BMP Stimulation of Alkaline Phosphatase Activity in Pluripotent Mouse C2C12 Cells is Inhibited by Dermatopontin,One of the Most Abundant Low Molecular Weight Proteins in Demineralized Bone Matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl₂, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of γ-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation

DNA-damaging antibiotics such as ciprofloxacin induce biofilm formation and the SOS response through autocleavage of SOS-repressor LexA in Pseudomonas aeruginosa. However, the biofilm-SOS connection remains poorly understood. It was investigated with 96-well and lipid biofilm assays. The effects of ciprofloxacin were examined on biofilm stimulation of the SOS mutant and wild-type strains. The stimulation observed in the wild-type in which SOS was induced was reduced in the mutant in which LexA was made non-cleavable (LexAN) and thus SOS non-inducible. Therefore, the stimulation appeared to involve SOS. The possible mechanisms of inducible biofilm formation were explored by subproteomic analysis of outer membrane fractions extracted from biofilms. The data predicted an inhibitory role of LexA in flagellum function. This premise was tested first by functional and morphological analyses of flagellum-based motility. The flagellum swimming motility decreased in the LexAN strain treated with ciprofloxacin. Second, the motility-biofilm assay was performed, which tested cell migration and biofilm formation. The results showed that wild-type biofilm increased significantly over the LexAN. These results suggest that LexA repression of motility, which is the initial event in biofilm development, contributes to repression of SOS-inducible biofilm formation.     

Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.     

结直肠癌蛋白质组学的研究进展

人类基因组计划(HGP)的完成,宣告了后基因组时代的到来,其中蛋白质组学已成为研究的重点.随着蛋白质组学研究的不断深入,现已形成一个"肿瘤蛋白质组学"的分支.肿瘤蛋白质组学可动态、整体、定量地观察肿瘤发生、进展中蛋白质种类、数量的改变,现已广泛用于各种肿瘤的研究.结直肠癌是人类的一种常见的恶性疾病,肿瘤蛋白质组学在寻找结直肠癌的肿瘤生物标记物,研究结直肠癌发生的分子机制和挖掘药物的抗癌靶点等方面得到了很好的应用.     

不同分期胃腺癌及癌旁组织蛋白质表达谱的研究

目的 建立II、IV期胃腺癌及癌旁胃组织的蛋白质表达谱并分析其表达的差异。方法 从胃腺癌II期和IV期患者各3例提取胃癌组织（CaII和CaIV）及胃旁组织（PII和PIV）中总蛋白,采用双向凝胶电泳获得CaII、CaIV、PII、PIV胃组织蛋白质表达谱,通过基质辅助激光解吸电离串联飞行时间质谱（MALDI-TOF/TOF）对主要差异蛋白进行分析。结果PII与CaII之间蛋白质表达有显著差异（达2.0倍以上）的蛋白点有28个,CaII3个蛋白点表达上调,25个蛋白点表达下调;PIV与CaIV之间蛋白质表达有显著差异（达2.0倍以上）的蛋白点有25个,CaIV这些蛋白点表达均下调。使用MALDI－TOF/TOF质谱成功鉴定到8个蛋白点在CaII和CaIV中的表达与PII、PIV均有显著差异,分别是谷胱甘肽巯基转移酶、内质网蛋白ERP29、SH3P、蛋白质膜联蛋白A4、肝脏型脂肪酸结合蛋白、磷酸丙糖异构酶、Transgelin和磷脂酰乙醇结合蛋白,这些蛋白大多与细胞增殖、细胞凋亡及信号转导相关。结论不同分期胃癌与其癌旁组织之间存在蛋白质表达差异,有助于从分子水平了解胃癌的发病机制,探索新的胃癌相关标志物和基因治疗靶点。     

In situ proteomic analysis by MALDI imaging identifies ubiquitin and thymosin-β4 as markers of malignant intraductal pancreatic mucinous neoplasms

PurposeIntraductal pancreatic mucinous neoplasms (IPMN) are precancerous cystic lesions. The aim was to investigate the in situ IPMN proteome using MALDI (Matrix-Assisted Laser Desorption/Ionisation) imaging and to characterize biomarkers associated with the grade of dysplasia.Experimental designFrozen human Branch duct -IPMN sections were selected according to dysplasia and proteomic analyses were performed by MALDI imaging to obtain mass spectra distribution. The most discriminating peaks were identified using tissue extraction and nanoLC-ESI-MS/MS. Identified peaks were validated in independent series of IPMN by immunochemistry on surgical specimens (tissue-microarrays (TMA), n = 45) and endoscopic ultrasound fine-needle aspiration (EUS FNA) samples (n = 25).ResultsBD-IPMN samples with low (n = 10) and high (n = 10) grades of dysplasia were analyzed. Differential spectra of proteins were found in the two groups with significantly different intensities (n = 15). The two peaks (intense in high grade IPMN) (m/z 8565 and 4747) were characterized as the monomeric ubiquitin (Mascot score = 319.22) and an acetylated fragment of thymosin-β4 (2–42) (Omssa score = 1.37 E−9). Validation on TMA and EUS FNA samples confirmed that ubiquitin was overexpressed in high grade dysplasia (p = 0.04 and p = 0.0004). Thymosin-β4 expression was confirmed on TMA by immunohistochemistry on high grade IPMN (p = 0.011).ConclusionUbiquitin and thymosin-β4 are overexpressed in IPMN with high grade dysplasia. Positive immunochemical staining on EUS-FNA material is a major argument in support of preventive resection.     

A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.