Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.     

Antigen retrieval for proteomic characterization of formalin-fixed and paraffin-embedded tissues

Formalin-fixed and paraffin-embedded tissues represent the vast majority of archived tissue. Access to such tissue specimens via shotgun-based proteomic analyses may open new avenues for both prospective and retrospective translational research. In this study, we evaluate the effects of fixation time on antigen retrieval for the purposes of shotgun proteomics. For the first time, we demonstrate the capability of a capillary isotachophoresis (CITP)-based proteomic platform for the shotgun proteomic analysis of proteins recovered from FFPE tissues. In comparison to our previous studies utilizing capillary isoelectric focusing, the CITP-based analysis is more robust and increases proteome coverage. In this case, results from three FFPE liver tissues yield a total of 4098 distinct Swiss-Prot identifications at a 1% false-discovery rate. To judge the accuracy of these assignments, immunohistochemistry is performed on a panel of 17 commonly assayed proteins. These proteins span a wide range of protein abundances as inferred from relative quantitation via spectral counting. Among the panel were 4 proteins identified by a single peptide hit, including three clusters of differentiation (CD) markers: CD74, CD117, and CD45. Because single peptide hits are often regarded with skepticism, it is notable that all proteins tested by IHC stained positive.     

Proteomic analysis of the inflamed intestinal mucosa reveals distinctive immune response profiles in Crohn's disease and ulcerative colitis

Although Crohn's disease (CrD) and ulcerative colitis (UC) share several clinical features, the mechanisms of tissue injury differ. Because the global cellular function depends upon the protein network environment as a whole, we explored changes in the distribution and association of mucosal proteins to define key events involved in disease pathogenesis. Endoscopic biopsies were taken from CrD, UC, and control colonic mucosa, and Multi-Epitope-Ligand-Cartographie immunofluorescence microscopy with 32 different Abs was performed. Multi-Epitope-Ligand-Cartographie is a novel, highly multiplexed robotic imaging technology which allows integrating cell biology and biomathematical tools to visualize dozens of proteins simultaneously in a structurally intact cell or tissue. In CrD, the number of CD3+CD45RA+ naive T cells was markedly increased, but only activated memory, but not naive, T cells expressed decreased levels of Bax, active caspase-3 or -8. In UC, only CD4+ T cells coexpressing NF-kappaB were caspase-8 and poly(ADP-ribose)-polymerase positive. Furthermore, the number of CD4+CD25+ T cells was elevated only in UC, whereas in CrD and controls, the number of these cells was similar. By using hub analysis, we also identified that the colocalization pattern with NF-kappaB+ and poly(ADP-ribose)-polymerase+ as base motifs distinguished CrD from UC. High-content proteomic analysis of the intestinal mucosa demonstrated for the first time that different T cell populations within the intestinal mucosa express proteins translating distinct biological functions in each form of inflammatory bowel disease. Thus, topological proteomic analysis may help to unravel the pathogenesis of inflammatory bowel disease by defining distinct immunopathogenic profiles in CrD and UC.     

Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation

The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent.     

Proteomic Profiling Identifies Distinct Protein Patterns in Acute Myelogenous Leukemia CD34+CD38- Stem-Like Cells

Acute myeloid leukemia (AML) is believed to arise from leukemic stem-like cells (LSC) making understanding the biological differences between LSC and normal stem cells (HSC) or common myeloid progenitors (CMP) crucial to understanding AML biology. To determine if protein expression patterns were different in LSC compared to other AML and CD34+ populations, we measured the expression of 121 proteins by Reverse Phase Protein Arrays (RPPA) in 5 purified fractions from AML marrow and blood samples: Bulk (CD3/CD19 depleted), CD34-, CD34+(CMP), CD34+CD38+ and CD34+CD38-(LSC). LSC protein expression differed markedly from Bulk (n=31 cases, 93/121 proteins) and CD34+ cells (n= 30 cases, 88/121 proteins) with 54 proteins being significantly different (31 higher, 23 lower) in LSC than in either Bulk or CD34+ cells. Sixty-seven proteins differed significantly between CD34+ and Bulk blasts (n=69 cases). Protein expression patterns in LSC and CD34+ differed markedly from normal CD34+ cells. LSC were distinct from CD34+ and Bulk cells by principal component and by protein signaling network analysis which confirmed individual protein analysis. Potential targetable submodules in LSC included the proteins PU.1(SP1), P27, Mcl1, HIF1α, cMET, P53, Yap, and phospho-Stats 1, 5 and 6. Protein expression and activation in LSC differs markedly from other blast populations suggesting that studies of AML biology should be performed in LSC.     

Protein changes in gastric epithelial cells RGM-1 in response to Helicobacter pylori infection

Helicobacter pylori-induced inflammation significantly increases the risk of gastric cancer. To investigate the role of H. pylori infection in gastric epithelial cell carcinogenesis, flow cytometry was used to analyze the apoptosis of gastric epithelial cells infected by H. pylori. Next, LTQ MS mass spectrometry (MS) was applied to identify protein changes in gastric epithelial cells infected with H. pylori, and then bioinformatics was adopted to analyze the cellular localization and biological function of differential proteins. LTQ MS/MS successfully identified identified 22 differential proteins successfully, including 20 host-cell proteins and two H. pylori bacterial proteins. Also, human proteins were located in all areas of cells and involved in various cell biological functions. The oncogene proteins p53, p16, and C-erbB-2 proteins in H. pylori-infected RGM-1 cells were remarkably increased from the analysis by Western blot analysis. H. pylori infection of gastric epithelial cells leads to changes in various protein components in the cell, and enhances the expression of oncogene proteins, thereby increasing the possibility of possibility of carcinogenesis of H. pylori infection.     

Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes

The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.     

A systematic characterization of mitochondrial proteome from human T leukemia cells

Global understanding of tissue-specific differences in mitochondrial signal transduction requires comprehensive mitochondrial protein identification from multiple cell and tissue types. Here, we explore the feasibility and efficiency of protein identification using the one-dimensional gel electrophoresis in combination with the nano liquid-chromatography tandem mass spectrometry (GeLC-MS/MS). The use of only 40 mug of purified mitochondrial proteins and data analysis using stringent scoring criteria and the molecular mass validation of the gel slices enables the identification of 227 known mitochondrial proteins (membrane and soluble) and 453 additional proteins likely to be associated with mitochondria. Replicate analyses of 60 mug of mitochondrial proteins on the faster scanning LTQ mass spectrometer validate all the previously identified proteins and most of the single hit proteins except the 81 single hit proteins. Among the identified proteins, 466 proteins are known to functionally participate in various processes such as respiration, tricarboxylic acid cycle (TCA cycle), amino acid and nucleotide metabolism, glycolysis, protection against oxidative stress, mitochondrial assembly, molecular transport, protein biosynthesis, cell cycle control, and many known cellular processes. The distribution of identified proteins in terms of size, pI, and hydrophobicity reveal that the present analytical strategy is largely unbiased and very efficient. Thus, we conclude that this approach is suitable for characterizing subcellular proteomes form multiple cells and tissues.     

Human T cells that are able to produce IL-17 express the chemokine receptor CCR6

Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.     

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.     

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity

The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.     

序列优化的小鼠IL-33基因在哺乳动物细胞中的有效分泌表达

目的: 白细胞介素(IL)-33具有重要的免疫调控作用,在疾病中扮演着重要的角色。本文旨在通过基因优化实现IL-33在哺乳动物细胞中的高效表达,为疾病机理研究以及疫苗免疫佐剂应用等提供基础。方法: 根据小鼠白细胞介素-33成熟肽(mIL-33)的氨基酸序列,以哺乳动物细胞基因表达密码子偏好性进行基因优化设计;化学合成优化的mIL-33基因片段,通过搭桥PCR将编码人CD8α信号肽的核酸序列分别与优化或未优化的mIL-33基因连接,并与绿色荧光蛋白(EGFP)基因分别构建到双表达单元质粒 pBudCE4.1的不同启动子下;重组质粒经 lipofectamine 3000 和PEI转染293FT 细胞;以 Western blot和ELISA检测重组蛋白的表达;收集表达的mIL-33刺激巨噬细胞Raw264.7,ELISA检测培养上清的TNFα水平,以证明IL-33的生物学活性。结果: 重组质粒经酶切鉴定及测序分析证实构建成功; lipofectamine 3000转染效率较PEI转染更高;Western blot和ELISA 结果显示密码子优化的mIL-33表达水平较未优化序列更高,在EF-1α启动子和CMV启动子指导下mIL-33在293FT 细胞表达水平相当,CD8α信号肽成功引导mIL-33的分泌,产物具生物学活性。结论: 密码子优化操作显著改善了 mIL-33在哺乳动物细胞中的表达水平,为进一步研究奠定了基础。     

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.     

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.