clarify the mechanism underlying this observation and found that during the development of drug resistance to adriamycin in MCF7 cells, the elevated expression of UCH-L1 coincides with the up-regulation of MDR1, CD147, MMP2, and MMP9 as well as increased cellular migration/invasion. Overexpression of UCH-L1 in MCF7 cells up-regulated MDR1, CD147, MMP2, and MMP9, which conferred MDR and promoted migration/invasion. On the other hand, silencing of UCH-L1 in MCF7/Adr cells led to the opposite effect. Immunohistochemistry in 203 breast cancer samples revealed that UCH-L1 expression is positively correlated with P-gp, CD147, MMP2, and MMP9 expression and standard tumor spread indicators. Kaplan-Meier survival analysis indicated a correlation between UCH-L1 expression and shorter recurrent and survival times. Moreover, UCH-L1-overexpressing clones treated with U0126 (an Erk1/2-specific inhibitor) significantly decreased the expression of MDR1, CD147, MMP2, and MMP9. These data indicate that UCH-L1 may assume a dual role, because it had intrinsic stimulatory effects on tumor migration/invasion and increased MDR. © 2015 Wiley Periodicals, Inc.

Inhibition of UCH-L1 in oligodendroglial cells results in microtubule stabilization and prevents α-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy.

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

&lt;i&gt;S&lt;/i&gt;-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells

Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels.

Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) delays Alzheimer's progression in vivo.

Deposition of amyloid β protein (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aβ is produced by β- and γ-cleavages of amyloid β precursor protein (APP). Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme that cleaves ubiquitin at its carboxyl terminal. Dysfunction of UCHL1 has been reported in neurodegenerative diseases. However, whether UCHL1 affects Aβ production and AD progression remains unknown. Here we report that UCHL1 interacts with APP and regulates Aβ production. UCHL1 increases free ubiquitin level and accelerates the lysosomal degradation of APP by promoting its ubiquitination. Furthermore, we demonstrate that overexpression of UCHL1 by intracranial injection of UCHL1-expressing rAAV reduces Aβ production, inhibits neuritic plaque formation and improves memory deficits in AD transgenic model mice. Our study suggests that UCHL1 may delay Alzheimer's progression by regulating APP degradation in a long-term fashion, and that overexpression of UCHL1 may be a safe and effective disease-modifying strategy to treat AD.

Increased levels of UCHL1 are a compensatory response to disrupted ubiquitin homeostasis in spinal muscular atrophy and do not represent a viable therapeutic target.

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. 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 neurone loss, muscle fibre atrophy and neuromuscular junction pathology were all quantified. Ubiquitin-like modifier activating enzyme 1 (Uba1) was then pharmacologically inhibited in neurones in vitro to examine the relationship between Uba1 levels and UCHL1 in SMA. 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. 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.

Development of surface plasmon resonance imaging biosensors for detection of ubiquitin carboxyl-terminal hydrolase L1

We have developed a new method for highly selective determination of the ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) concentration using a surface plasmon resonance imaging (SPRI) technique and two different biosensors. UCH-L1 was captured from a solution by immobilized specific rabbit monoclonal antibody or specific LDN-57444 inhibitor due to formation of receptor–UCH-L1 complex on the biosensor surface. The analytically useful dynamic response range of both biosensors is between 0.1 and 2.5ng/ml. The detection limit is 0.06ng/ml for the biosensor with antibody and 0.08ng/ml for the biosensor with inhibitor. Biosensors based on both antibody and inhibitor were found to be suitable for quantitative determination of the UCH-L1 and exhibit good tolerance to the potential interferents. Both biosensors gave comparable results in the range of 0 to 0.20ng/ml for plasma samples and 0.30 to 0.49ng/ml for cerebrospinal fluid samples. To validate the new methods, comparative determination of UCH-L1 by the commercial enzyme-linked immunosorbent assay (ELISA) kit was performed. In general, in terms of UCH-L1 concentration, a good correlation between SPRI and ELISA was found. The developed biosensors can be used successfully for the determination of UCH-L1 in body fluids.

Comparison of the performances of copeptin and multiple biomarkers in long-term prognosis of severe traumatic brain injury

Enhanced blood levels of copeptin correlate with poor clinical outcomes after acute critical illness. This study aimed to compare the prognostic performances of plasma concentrations of copeptin and other biomarkers like myelin basic protein, glial fibrillary astrocyte protein, S100B, neuron-specific enolase, phosphorylated axonal neurofilament subunit H, Tau and ubiquitin carboxyl-terminal hydrolase L1 in severe traumatic brain injury. We recruited 102 healthy controls and 102 acute patients with severe traumatic brain injury. Plasma concentrations of these biomarkers were determined using enzyme-linked immunosorbent assay. Their prognostic predictive performances of 6-month mortality and unfavorable outcome (Glasgow Outcome Scale score of 1–3) were compared. Plasma concentrations of these biomarkers were statistically significantly higher in all patients than in healthy controls, in non-survivors than in survivors and in patients with unfavorable outcome than with favorable outcome. Areas under receiver operating characteristic curves of plasma concentrations of these biomarkers were similar to those of Glasgow Coma Scale score for prognostic prediction. Except plasma copeptin concentration, other biomarkers concentrations in plasma did not statistically significantly improve prognostic predictive value of Glasgow Coma Scale score. Copeptin levels may be a useful tool to predict long-term clinical outcomes after severe traumatic brain injury and have a potential to assist clinicians.

Serum biomarkers to evaluate the integrity of the neurovascular unit

Biomarkers have the potential to enable the clinicians to screen infants for brain injury, monitor progression of disease, identify injured brain regions, assess efficacy of neuroprotective therapies, and offer hope to identify the timing of the injury, thus shedding light on the potential pathophysiology and the most effective therapy. Currently, clinicians do not routinely use biomarkers to care for neonates with Neonatal Encephalopathy (NE) and brain injury due to prenatal hypoxia–asphyxia. This review will cover potential biomarkers of the neurovascular unit in the setting of NE that (i) can help assess the degree or severity of encephalopathy at birth; (ii) can help monitor progression of disease process and efficacy of neuroprotective therapy; (iii) can help assess neurodevelopmental outcome. These biomarkers will be summarized in two categories: 1) Specific biomarkers targeting the neurovascular unit such as glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), S100B, and neuron specific enolase (NSE) and 2) general inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1b (IL-1b), and pNF-H, among others.

Prognostic significance of plasma copeptin detection compared with multiple biomarkers in intracerebral hemorrhage

BackgroundHigher plasma copeptin concentrations have been associated with poor clinical outcomes after intracerebral hemorrhage. This study was designed to compare plasma concentrations of copeptin and other biomarkers like myelin basic protein, glial fibrillary astrocyte protein, S100B, neuron-specific enolase, phosphorylated axonal neurofilament subunit H, tau and ubiquitin carboxyl-terminal hydrolase L1 for analysis of their prognostic prediction. MethodsWe measured plasma concentrations of these biomarkers in 118 healthy controls and in 118 acute patients with a comparison analysis for their prediction of 6-month mortality and unfavorable outcome (modified Rankin Scale score>2). ResultsPlasma concentrations of these biomarkers were statistically significantly higher in all patients than in healthy controls, in non-survivors than in survivors and in patients with unfavorable outcome than with favorable outcome. Areas under receiver operating characteristic curves of plasma concentrations of these biomarkers were similar to those of the National Institute of Health Stroke Scale score for prognostic prediction. Plasma copeptin concentration statistically significantly improved the prognostic predictive value of the National Institute of Health Stroke Scale score, but other biomarkers did not. ConclusionsCopeptin may help in the prediction of long-term clinical outcomes after intracerebral hemorrhage.

Glutathione-mediated reversibility of covalent modification of ubiquitin carboxyl-terminal hydrolase L1 by 1,2-naphthoquinone through Cys152, but not Lys4

Covalent modification of cellular proteins by electrophiles affects electrophilic signal transduction and the dysfunction of enzymes that is involved in cytotoxicity. We have recently found a unique reaction which restores glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that has been modified by 1,2-naphthoquinone (1,2-NQ) through a glutathione (GSH)-dependent S-transarylation reaction. We report here that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) undergoes the same reaction. Exposure of human neuroblastoma SH-SY5Y cells to 1,2-NQ after pretreatment with buthionine sulfoximine (BSO) to deplete GSH resulted in an enhancement of covalent modification of UCH-L1 by 1,2-NQ. With recombinant human UCH-L1, we demonstrated that UCH-L1 underwent arylation by 1,2-NQ through Cys152 and Lys4, thereby decreasing its catalytic activity. Addition of GSH to an incubation mixture of 1,2-NQ-UCH-L1 adduct partially restored this decline in enzyme activity which was accompanied by decreased covalent attachment of 1,2-NQ, together with production of 1,2-NQ-GSH adduct. UCH-L1 in which Lys4 was mutated exhibited a lower level of covalent modification and enzyme inhibition, but completely recovered after addition of GSH. Taken together, these results suggest that Cys152 modification in UCH-L1 by 1,2-NQ is reversible via GSH-mediated S-transarylation reaction whereas Lys4 modification by 1,2-NQ is irreversible by GSH. Because UCH-L1 dysfunction has been associated with neurodegeneration, the electrophilic modification of Lys rather than Cys in UCH-L1 may be implicated in such neurodegenerative diseases.

Ubiquitin C-terminal hydrolase L1 interacts with choline transporter in cholinergic cells

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme, which is highly expressed in neuronal cells. Previous studies have indicated that UCHL1 is involved in cognitive function, neurodegenerative diseases, and neuromuscular junction development. Acetylcholine (Ach) is a critical neurotransmitter in these functions. Yet, the effect of UCHL1 on the cholinergic system has not been reported. In this study, using a cholinergic neuronal cell line, SN56, as an invitro model, we detected the physical interaction of UCHL1 and high affinity choline transporter (CHT), which is a key protein regulating Ach re-synthesis. Reduction of UCHL1 by siRNA gene knockdown significantly increased poly-ubiquitinated CHT and decreased native CHT protein level, but did not affect CHT mRNA expression. Biotinylation assay showed that UCHL1 is localized only in the cytosol of the cells and that the gene knockdown of UCHL1 significantly reduced cytosolic CHT but had no significant effect on membrane CHT level. These data provide novel and potentially important evidence that UCHL1 may play a role in the regulation of cholinergic function by affecting CHT ubiquitination and degradation.

Biomarkers for Severity of Neonatal Hypoxic-Ischemic Encephalopathy and Outcomes in Newborns Receiving Hypothermia Therapy

To evaluate serum neuronal and inflammatory biomarkers to determine whether measurements of umbilical cords at birth can stratify severity of hypoxic-ischemic encephalopathy (HIE), whether serial measurements differ with hypothermia-rewarming, and whether biomarkers correlate with neurological outcomes. This is a prospective cohort of inborn term newborns with varying degrees of HIE by neurological assessment. Neuronal glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1, and inflammatory cytokines were measured in serum from umbilical artery at 6-24, 48, 72, and 78 hours of age. Neurodevelopmental outcomes (Bayley Scales of Infant and Toddler Development-III scales) were performed at 15-18 months. Twenty neonates had moderate (n = 17) or severe (n = 3) HIE and received hypothermia; 7 had mild HIE and were not cooled. At birth, serum GFAP and ubiquitin carboxyl-terminal hydrolase L1 increased with the severity of HIE (P < .001), and serial GFAP remained elevated in neonates with moderate to severe HIE. Interleukin (IL)-6, IL-8, and vascular endothelial growth factor were greater at 6-24 hours in moderate to severe vs mild HIE (P < .05). The serial values were unaffected by hypothermia-rewarming. Elevated GFAP, IL-1, IL-6, IL-8, tumor necrosis factor, interferon, and vascular endothelial growth factor at 6-24 hours were associated with abnormal neurological outcomes. The severity of the hypoxic-ischemic injury can be stratified at birth because elevated neuronal biomarkers in cord serum correlated with severity of HIE and outcomes.

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.

Mass Spectrometry Assessment of Ubiquitin Carboxyl-Terminal Hydrolase L1 Partitioning between Soluble and Particulate Brain Homogenate Fractions

Partitioning of specific proteins between soluble and insoluble forms because of aggregation, membrane attachment, and (or) association with senile plaques and neurofibrillary tangles is a major feature of several neurodegenerative disorders, including Alzheimer's disease (AD). Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is an example of a neuron-specific protein which displays two different dimerization-dependent catalytic activities and can be farnesylated for membrane attachment, oxidized, and truncated. Decreased levels of soluble UCH-L1 are inversely proportional to the number of neurofibrillary tangles. Further assessment of a link between UCH-L1 function and the pathogenesis of AD requires an analytical method to separately quantify different UCH-L1 forms. In the present study, we have developed a multiple reaction monitoring (MRM) assay to measure UCH-L1 in the high-speed supernatant and pellet of frontal cortex homogenate. The well-characterized (15)N-labeled quantification concatamer (QconCAT) carrying prototypic tryptic peptides of UCH-L1 was used as an internal standard. The composed protocol of frontal cortex processing includes solubilization and reduction/alkylation of proteins in the presence of 1% sodium dodecyl sulfate (SDS) and following with desalting/delipidation of the sample by chloroform/methanol precipitation with extra water washing of the protein pellet. The measurements were performed for frontal cortex samples from control and severe AD donors. The proposed workflow can be recommended for quantification of partitioning of other proteins of interest.

Biomarkers for the Clinical Differential Diagnosis in Traumatic Brain Injury-A Systematic Review

Rapid triage and decision-making in the treatment of traumatic brain injury (TBI) present challenging dilemma in "resource poor" environments such as the battlefield and developing areas of the world. There is an urgent need for additional tools to guide treatment of TBI. The aim of this review is to establish the possible use of diagnostic TBI biomarkers in (1) identifying diffuse and focal brain injury and (2) assess their potential for determining outcome, intracranial pressure (ICP), and responses to therapy. At present, there is insufficient literature to support a role for diagnostic biomarkers in distinguishing focal and diffuse injury or for accurate determination of raised ICP. Presently, neurofilament (NF), S100β, glial fibrillary acidic protein (GFAP), and ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) seemed to have the best potential as diagnostic biomarkers for distinguishing focal and diffuse injury, whereas C-tau, neuron-specific enolase (NSE), S100β, GFAP, and spectrin breakdown products (SBDPs) appear to be candidates for ICP reflective biomarkers. With the combinations of different pathophysiology related to each biomarker, a multibiomarker analysis seems to be effective and would likely increase diagnostic accuracy. There is limited research focusing on the differential diagnostic properties of biomarkers in TBI. This fact warrants the need for greater efforts to innovate sensitive and reliable biomarkers. We advocate awareness and inclusion of the differentiation of injury type and ICP elevation in further studies with brain injury biomarkers.

Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response

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.