Ubiquitin Carboxyl-terminal Hydrolase Isozyme L1 Research Articles

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.

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines

Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

Proteomic analysis of the hippocampus in naïve and ischemic-preconditioned rat

The hippocampus exhibits regional differences in vulnerability to ischemia, wherein pyramidal cells in the CA1 region are vulnerable to ischemia while pyramidal cells in the CA3 region and granule cells in the dentate gyrus (DG) region are relatively ischemia resistant. However, pyramidal cells in the CA1 region reportedly exhibit ischemic tolerance following exposure to a brief non-lethal period of ischemia known as ischemic preconditioning. In this study, we used proteomic analysis to examine the difference in protein expression between naïve rat CA1 and CA3/DG regions, as well as the altered protein expression in the CA1 region after 3min of ischemic preconditioning. Proteomic analysis identified ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), glutathione S-transferase μ5 (GSTμ5), glutamine synthetase (GS), and dynamin-1 as proteins with differential expression levels in naïve CA1 and CA3/DG regions. The difference in expression levels of GSTμ5 and GS between these two regions was further confirmed by western blot. Our analysis also identified aconitase2, α-tubulin, protein-l-isoaspartate O-methiltransferase (PIMT), and voltage-dependent anion channel 1 (VDCA1) as proteins with down-regulated expression levels in the CA1 region following 3min ischemic preconditioning. The decrease in the expression of aconitase2 was also confirmed by western blot and immunohistochemical staining. The present results suggest that GSTμ5 and GS may be associated with ischemia-resistance in the CA3/DG region and that aconitase2 may play a part in the ischemic tolerance in the CA1 region.

Proteomic Analysis of Protein Expression Throughout Disease Progression in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. Mice in the transgenic AβPPswe/PS1dE9 mouse line express a chimeric mouse/human amyloid-β protein precursor (Mo/HuAβPP695swe) and mutant human presenilin 1 (PS1-dE9) associated with early-onset AD. Knowing the protein expression in these mice may offer better understanding of the pathological changes in AD. In this study, we used two-dimensional gel electrophoresis combined with mass spectrometry techniques to compare protein expression in AβPPswe/PS1dE9 mice with age-matched wild-type mice throughout the disease progression. We identified 15 proteins that were significantly different between the AβPPswe/PS1dE9 mice and age-matched controls and also changed with disease development. Among those, the expression levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times higher than those in normal mice: DCC-interacting protein 13-beta, serum albumin, creatine kinase B-type, heat shock 70 kDa protein 1A, T-complex protein 1 subunit beta, adenylate kinase isoenzyme 1, pyruvate dehydrogenase E1 component subunit beta mitochondrial, and V-type proton ATPase catalytic subunit A. Levels of the following proteins in AβPPswe/PS1dE9 mice were at least 1.5 times lower than those in normal mice: dihydropyrimidinase-related protein 2, actin cytoplasmic 2, isoform 1 of V-type proton ATPase catalytic subunit, tubulin alpha-1C chain, F-actin-capping protein subunit alpha-2, ubiquitin carboxyl-terminal hydrolase isozyme L1, and actin cytoplasmic 1. These proteins are involved in regulating various cellular functions, including cytoskeletal structure, energy metabolism, synaptic components, and protein degradation. These findings indicate altered protein expression in the pathogenesis of AD and illuminate novel therapeutic avenues for treatment in AD.

Proteomic analysis of β-asarone induced cytotoxicity in human glioblastoma U251 cells

Though rhizoma acori graminei (RAG) is frequently prescribed in formulations for brain tumor in traditional Chinese medicine, the potential mechanisms are still unclear. The aim of this study is to determine the effect of β-asarone, a major component in the volatile oil of RAG, against brain tumor and elucidate the underlying molecular mechanisms. The results showed that β-asarone significantly inhibited the cell viability of human glioblastoma U251 cells. Moreover, YO-PRO-1/PI staining revealed that cells treated with β-asarone underwent apoptotic and necrotic death. Then, the two-dimensional gel electrophoresis (2-DE)-based proteomics was applied to investigate the different protein profiles of U251 cells treated with vehicle or β-asarone. Sixteen proteins affected by β-asarone were successfully identified by MALDI-TOF/TOF mass spectrometry. Gene ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. Importantly, four proteins (heterogeneous nuclear ribonucleoprotein H1 (H), isoform CRA_b, heterogeneous nuclear ribonucleoprotein A2/B1, isoform CRA_a, ubiquitin carboxyl-terminal hydrolase isozyme L1 and cathepsin D) acting as either oncoproteins or tumor suppressors draw our special attention. Finally, the effect of β-asarone on these four genes was confirmed at transcriptional level by semi-quantitative RT-PCR. Collectively, a variety of proteins affected by β-asarone were identified by 2-DE coupled with MALDI-TOF/TOF MS/MS analysis. Four potential protein targets were proposed, which will enable a better understanding of the anti-tumor activity of β-asarone.

Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin.

The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN-/-) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

Identification of Site-Specific Stroke Biomarker Candidates by Laser Capture Microdissection and Labeled Reference Peptide.

The search to date for accurate protein biomarkers in acute ischemic stroke has taken into consideration the stage and/or the size of infarction, but has not accounted for the site of stroke. In the present study, multiple reaction monitoring using labeled reference peptide (LRP) following laser capture microdissection (LCM) is used to identify site-specific protein biomarker candidates. In middle cerebral artery occlusion (MCAO) rat models, both intact and infarcted brain tissue was collected by LCM, followed by on-film digestion and semi-quantification using triple-quadrupole mass spectrometry. Thirty-four unique peptides were detected for the verification of 12 proteins in both tissue homogenates and LCM-captured samples. Six insoluble proteins, including neurofilament light polypeptide (NEFL), alpha-internexin (INA), microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), myelin proteolipid protein (PLP) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), were found to be site-specific. Soluble proteins, such as neuron-specific enolase (NSE) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and some insoluble proteins, including neurofilament heavy polypeptide (NEFH), glial fibrillary acidic protein (GFAP), microtubule-associated protein tau (MAPT) and tubulin β-3 chain (TUBB3), were found to be evenly distributed in the brain. Therefore, we conclude that some insoluble protein biomarkers for stroke are site-specific, and would make excellent candidates for the design and analysis of relevant clinical studies in the future.

DJ-1 in Endometrial Cancer: A Possible Biomarker to Improve Differential Diagnosis Between Subtypes

The objectives of this study were to characterize the well-defined endometrial cancer (EC) type I (endometrioid [EEC] G1-G2) versus the prototype of EC type II (serous [ESC]) and to evaluate the expression of specific biomarkers differentially expressed between 2 well-defined types, in those EC subtypes (such as EEC G3) disputed between types I and II. Data from 25 patients (10 EEC G1-G2, 8 EEC G3, 5 ESC, and 2 clear cell) submitted to the surgical treatment were collected. Two-dimensional electrophoresis and mass spectrometry (MS) analysis were performed on 5 EEC G1-G2 and 5 healthy endometrial samples of the same patients. Differentially expressed proteins, such as DJ-1, were validated by Western blot. In patients with EEC G1-G2, serum levels of DJ-1, an overexpressed oncoprotein related to EC pathogenesis and progression, were evaluated and then compared with levels identified in patients with ESC and healthy controls. The DJ-1 immunohistochemical (IHC) staining was performed on neoplastic and healthy endometrium collected from the same patients. The 8 stored samples of EEC G3 were submitted to DJ-1 IHC assays. The 2-dimensional electrophoresis analysis identified 1040 protein spots differentially expressed in EEC G1-G2 compared with healthy endometrium. Forty-two spots were subjected to liquid chromatography-MS/MS analysis. Thirty-three up-regulated (like an annexin 2 [ANXA2] shorter isoform, CAPG [macrophage-capping protein], DJ-1/PARK7) and 9 down-regulated (like calreticulin and ubiquitin carboxyl-terminal hydrolase isozyme L1) proteins were identified and validated by Western blot. A significant increase in serum DJ-1 levels of EEC G1-G2 versus the healthy controls and in ESC versus EEC patients was observed. DJ-1 IHC score was significantly higher in ESC versus those EEC G1-G2. In 3 cases of EEC G3, the DJ-1 expression was similar to the ESC subtype. The identification of proteins, such as DJ-1, differentially expressed, between well-defined EC types I and II allows to make a subtype-specific presurgical diagnosis and help surgeon to safely preoperatively choose a proper surgical treatment.

High and Low Doses of Ionizing Radiation Induce Different Secretome Profiles in a Human Skin Model

It is postulated that secreted soluble factors are important contributors of bystander effect and adaptive responses observed in low dose ionizing radiation. Using multidimensional liquid chromatography-mass spectrometry based proteomics, we quantified the changes of skin tissue secretome – the proteins secreted from a full thickness, reconstituted 3-dimensional skin tissue model 48 hr after exposure to 3, 10 and 200 cGy of X-rays. Overall, 135 proteins showed statistical significant difference between the sham (0 cGy) and any of the irradiated groups (3, 10 or 200 cGy) on the basis of Dunnett adjusted t-test; among these, 97 proteins showed a trend of downregulation and 9 proteins showed a trend of upregulation with increasing radiation dose. In addition, there were 21 and 8 proteins observed to have irregular trends with the 10 cGy irradiated group either having the highest or the lowest level among all three radiated doses. Moreover, two proteins, carboxypeptidase E and ubiquitin carboxyl-terminal hydrolase isozyme L1 were sensitive to ionizing radiation, but relatively independent of radiation dose. Conversely, proteasome activator complex subunit 2 protein appeared to be sensitive to the dose of radiation, as rapid upregulation of this protein was observed when radiation doses were increased from 3, to 10 or 200 cGy. These results suggest that different mechanisms of action exist at the secretome level for low and high doses of ionizing radiation.

Protective and toxic roles of dopamine in Parkinson's disease

The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ-1 (PARK7), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1), serine/threonine-protein kinase (PINK-1), leucine-rich repeat kinase 2 (LRRK2), cation-transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o-quinone, aminochrome and 5,6-indolequinone are formed. However, both dopamine o-quinone and 5,6-indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o-quinone, aminochrome and 5,6-indolequinone polymerization to neuromelanin and (iii) two-electron reduction of aminochrome catalyzed by DT-diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone plays an important role in neurodegeneration in Parkinson's disease since they induce mitochondria and protein degradation dysfunction; formation of neurotoxic alpha synuclein protofibrils and oxidative stress. However, the cells have a protective system against dopamine oxidation composed by dopamine uptake mediated by Vesicular monoaminergic transporter-2 (VMAT-2), neuromelanin formation, two-electron reduction and GSH-conjugation mediated by Glutathione S-transferase M2-2 (GSTM2).

Activation of the Ubiquitin Proteasome Pathway in a Mouse Model of Inflammatory Myopathy: A Potential Therapeutic Target

Myositis is characterized by severe muscle weakness. We and others have previously shown that endoplasmic reticulum (ER) stress plays a role in the pathogenesis of myositis. The present study was undertaken to identify perturbed pathways and assess their contribution to muscle disease in a mouse myositis model. Stable isotope labeling with amino acids in cell culture (SILAC) was used to identify alterations in the skeletal muscle proteome of myositic mice in vivo. Differentially altered protein levels identified in the initial comparisons were validated using a liquid chromatography tandem mass spectrometry spike-in strategy and further confirmed by immunoblotting. In addition, we evaluated the effect of a proteasome inhibitor, bortezomib, on the disease phenotype, using well-standardized functional, histologic, and biochemical assessments. With the SILAC technique we identified significant alterations in levels of proteins belonging to the ER stress response, ubiquitin proteasome pathway (UPP), oxidative phosphorylation, glycolysis, cytoskeleton, and muscle contractile apparatus categories. We validated the myositis-related changes in the UPP and demonstrated a significant increase in the ubiquitination of muscle proteins as well as a specific increase in ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1) in myositis, but not in muscle affected by other dystrophies or normal muscle. Inhibition of the UPP with bortezomib significantly improved muscle function and also significantly reduced tumor necrosis factor α expression in the skeletal muscle of mice with myositis. Our findings indicate that ER stress activates downstream UPPs and contributes to muscle degeneration and that UCHL-1 is a potential biomarker for disease progression. UPP inhibition offers a potential therapeutic strategy for myositis.

Glyoxalase 1 as a candidate for indicating the metastatic potential of SN12C human renal cell carcinoma cell clones

Three clones with differential metastatic potential were established from the parental SN12C human renal cell carcinoma (HRCC). We previously reported that in the two high metastatic SN12C clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH‑L1) showed decreased expression by using two-dimensional electrophoresis (2‑DE) covering a pH range (pH3.0‑10.0) followed by liquid chromatography‑tandem mass spectrometry. However, in the case of the low metastatic clone, the spot volume for UCH‑L1 was almost the same as for the parental SN12C. In the present study, we found one protein spot which was correlated with the metastatic potential of SN12C clones by using 2‑DE over a narrow pH range (pH4.0‑7.0). The protein glyoxalase1 (GLO1) appeared to be directly proportional to the metastatic potential of the SN12C clones. GLO1 was the only protein which consistently varied according to the metastatic potentials of SN12C clones. GLO1 was increased in high metastatic cell lines by western blot analysis. These findings suggest that GLO1 is associated with the metastatic potential of SN12C HRCC clones. We expanded our experimental range to include clones of scirrhous gastric cancer cell lines (OCUM‑2M, OCUM‑2D and OCUM‑2MLN) and similar results were obtained, thereby further strengthening our original findings.

Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)-induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR-181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform-specific interacting proteins in HPC mouse brain. In this study, the role of miR-181b in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR-181b expression levels significantly decreased in mouse brain following MCAO and in OGD-treated N2A cells. Up- and downregulation of miR-181b by transfection of pre- or anti-miR-181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD-treated N2A cells. By using a T7 promoter-driven control dual luciferase assay, we confirmed that miR-181b could bind to the 3'-untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR-181b antagomir reduced caspase-3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti-miR-181b-mediated neuroprotection against OGD-induced N2A cell injury in vitro. These results suggest that the downregulated miR-181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.

Differential expression of peroxiredoxin 6, annexin A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1 in testis of rat fetuses after maternal exposure to di-n-butyl phthalate

ObjectivesTo isolate and identify differentially expressed proteins in testis of rat fetuses after maternal exposure to di-n-butyl phthalate (DBP). MethodsPregnant rats were daily treated by gavage with 1ml/kg corn oil or 750mg/kg DBP from GD14 to GD18. We used the technique of proteomic analysis to compare the testis protein patterns obtained by two-dimensional gel electrophoresis from fetal rats of gestation day 19. ResultsWe found significant differences in protein spot intensities compared to control. Subsequently several of these variant protein spots were identified by mass spectrometry. Peroxiredoxin 6 (Prdx6), annexin A5 (AnxA5) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UchL1) were three of them, the differential expression of which were confirmed by western blotting. Further, immunohistochemical analyses of fetal rat testes sections were made to determine the cellular distribution of these proteins, consequently strong Prdx6 and AnxA5 stainings were found primarily in Leydig cells, while a weak UchL1 staining was found primarily in spermatogonium. ConclusionsThe present study had found several differentially regulated proteins and demonstrated the differential expression of Prdx6, AnxA5 and UchL1 in fetal rat testis after maternal exposure to DBP, when compared with controls. Combining the cellular location of these proteins and their function in other tissues, the results of this study indicated that oxidative injury and abnormal apoptotic regulation might participate the formation of testicular dysgenesis in fetuses of dams exposed to DBP.

Identification of biomarkers for endometriosis in eutopic endometrial cells from patients with endometriosis using a proteomics approach

Endometriosis is a gynecological disease defined as the presence of endometrial tissue outside the uterine cavity, which is caused by various factors. Proteomic analysis of two sets of eutopic endometrial cells collected from the menstrual blood of females with (n=6; n=3) or without (n=6; n=3) endometriosis was performed to identify novel potential biomarkers for endometriosis. The data revealed that samples from endometriosis patients had stem cell characteristics, as they had higher mRNA expression levels of octamer-binding transcription factor 4 (Oct-4), C-X-C chemokine receptor type 4 (CXCR4), SRY-box containing gene2 (SOX2) and mesenchymal-epithelial transition factor (MET) compared with that of the normal controls. Three proteins, collapsin response mediator protein 2 (CRMP2), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) and myosin regulatory light polypeptide 9 (MYL9), were simultaneously identified from the two sets of samples from females with or without endometriosis by two-dimensional electrophoresis (2-DE). A difference in CRMP2 expression was confirmed with western blotting. Taken together, the results suggest that CRMP2 plays a role in the pathogenesis of endometriosis.

Proteomic analysis and brain‐specific systems biology in a rodent model of penetrating ballistic‐like brain injury

Proteomics and systems biology have significantly contributed to biomarker discovery in the field of brain injury. This study utilized 2D-DIGE-PMF-MS as a preliminary screen to detect biomarkers in a rat model of penetrating ballistic-like brain injury (PBBI). Brain-specific systems biology analysis of brain tissue identified 386 proteins having a fold change of more than 2, of which 321 proteins were increased and 65 were decreased 24 h after PBBI compared to sham controls. The majority of upregulated proteins were cytoskeletal (10.5%), nucleic acid binding (9.3%), or kinases (8.9%). Most proteins were involved in protein metabolism (22.7%), signal transduction (20.4%), and development (9.6%). Pathway analysis indicated that these proteins were involved in neurite outgrowth and cell differentiation. Semiquantitative Western blotting of 6, 24, 48, and 72 h after PBBI indicated ubiquitin carboxyl-terminal hydrolase isozyme L1 (a proposed traumatic brain injury biomarker in human clinical trials), tyrosine hydroxylase, and syntaxin-6 were found to be consistently elevated in brain tissue and cerebral spinal fluid after PBBI compared to sham controls. Combining proteomics and brain-specific systems biology can define underlying mechanisms of traumatic brain injury and provide valuable information in biomarker discovery that, in turn, may lead to novel therapeutic targets.

Proteomics of rat hypothalamus, hippocampus and pre-frontal/frontal cortex after central administration of the neuropeptide PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts pleiotropic functions, acting as a hypophysiotropic factor, a neurotrophic and a neuroprotective agent. The molecular pathways activated by PACAP to exert its physiological roles in brain are incompletely understood. In this study, adrenocorticotropic hormone (ACTH), prolactin, luteinising hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), brain-derived neurotrophic factor and corticosterone blood levels were determined before and 20, 40, 60, and 120 min after PACAP intracerebroventricular administration. PACAP treatment increased ACTH, corticosterone, LH and FSH blood concentrations, while it decreased TSH levels. A proteomics investigation was carried out in hypothalamus, hippocampus and pre-frontal/frontal cortex (P/FC) using 2-dimensional gel electrophoresis at 120 min, the end-point suggested by studies on PACAP hypophysiotropic activities. Spots showing statistically significant alterations after PACAP treatment were identified by Matrix-assisted laser desorption/ionization-Time of flight mass spectrometry. Identified proteins were consistent with PACAP involvement in different molecular processes in brain. Altered expression levels were observed for proteins involved in cytoskeleton modulation and synaptic plasticity: actin in the hypothalamus; stathmin, dynamin, profilin and cofilin in hippocampus; synapsin in P/FC. Proteins involved in cellular differentiation were also modulated: glutathione-S-transferase α and peroxiredoxin in hippocampus; nucleoside diphosphate kinase in P/FC. Alterations were detected in proteins involved in neuroprotection, neurodegeneration and apoptosis: ubiquitin carboxyl-terminal hydrolase isozyme L1 and heat shock protein 90-β in hypothalamus; α-synuclein in hippocampus; glyceraldehyde-3-phosphate dehydrogenase and prohibitin in P/FC. This proteomics study identified new proteins involved in molecular mechanisms mediating PACAP functions in the central nervous system.

Proteomic investigation of a neural substrate intimately related to brain death

Our current understanding on brain death remains limited despite its clinical importance. This study evaluated whether the proteome expressed in the rat rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is uniquely different from other brain areas, using the cerebral cortex, which is defunct under persistent vegetative state for comparison. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase, exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits α and β in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit α type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.

Proteomic analysis of cisplatin resistance in human ovarian cancer using 2-DE method

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for human ovarian cancer. However, overcoming drug resistance has become an important issue in cancer chemotherapy. In this study, we performed 2-DE and ESI-Q-TOF MS/MS analysis to identify differential proteins expression between cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780-CP) ovarian cancer cell lines. Of the 14 spots identified as differentially expressed (±over twofold, P < 0.05) between the two cell lines, ten spots (corresponding to ten unique proteins) were positively identified by ESI-Q-TOF MS/MS analysis. These proteins include capsid glycoprotein, fructose-bisphosphate aldolase C, heterogeneous nuclear ribonucleoproteins A2/B1, putative RNA-binding protein 3, Ran-specific GTPase-activating protein, ubiquitin carboxyl-terminal hydrolase isozyme L1, stathmin, ATPSH protein, chromobox protein homolog3 and phosphoglycerate kinase 1. The proteins identified in this study would be useful in revealing the mechanisms underlying cisplatin resistance and also provide some clues for further research.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.