Proteomic surveillance of autoantigens in relapsing polychondritis

Relapsing polychondritis (RP) is a systemic inflammatory disease, in which autoimmunity to cartilage-related components is thought to be involved in its pathogenesis. However, the autoimmune profile in RP has not been studied fully. We therefore investigated autoantibodies/autoantigens in RP comprehensively, by 2-dimensional electrophoresis (2DE), subsequent western blotting (WB) and mass spectrometry, using cell-extracted proteins as the antigen source. As a result, we detected 15 autoantigens on 2DE-WB, and further identified five of them. On average, one RP serum recognized approximately 8 out of the 15 autoantigens. Frequencies of the autoantibodies to the 5 identified antigens of tubulin alpha ubiquitous/6, vimentin, alpha enolase, calreticulin, and colligin-1/-2 were 91%, 46%, 36%, 82%, and 36%, respectively. ELISA using recombinant proteins for them revealed that frequencies of the autoantibodies to tubulin alpha ubiquitous, vimentin, alpha enolase, calreticulin, and colligin-1 were 36%, 64%, 46%, 27%, and 18%, respectively. Our data demonstrated that the autoimmune reaction was not restricted to cartilagerelated components, rather a variety of autoimmune responses occurred in patients with RP, which may be involved in the pathophysiology of RP. In addition, the proteomic approach using cell-extracted proteins would be a powerful way to investigate autoantigens.     

The relationship between serum prolactin levels and disease activity in patients with Behcet's disease

The aim of this study was to determine the serum levels of prolactin in patients with Behcet's disease and to evaluate its correlation with disease activity. Serum prolactin levels were measured by a chemiluminescence method in 32 patients with Behcet's disease and compared with 20 age-and sex-matched healthy controls. The patients with Behcet's disease were subdivided into two groups according to disease activity: active (18 patients; 13 men and five women, average age 34.0 +/- 6.5 (28-48) years), and inactive (14 patients; 10 men and four women, average age 32.7 +/- 3.1 (22-49 years). Patients with active Behcet's disease had higher serum prolactin levels than the inactive and control groups. Prolactin levels in patients with active Behcet's disease differed significantly from the healthy control subjects (p < 0.05) only, but not the inactive group. Four patients out of 32 (12.5%) Behcet's disease patients showed mild hyperprolactinemia. All four of these cases were from the active Behcet's disease group. Prolactin levels were correlated with ESR (p < 0.05) and CRP (p < 0.05) levels in the active BD group, but not in the inactive BD and control groups. Our results suggest a possible role for this immunoregulatory hormone in the disease expression and pathogenesis of Behcet's disease.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

Identification of candidate antigen in Whipple's disease using a serological proteomic approach

Whipple's disease (WD) is a chronic multisystemic infection, caused by Tropheryma whipplei, a Gram-positive rod. Recently, a reliable method has been developed for cultivating T. whipplei in vitro. This together with the availability of complete genome sequence of T. whipplei prompted us to initiate proteome analysis of T. whipplei. The objective of the present study was to identify candidate proteins for serological diagnosis of WD. Immunoreactivities of sera collected from 18 patients with WD were compared with those of 24 control subjects who did not have WD. For this, we used 2-DE, immunoblotting, and MS. In total, we identified 23 candidate antigenic proteins. These included a subset of six proteins, each of which was found significantly more frequently in cases as compared to their controls. The remaining 17 proteins were found exclusively in cases. The methods we used in the current study enabled us to identify candidate antigens that, in our view, might be useful for serological diagnosis of WD.     

Identification of skeletal muscle autoantigens by expression library screening using sera from autoimmune rippling muscle disease (ARMD) patients

Novel forms of contractile regulation observed in skeletal muscle are evident in neuromuscular diseases like rippling muscle disease (RMD). Previous studies of an autoimmune form of RMD (ARMD) identified a very high molecular weight skeletal muscle protein antigen recognized by ARMD patient antisera. This study utilized ARMD and myasthenia gravis (MG) patient antisera, to screen a human skeletal muscle cDNA library that subsequently identified proteins that could play a role in ARMD. Based on nucleotide sequence analysis, three distinct ARMD antigens were identified: titin Isoform N2A, ATP synthase 6, and PPP1R3 (protein phosphatase 1 regulatory subunit 3). The region of titin identified by ARMD antisera is distinct from the main immunogenic region (MIR) recognized by classical MG antibodies. Sera from classical MG patient identifies an expressed sequence corresponding to the titin MIR. Although the mechanism of antibody penetration is not known, previous studies have shown that rippling muscle antibodies affect the contractile machinery of myofibers resulting in mechanical sensitivity. Titin's role as a modulator of muscle contractility makes it a potential target in understanding muscle mechanosensitive regulation.     

Proteomic analysis of the neuroprotective mechanisms of acupuncture treatment in a Parkinson's disease mouse model

Acupuncture is frequently used as an alternative therapy for Parkinson's disease (PD), and it attenuates dopaminergic (DA) neurodegeneration in the substantia nigra (SN) in PD animal models. Using proteomic analysis, we investigated whether acupuncture alters protein expression in the SN to favor attenuation of neuronal degeneration. In C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day), intraperitoneal (i.p.) for 5 days, 2 or 100 Hz electroacupuncture (EA) was applied at the effective and specific acupoint, GB34, once a day for 12 consecutive days from the first MPTP treatment. Both treatments in MPTP mice led to restoration of behavioral impairment and rescued tyrosine hydroxylase (TH)-positive DA neurodegeneration. Using peptide fingerprinting MS, we identified changes in 22 proteins in the SN following MPTP treatment, and nine of these proteins were normalized by EA. They were involved in cell death regulation, inflammation, or restoration from damage. The levels of cyclophilin A (CypA), which is a neuroprotective agent, were unchanged by MPTP treatment but were increased in MPTP-EA mice. These results suggest that acupoint GB34-specific EA changes protein expression profiles in the SN in favor of DA neuronal survival in MPTP-treated mice, and that EA treatment may be an effective therapy for PD patients.     

Common MEFV gene mutations in Turkish patients with Behcet's disease

Behcet's disease (BD) is a chronic systemic inflammatory disorder whose etiology has not been fully established yet. The MEditerranean FeVer (MEFV) gene has been identified as the cause of Familial Mediterranean Fever (FMF). BD shows similarities with FMF, in terms of clinical findings and treatments, as well as their geographical and ethnic co-occurrence. In this study we investigated common MEFV gene mutation frequencies in Turkish patients with BD in an area of Turkey where both diseases are frequently encountered. We screened 207 BD patients who had no symptoms and family history for FMF and 200 healthy subjects for five common MEFV gene mutations (E148Q, M680I, M694V, V726A, P369S) and clinical features. Seventy-five patients were found to carry a single MEFV mutation, and six patients were compound heterozygous. The difference in the frequency of the MEFV mutation between the BD and control groups was statistically significant (p < 0.001, odds ratio [OR] 2.74, 95% confidence interval [CI] 1.75–4.29). The frequencies of E148Q and M680I mutations were significantly higher in the BD group (p = 0.001, p = 0.046, respectively). The frequency of uveitis was significantly lower in patients with the mutation than in patients without the mutation (p = 0.029, OR 0.54, 95% CI 0.30–0.98). There was no statistical significance between carriers and non-carriers with respect to gender and other manifestations of BD. The frequency of the MEFV mutation was significantly higher in patients with BD compared to the healthy control group. Based on our results, MEFV mutations appear to have a role in the pathogenesis of BD.     

Effects of stem cell therapy on protein profile of parkinsonian rats using an18O‐labeling quantitative proteomic approach

The application of neural stem cell (NSC) research to neurodegenerative diseases has led to promising clinical trials. Currently, NSC therapy is most promising for Parkinson's disease (PD). We conducted behavioral tests and immunoassays for the profiling of a PD model in rats to assess the therapeutic effects of NSC treatments. Further, using a multiple sample comparison workflow, combined with ¹⁸O‐labeled proteome mixtures, we compared the differentially expressed proteins from control, PD, and NSC‐treated PD rats. The results were analyzed bioinformatically and verified by Western blot. Based on our initial findings, we believe that the proteomic approach is a valuable tool in evaluating the therapeutic effects of NSC transplantation on neurodegenerative disorders.     

Platelet biomarkers for a descending cognitive function: A proteomic approach

Memory loss is the most common clinical sign in Alzheimer''s disease (AD); thus, searching for peripheral biomarkers to predict cognitive decline is promising for early diagnosis of AD. As platelets share similarities to neuron biology, it may serve as a peripheral matrix for biomarkers of neurological disorders. Here, we conducted a comprehensive and in‐depth platelet proteomic analysis using TMT‐LC‐MS/MS in the populations with mild cognitive impairment (MCI, MMSE = 18–23), severe cognitive impairments (AD, MMSE = 2–17), and the age‐/sex‐matched normal cognition controls (MMSE = 29–30). A total of 360 differential proteins were detected in MCI and AD patients compared with the controls. These differential proteins were involved in multiple KEGG pathways, including AD, AMP‐activated protein kinase (AMPK) pathway, telomerase RNA localization, platelet activation, and complement activation. By correlation analysis with MMSE score, three positively correlated pathways and two negatively correlated pathways were identified to be closely related to cognitive decline in MCI and AD patients. Partial least squares discriminant analysis (PLS‐DA) showed that changes of nine proteins, including PHB, UQCRH, CD63, GP1BA, FINC, RAP1A, ITPR1/2, and ADAM10 could effectively distinguish the cognitively impaired patients from the controls. Further machine learning analysis revealed that a combination of four decreased platelet proteins, that is, PHB, UQCRH, GP1BA, and FINC, was most promising for predicting cognitive decline in MCI and AD patients. Taken together, our data provide a set of platelet biomarkers for predicting cognitive decline which may be applied for the early screening of AD.     

Proteomic architecture of frailty across the spectrum of cardiovascular disease

While frailty is a prominent risk factor in an aging population, the underlying biology of frailty is incompletely described. Here, we integrate 979 circulating proteins across a wide range of physiologies with 12 measures of frailty in a prospective discovery cohort of 809 individuals with severe aortic stenosis (AS) undergoing transcatheter aortic valve implantation. Our aim was to characterize the proteomic architecture of frailty in a highly susceptible population and study its relation to clinical outcome and systems-wide phenotypes to define potential novel, clinically relevant frailty biology. Proteomic signatures (specifically of physical function) were related to post-intervention outcome in AS, specifying pathways of innate immunity, cell growth/senescence, fibrosis/metabolism, and a host of proteins not widely described in human aging. In published cohorts, the “frailty proteome” displayed heterogeneous trajectories across age (20–100 years, age only explaining a small fraction of variance) and were associated with cardiac and non-cardiac phenotypes and outcomes across two broad validation cohorts (N > 35,000) over ≈2–3 decades. These findings suggest the importance of precision biomarkers of underlying multi-organ health status in age-related morbidity and frailty.     

Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl-terminal hydrolase L-1, reveals oxidation of key proteins

Ubiquitin carboxyl-terminal hydrolase L-1 (UCH L-1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L-1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L-1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L-1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor alpha/ATP synthase and neurofilament-L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.     

Cologastric fistula with a foreign body in a patient with Crohn's disease

Although the medical management of fistulizing Crohn's disease is improving, a subset of patients does not respond to maximal medical therapy and is referred for surgical consultation. We report a case of Crohn's colitis with an ingested foreign body resulting in a cologastric fistula. The patient underwent segmental colectomy and takedown of the cologastric fistula. At the time of laparotomy, the foreign body was found in the fistulous colonic segment. The presence of an ingested foreign body likely contributed to a rare fistula that was refractory to medical management.     

Differential expression of synaptic proteins in unilateral 6‐OHDA lesioned rat model—A comparative proteomics approach

Parkinson's disease (PD) is characterized as a movement disorder due to lesions in the basal ganglia. As the major input region of the basal ganglia, striatum plays a vital role in coordinating movements. It receives afferents from the cerebral cortex and projects afferents to the internal segment of the globus pallidus and substantia nigra pars reticulate. Additionally, accumulating evidences support a role for synaptic dysfunction in PD. Therefore, the present study explores the changes in protein abundance involved in synaptic disorders in unilateral lesioned 6‐OHDA rat model. Based on ¹⁸O/¹⁶O‐labeling technique, striatal proteins were separated using online 2D‐LC, and identified by nano‐ESI‐quadrupole‐TOF. A total of 370 proteins were identified, including 76 significantly differentially expressed proteins. Twenty‐two downregulated proteins were found in composition of vesicle, ten of which were involved in neuronal transmission and recycling across synapses. These include N‐ethylmaleimide‐sensitive fusion protein attachment receptor proteins (SNAP‐25, syntaxin‐1A, syntaxin‐1B, VAMP2), synapsin‐1, septin‐5, clathrin heavy chain 1, AP‐2 complex subunit beta, dynamin‐1, and endophilin‐A1. Moreover, MS result for syntaxin‐1A was confirmed by Western blot analysis. Overall, these synaptic changes induced by neurotoxin may serve as a reference for understanding the functional mechanism of striatum in PD.     

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild‐type rats

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation‐prone proteins such as αSN, which is central in the development of Parkinson''s disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT‐positive aggregates that contain higher‐order β‐sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross‐seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.     

Effects of platelets on the protein expression in aortic segments: A proteomic approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor‐α (TNF‐α)‐preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton‐related proteins expression was modified by PRP. However, when PRP was coincubated with TNF‐α pre‐stimulated aortic segments lesser number of cytoskeleton‐related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic‐related proteins. However, in TNF‐α‐preincubated segments the presence of PRP upexpressed glyceraldehyde‐3‐phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF‐α‐preincubated segments the expression of fructose 1,6‐bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α‐chain. In addition, the activity of fructose 1,6‐bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall. J. Cell. Biochem. 111: 889–898, 2010. © 2010 Wiley‐Liss, Inc.     

Proteomic identification of nitrated proteins in Alzheimer's disease brain

Nitration of tyrosine in biological conditions represents a pathological event that is associated with several neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease (AD). Increased levels of nitrated proteins have been reported in AD brain and CSF, demonstrating the potential involvement of reactive nitrogen species (RNS) in neurodegeneration associated with this disease. Reaction of NO with O2- leads to formation of peroxynitrite ONOO-, which following protonation, generates cytotoxic species that oxidize and nitrate proteins. Several findings suggest an important role of protein nitration in modulating the activity of key enzymes in neurodegenerative disorders, although extensive studies on specific targets of protein nitration in disease are still missing. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. We previously applied a proteomics approach to determine specific targets of protein oxidation in AD brain, by successfully coupling immunochemical detection of protein carbonyls with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry analysis. In the present study, we extend our investigation of protein oxidative modification in AD brain to targets of protein nitration. The identification of six targets of protein nitration in AD brain provides evidence to the importance of oxidative stress in the progression of this dementing disease and potentially establishes a link between RNS-related protein modification and neurodegeneration.     

Protein interaction network for Alzheimer's disease using computational approach

Alzheimer''s disease (AD) is the most common form of dementia. It is the sixth leading cause of death in old age people. Despite recent advances in the field of drug design, the medical treatment for the disease is purely symptomatic and hardly effective. Thus there is a need to understand the molecular mechanism behind the disease in order to improve the drug aspects of the disease. We provided two contributions in the field of proteomics in drug design. First, we have constructed a protein-protein interaction network for Alzheimer''s disease reviewed proteins with 1412 interactions predicted among 969 proteins. Second, the disease proteins were given confidence scores to prioritize and then analyzed for their homology nature with respect to paralogs and homologs. The homology persisted with the mouse giving a basis for drug design phase. The method will create a new drug design technique in the field of bioinformatics by linking drug design process with protein-protein interactions via signal pathways. This method can be improvised for other diseases in future.     

Proteomic profiling of proteins associated with lymph node metastasis in colorectal cancer

Lymph node metastasis (LNM) is associated with poor prognosis in colorectal cancer (CRC). The presence or absence of lymph node metastases is a strong independent prognostic factor for CRC survival. Investigation of proteins associated with the process of lymph node metastasis (LNM) is crucial for understanding of the molecular mechanisms underlying the LNM process and for predicting the CRC prognosis. In the present study, proteins from CRC tissues and adjacent normal mucosa (NMC) were examined using two‐dimensional gel electrophoresis coupled with MALDI‐TOF‐MS. The expression levels of Ferritin Heavy Chain (FHC) were decreased in LNM CRC as compared to those in non‐LNM CRC, while the expression of Cathepsin D and Ubiquitin C‐terminal hydrolase‐L1 (UCH‐L1) were increased in LNM CRC. The results were confirmed by Western blotting and immunohistochemical staining. Furthermore, in vitro cell invasion assay showed that the overexpression of UCH‐L1 through gene transfection increased the invasive ability of HCT8 cells, suggesting that UCH‐L1 is not only a biomarker for LNM in CRC, but also a functional protein that may play a significant role in cell migration. The proteins identified in the present study should further our understanding of the LNM process of CRC and may become useful markers for diagnosis and targets for therapeutic interventions. J. Cell. Biochem. 110: 1512–1519, 2010. © 2010 Wiley‐Liss, Inc.     

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease

Parkinson's disease (PD) is an age‐related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α‐synuclein‐containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN‐induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early‐onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy‐lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2‐D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6‐month‐old PINK1‐deficient mice and wild‐type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD.