Aims/hypothesis The aim of this study was to compare the protein profile of vitreous fluid from diabetic patients with proliferative diabetic retinopathy (PDR) with that from nondiabetic patients with idiopathic macular holes (MH). The mRNA of proteins differentially produced was also assessed in the retinas from diabetic and non-diabetic donors. Materials and methods Vitreous humour from type 1 diabetic patients with PDR (n=8) and from non-diabetic patients with MH (n=10) closely matched in terms of age were studied. The comparative proteomic analysis was performed using fluorescence-based difference gel electrophoresis (DIGE). Differentially produced proteins (abundance ratio >1.4, p<0.05) were identified by mass spectrometry. Expressions of mRNA were measured by real-time RT-PCR in retinas from ten human eyes obtained at post-mortem (five eyes from diabetic subjects and five eyes from non-diabetic subjects). Results Eight proteins were highly produced in PDR patients in comparison with non-diabetic subjects: zinc-α 2 -glycoprotein (ZAG), apolipoprotein (apo) A1, apoH, fibrinogen A, and the complement factors C3, C4b, C9 and factor B). We found three proteins that were underproduced in PDR subjects: pigment epithelial derived factor (PEDF), interstitial retinol-binding protein (IRBP) and inter-α-trypsin inhibitor heavy chain (ITIH2). There was no overlap in the vitreous levels of the above-mentioned proteins between PDR patients and non-diabetic control subjects. The differential production of ZAG, C3, factor B, PEDF and IRBP was further confirmed by western blot, and was in agreement with mRNA levels detected in the retina. Conclusions/interpretation Proteomic analysis by DIGE, which permits an accurate quantitative comparison, was useful in identifying new potential candidates involved in the pathogenesis of PDR.

BackgroundUnderstanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1Ser428 becomes phosphorylated in a RAS-Erk1/2-p90RSK pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown.Methodology/Principal FindingsUsing the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1Ser428 (Ser431 in the mouse) is constitutively phosphorylated in BRAFV600E mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAFV600E mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAFV600E mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1Ser428 phosphorylation. Notably, the inhibition of the RAS pathway in BRAFV600E mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKα metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1.Conclusions/SignificanceThese data demonstrate that growth factor treatment and in particular oncogenic BRAFV600E induces the uncoupling of LKB1-AMPKα complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.

Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program.Metastasis, the leading cause of death in cancer patients, is a multistep process that allows selected cells to move from the primary tumor and establish secondary tumors in different organs. Metastatic cells are endowed with specific abilities in order to escape from the initial tumor, survive in circulation, arrest in a distant capillary, extravasate, and grow in a remote site. Increased cell migration is required to fulfill several steps of the metastatic program (1).The initial characterization of cell surface and extracellular zinc endopeptidases showed that they can degrade components of the extracellular matrix (2). This finding along with the fact that, to invade neighboring tissue, migrating malignant cells induce the degradation of the extracellular matrix, led to an intuitive but simplistic hypothesis. Metalloproteases would contribute to tumor progression through the degradation of extracellular structural components. Following up on this view, the therapeutic value of several nonspecific metalloprotease inhibitors was assayed in clinical trials on cancer patients. Unfortunately, these trials failed, showing that the degradation of the extracellular matrix does not explain the role of zinc-dependent metalloproteases in human tumors (3).Extracellular metalloproteases are classified in large families including the matrix metalloproteases, the ADAMs (proteins containing a disintegrin and a metalloprotease domain), and the ADAMTS (ADAMs with thrombospondin motifs) (4). Contradicting the initial hypothesis, it has become recently clear that individual matrix metalloproteases as well as ADAMs and ADAMTSs fulfill far more sophisticated roles. They show restricted specificity, and their function depends on the substrates they cleave. Whereas some metalloproteases are protumorigenic, others act as tumor suppressors (5). For example, ADAM17 contributes to ...

This article is available online at http://www.jlr.org LDL, the main cholesterol carrier in plasma, is composed of approximately 75% lipid (mainly cholesterol) and 25% protein. The major protein in LDL is apolipoprotein B-100 (apoB-100), a protein of 550 kDa that accounts for more than 95% of the total protein mass in LDL ( 1 ). However, minor amounts of other apolipoproteins associated to LDL have been reported. Several of these proteins, such as apoE, apoC-III, and platelet-activating factor acetylhydrolase (PAF-AH), have important roles in LDL metabolism and modulate its atherogenicity despite their low concentration ( 2-4 ). Several studies have made a proteomic approach using 2D-electrophoresis or SELDI-based analysis to detect minor proteins in LDL ( 5-9 ), including apoE, apoC-III, apoC-II, apoA-I, apoA-IV, apoM, apoJ, serum amyloid A4 (SAA4), calgranulin A, lysozyme C, apoD, apoH, ␣ 1-antitrypsin, orosomucoid-1, paraoxonase-1, retinol binding protein, and prenylcysteine lyase-1.LDL is not a homogeneous entity but a group of particles that differs in density, size, electric charge, and composition. It is well established that small, dense LDL particles are more atherogenic than large, buoyant particles ( 10 ), and both of these subfractions differ in their protein content ( 6 ). Another property of LDL that confers enhanced atherogenicity is increased electronegative charge. Electronegative LDL [LDL( Ϫ )] is a minor subfraction of plasma LDL that is pro-infl ammatory and induces apoptosis in endothelial cells and leukocytes ( 11-13 ). Its relative proportion is increased in subjects Abstract Low density lipoprotein is a heterogeneous group of lipoproteins that differs in lipid and protein composition. One copy of apolipoprotein (apo)B accounts for over 95% of the LDL protein, but the presence of minor proteins could disturb its biological behavior. Our aim was to study the content of minor proteins in LDL subfractions separated by anion exchange chromatography. 29 July 2010. Published, JLR Papers in Press, August 10, 2010 DOI 10.1194 Abbreviations: apo, apolipoprotein; BHT, butylated hydroxytoluene; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; GGE, non-denaturing gradient gel electrophoresis; LDL(+), electropositive low-density lipoprotein; LDL( Ϫ ), electronegative low-density lipoprotein; Lp(a), lipoprotein (a); LRP2, low-density lipoprotein receptor-related protein 2; PAF-AH, platelet-activating factor-acetylhydrolase; SAA4, serum amyloid A4; TG, triglyceride; TTBS, Tweencontaining tris buffer saline. Supplementary key words apolipoproteins • atherosclerosis • modifi ed LDL This work was supported by Ministerio de Sanidad/Instituto de Salud Carlos III/FIS (ISCIII/FIS) Grants PI060500, CP040110 (S.B.), and CP060220 (JLS-Q.); and Ministerio de Educación y Ciencia Grant AP2004-1468 (C.B). The Proteomics Laboratory at Vall d'Hebron Research Institute is a member of the Instituto Nacional de Proteómica (ProteoRed) funded by Fundación Genoma España. CIBER de Diabetes y Enfermedades M...

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.

Objective. To identify the peptide anchor motif for the rheumatoid arthritis (RA)-related HLA allele, DR10, and find shared natural ligands or sequence similarities with the other disease-associated alleles, DR1 and DR4.Methods. The HLA-DR10-associated peptides were purified, and a proportion of these natural ligands were de novo sequenced by mass spectrometry. Based on crystallographic structures, the complexes formed by peptide influenza virus hemagglutinin HA306-318 with DR1, DR4, and DR10 were modeled, and binding scores were obtained.Results. A total of 238 peptides were sequenced, and the anchor motif of the HLA-DR10 peptide repertoire was defined. A large proportion of the DR10-associated peptides had the structural features to bind DR1 and DR4 but were theoretical nonbinders to the negatively associated alleles DR15 and DR7. Among the sequenced ligands, 10 had been reported as ligands to other RA-associated alleles. Modeling data showed that peptide HA306-318 can bind DR1, DR4, and DR10 with similar affinities.Conclusion. The data show the presence of common peptides in the repertoires of RA-associated HLA alleles. The combination of the shared epitope present in DR1, DR4, and DR10 together with common putative arthritogenic peptide(s) could influence disease onset or outcome.

Although stroke is among the most common causes of death and chronic disability worldwide, the proteome of the ischemic human brain remains unknown. Only a few studies have investigated the ischemic brain proteome in rodent stroke models. We performed a proteomic study of the human brain after ischemic stroke using a 2-dimensional differential gel electrophoresis-based proteomic approach. In brain samples from 6 deceased stroke patients and 3 control subjects, there was an average of 1,442 ± 231 protein spots in the gels. Changes of at least 1.5-fold in the relative expression of 132 protein spots between different cerebral areas (infarct core, peri-infarct, and contralateral tissue) were identified (p < 0.05); 39 of these were successfully identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the identified protein spots, we validated the results of 10 proteins by Western blot and determined the cellular localization in brain parenchyma for 3 of the identified proteins: dihydropyrimidinase-related protein 2, vesicle-fusing ATPase, and Rho dissociation inhibitor 1. These results contribute to understanding the processes that follow cerebral ischemia; moreover, some of the identified proteins may be therapeutic targets or biologic markers for determining the diagnosis and prognosis of stroke.

Background Diabetic macular edema is the main cause of visual impairment in diabetic patients. The aim of the present study was to explore the differential proteomic pattern of the vitreous fluid from diabetic macular edema patients by means of fluorescence-based difference gel electrophoresis (DIGE).