Evolution of Specificity in Protein-Protein Interactions

Hub proteins are proteins that maintain promiscuous molecular recognition. Because they are reported to play essential roles in cellular control, there has been a special interest in the study of their structural and functional properties, yet the mechanisms by which they evolve to maintain functional interactions are poorly understood. By combining biophysical simulations of coarse-grained proteins and analysis of proteins-complex crystallographic structures, we seek to elucidate those mechanisms. We focus on two types of hub proteins: Multi hubs, which interact with their partners through different interfaces, and Singlish hubs, which do so through a single interface. We show that loss of structural stability is required for the evolution of protein-protein-interaction (PPI) networks, and it is more profound in Singlish hub systems. In addition, different ratios of hydrophobic to electrostatic interfacial amino acids are shown to support distinct network topologies (i.e., Singlish and Multi systems), and therefore underlie a fundamental design principle of PPI in a crowded environment. We argue that the physical nature of hydrophobic and electrostatic interactions, in particular, their favoring of either same-type interactions (hydrophobic-hydrophobic), or opposite-type interactions (negatively-positively charged) plays a key role in maintaining the network topology while allowing the protein amino acid sequence to evolve.     

CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.     

Suppression by fucoidan of liver fibrogenesis via the TGF-β/Smad pathway in protecting against oxidative stress

Fucoidan, a sulfated polysaccharide extracted from various types of brown seaweed, possesses a wide range of pharmacological properties. We investigated the protective effect of fucoidan on dimethylnitrosamine-induced liver fibrogenesis in rats and its mechanism. Liver fibrosis was induced by injecting DMN (10 mg/kg, 3 times per week, I.P.) for 4 weeks, and fucoidan was simultaneously administered (100 mg/kg, 3 times per week, P.O.). The anti-oxidative and anti-inflammatory effects of fucoidan were observed by relative mediators. Fucoidan improved liver fibrosis by inhibiting the expression of transforming growth factor beta 1 (TGF-β(1))/Smad3 and the tissue inhibitor of metalloproteinase 1 (TIMP-1), and increasing the expression of metalloproteinase-9 (MMP-9). Fucoidan also significantly decreased the accumulation of the extracellular matrix (ECM) and collagen. These results suggest that fucoidan had an anti-fibrotic effect, which was exerted by inhibiting the TGF-β/Smad pathway, as well as anti-oxidative and anti-inflammatory effects.     

Proteomic analysis of differential protein expression in response to epidermal growth factor in neonatal porcine pancreatic cell monolayers

We have proposed that porcine neonatal pancreatic cell clusters (NPCCs) may be a useful alternative source of cells for islet transplantation, and that monolayer cultures might provide an opportunity to manipulate the cells before transplantation. In addition we previously identified 10 genes up-regulated by epidermal growth factor (EGF) in cultured porcine NPCC monolayers. We have now analyzed the intracellular signaling pathways activated by EGF and searched for proteins differentially expressed following EGF treatment of the monolayers, using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). EGF treatment resulted in phosphorylation of both Erk 1/2 and Akt, as well as increased cell proliferation. Five unknown and 13 previously identified proteins were differentially expressed in response to EGF. EGF treatment increased the expression of several structural proteins of epithelial cells, such as cytokeratin 19 and plakoglobin, whereas vimentin, the intermediate filament protein of mesenchymal cells, and non-muscle myosin alkali chain isoform 1, decreased. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 factor, which promotes epithelial cell proliferation, and hemoglobin alpha I & II also increased, whereas cyclin A1, immunoglobulin heavy chain, apolipoprotein A1, 5,10-ethylenetetrahydrofolated reductase (5,10-MTHFR), angiotensin-converting enzyme 2 (ACE2), co-lipase II precursor, and NAD+ isocitrate dehydrogenase (NAD+ IDH) alpha chain proteins decreased. Our results show that EGF stimulates proliferation of pancreatic epithelial cells by simultaneously activating the MAPK and PI-3K pathways. HnRNP A2/B1, hemoglobin, cyclin A1, and ACE2 may play roles in the proliferation of epithelial cells in response to EGF.     

Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis

We have previously shown that overexpressed chicken pp60c-src has retarded mobility, novel serine/threonine phosphorylation, and enhanced kinase activity during NIH 3T3 cell mitosis. Here we show that novel mitotic phosphorylations occur at Thr 34, Thr 46, and Ser 72. The possibility, previously raised, that Ser 17 is dephosphorylated during mitosis is excluded. The phosphorylated sites lie in consensus sequences for phosphorylation by p34cdc2, the catalytic component of maturation promoting factor (MPF). Furthermore, highly purified MPF from metaphase-arrested Xenopus eggs phosphorylated both wild-type and kinase-defective pp60c-src at these sites. Altered phosphorylation alone is sufficient to account for the large retardation in mitotic pp60c-src electrophoretic mobility: phosphorylation of normal pp60c-src by MPF retarded mobility and dephosphorylation of mitotic pp60c-src restored normal mobility. These results suggest that pp60c-src is one of the targets for MPF action, which may account in part for the pleiotropic changes in protein phosphorylation and cellular architecture that occur during mitosis.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Clinical relevance of infections with zoonotic and human oral species of <Emphasis Type="Italic">Campylobacter</Emphasis>

Genus Campylobacter has been recognized as a causative bacterial agent of animal and human diseases. Human Campylobacter infections have caused more concern. Campylobacters can be classified into two groups in terms of their original host: zoonotic and human oral species. The major zoonotic species are Campylobacter jejuni and Campylobacter coli, which mostly reside in the intestines of avian species and are transmitted to humans via consumption of contaminated poultry products, thus causing human gastroenteritis and other diseases as sequelae. The other campylobacters, human oral species, include C. concisus, C. showae, C. gracilis, C. ureolyticus, C. curvus, and C. rectus. These species are isolated from the oral cavity, natural colonization site, but have potential clinical relevance in the periodontal region to varying extent. Two species, C. jejuni and C. coli, are believed to be mainly associated with intestinal diseases, but recent studies suggested that oral Campylobacter species also play a significant role in intestinal diseases. This review offers an outline of the two Campylobacter groups (zoonotic and human oral), their virulence traits, and the associated illnesses including gastroenteritis.