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BBA - General Subjects (v.1800, #6)
Cell migration—The role of integrin glycosylation
by Marcelina E. Janik; Anna Lityńska; Pierre Vereecken (pp. 545-555).
Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell–ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding.The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation.A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.
Keywords: Abbreviations; APC protein; adenomatous polyposis coli protein; ATRA; all-trans retinoic acid; BM; basal membrane; CMP-NeuAc; cytidine monophosphate-sialic acid; Csk; C-terminal Src kinase; ECM; extracellular matrix; EGF; epidermal growth factor; ER; endoplasmic reticulum; ERK; extracellular signal regulated kinase; FA; focal adhesion; FAK; focal adhesion kinase; FN; fibronectin; Fut8; α1,6-fucosyltransferase; Gal; galactose; GlcNAc; N; -acetylglucosamine; GnT; N; -acetylglucosaminyltransferase; GSK3β; glycogen synthase kinase-3β; GTP; guanosine triphosphate; HGF; hepatocyte growth factor; ILK; integrin linked kinase; JEB; junctional epidermolysis bullosa; JNK; c-Jun protein kinase; LEF-1; lymphoid enhancer-binding factor 1; LN-332; laminin 332; MAA; Maackia amurensis; agglutinin; Man; mannose; MAPK; mitogen-activated protein kinase; MMP; matrix metalloproteinase; MT1-MMP; membrane type 1 matrix metalloproteinase; p130Cas; Crk-associated substrate; PAK; p21 activated kinase; PHA-L; Phaseolus vulgaris; leucoagglutinin; PI3K; phosphatidylinositol 3-kinase; PKC; protein kinase C; PYK2; proline-rich tyrosine kinase-2; RGD; arginine-glycin-aspartic acid; RTK; receptor tyrosine kinase; SNA; Sambucus nigra; lectin; ST6GalI; α2-6 sialyltransferase I; TCF; T-cell factor; TGF-β; 1; transforming growth factor-β; 1; VN; vitronectinMigration; α; 3; β; 1; integrin; α; 5; β; 1; integrin; α; v; β; 3; integrin; N; -oligosaccharides
Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure
by Amir Y. Mirarefi; Sébastien Boutet; Subramanian Ramakrishnan; Andor J. Kiss; Chi-Hing C. Cheng; Arthur L. DeVries; Ian K. Robinson; Charles F. Zukoski (pp. 556-564).
The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (α, β, and γ). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37°C) along with several lenses from Antarctic fish (−2°C) and subtropical bigeye tuna (18°C).Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure.We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of α-crystallins.The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller γ-crystallins in the void space of α-crystallins.Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.
Keywords: Crystallin; Lens; Liquid–liquid phase separation; Transparency; Short-range order; Small-angle X-ray scattering (SAXS); Antarctica
Aberrant RNA splicing in RHD 7-9 exons of DEL individuals in Taiwan: A mechanism study
by Hsiang-Chun Liu; Hock-Liew Eng; Yu-Fen Yang; Ya-Hui Wang; Kuan-Tsou Lin; Hua-Lin Wu; Tsun-Mei Lin (pp. 565-573).
The Rh blood D group provides a clinically important model of aberrant splicing with skipped exons. Approximately 30% of serologically D-negative Chinese individuals have an intact RHD gene (DEL phenotype) and induce allo-immunization in transfusions. The RHD1227GNA polymorphism occurs in >95% DEL phenotype of Asian descent. The effects of RHD 1227A and a novel allele on exon 9 splicing were examined.Amplified DEL RNA products revealed that 3 transcripts involved skipping of exons 8-9, exon 9, or exon 9 with an inserted 170-bp cryptic exon located between exons 7 and 8. A novel, single nucleotide polymorphism was identified in the 7th intron, (IVS7) 923C>T, and present in all DEL patients. The odds ratio of RHD1227G>A allele with DEL phenotype was 2711. Splicing analysis of transcripts from minigenes containing the 1227GNA allele, but not the (IVS7) 923C>T allele, demonstrated aberrant exon 9 skipping.A combined haplotype of 1227G>A and IVS7 923C>T alleles was apparent in >95% DEL Chinese individuals. RHD1227A mutation significantly increased aberrant mRNA splicing, producing a hybrid RHD mRNA lacking exon 9. These results provide a molecular basis of the DEL phenotype in the Chinese population.
Keywords: RHD; gene; Single nucleotide polymorphism; DEL; Splicing mutation
Galactose specific adhesin of enteroaggregative E. coli induces IL-8 secretion via activation of MAPK and STAT-3 in INT-407 cells
by Atul Goyal; Monica Konar; Akanksha Setia; Anil Narang; Sujata Ghosh (pp. 574-579).
Enteroaggregative Escherichia coli (EAEC) is one of the most common bacterial pathogens associated with the etiology of persistent diarrhea. A characteristic feature of EAEC-pathogenesis is the induction of profound inflammatory response in the intestinal epithelium. The present study was designed to investigate the underlying mechanism of inflammatory responses induced by a novel galactose specific adhesin of T7 strain of EAEC (EAEC-T7) in human intestinal epithelial cell line (INT-407).INT-407 cells were stimulated with the adhesin in the absence and presence of anti-adhesin (IgGAD)/d-galactose/H7/staurosporin (inhibitor of PKC)/PD098059 (inhibitor of MEK)/SB203580 (inhibitor of p38-MAPkinase)/AG490 (inhibitor of JAK (-2,-3)/STAT-3 pathway). The expression of activated Raf-1, MEK-1, ERK1/2, JNK, p38-MAPK and STAT-3 was analyzed by Western immunoblot. Release of interleukin-8 (IL-8) was measured by ELISA.The adhesin was found to induce activation of Raf-1, MEK-1, ERK1/2, p38-MAPK and STAT-3, which was reduced in the presence of IgGAD/d-galactose. The activation of Raf-1 was found to be attenuated in the presence of H7/staurosporin. The expression of phosphorylated STAT-3 was downregulated in the presence of AG490 and PD098059. Further, the adhesin induced IL-8 secretion was reduced in the presence of the inhibitors of MEK (PD098059), p38-MAPK (SB203580) and JAK (-2,-3)/STAT-3 pathway (AG490).We propose that STAT-3 activation is quintessential for the galactose specific adhesin induced IL-8 secretion by INT-407 cells and must occur in concert with the activation of ERK1/2.Our contribution regarding the galactose specific adhesin mediated signaling leads to an improved understanding of the EAEC-pathogenesis and may provide novel therapeutic approaches to combat EAEC infection.
Keywords: EAEC; Adhesin; Signaling; MAPK; STAT-3; IL-8
Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration
by Yolanda M. Fortenberry; Stephanie Brandal; Ryan C. Bialas; Frank C. Church (pp. 580-590).
Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer.We found that PCI inhibits cathepsin L with an inhibition rate (k2) of 3.0×105M−1s−1. Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells.Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L.Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.
Keywords: Abbreviations; PCI; protein C inhibitor; AT; antithrombin; serpins; serpin protease inhibitors; SI; stoichiometry of inhibition; RCL; reactive center loop; DTT; dithiothreitolSerpins; Protein C inhibitor; siRNA; Cathepsins; Heparin; Breast cancer cells; Cell migration
Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade
by Nibedita Mahata; Debasis Pore; Amit Pal; Manoj K. Chakrabarti (pp. 591-598).
IP3-mediated calcium mobilization from intracellular stores activates and translocates PKC-α from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-α to membrane from cytosol in the E scherichia coli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205.Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole).STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-α translocation to the membrane.These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-α movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane.In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-α to membrane.
Keywords: Abbreviations; cGMP; cyclic GMP; STa; E. coli; heat-stable enterotoxin; PKC-α; protein kinase C-α; PMA; phorbol 12-myristate-13-acetate; BSA; bovine serum albumin; Ca; 2+; calcium Escherichia coli; heat-stable enterotoxin; Cytoskeleton; Cytochalasin D; PKC-α; COLO-205 cell line
Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride
by Arivazhagan Rajendran; Chunxia Zhao; Burki Rajendar; Viruthachalam Thiagarajan; Yusuke Sato; Seiichi Nishizawa; Norio Teramae (pp. 599-610).
We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA.The combination of experimental and theoretical methods was used.The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by31P NMR experiments. The thermodynamics of the ligand–nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory.Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling.The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and we believe that our studies will bring a new insight into such phenomena.
Keywords: Abbreviations; SNP; single nucleotide polymorphism; AP; abasic; DCPC; 3,5-diamino-6-chloro-2-pyrazinecarbonitrile; TDDFT; time dependent density functional theory; Spacer C3; Spacer phosphoramidite C3; ITC; isothermal titration calorimetry; CV; cyclic voltammetry; MM; molecular mechanics; HOMO; highest occupied molecular orbital; LUMO; lowest unoccupied molecular orbital; CT; charge transferSingle nucleotide polymorphism; Nucleobase recognition; Abasic site; Flanking bases; Amiloride; Molecular modeling; TDDFT
Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFβ1
by Catriona Clarke; Hussam Baghdadi; Alexander F. Howie; J. Ian Mason; Simon W. Walker; Geoffrey J. Beckett (pp. 611-618).
Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor β1 (TGFβ1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated.In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFβ1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells.We found that in response to treatment with TGFβ1, C3A cells produced mRNA encoding the pro-αI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase.Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFβ1, possibly as part of an epithelial to mesenchymal transition.These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.
Keywords: Selenium; Non-alcoholic steatohepatitis; Cytokine; Epithelial mesenchymal transition; Fatty acid; Transforming growth factor β1
Ras signalling regulates differentiation and UCP1 expression in models of brown adipogenesis
by Maria Murholm; Karen Dixen; Jacob B. Hansen (pp. 619-627).
The Ras/Raf/MEK/ERK pathway has been recognised as an important signalling module in adipogenesis and adipocyte function, but whether it promotes or inhibits the formation of fat cells has not been reconciled.Here we investigate the significance of Ras signalling intensity on two unrelated models of mouse brown adipocyte differentiation.A constitutively active H-Ras mutant (Ras V12) caused a complete block of adipose conversion, as manifested by a lack of both lipid accumulation and induction of adipocyte gene expression. The Ras V12-mediated impediment of differentiation was inefficiently rescued by forced expression of the adipogenic transcription factors C/EBPα and PPARγ. However, the defective differentiation was alleviated by MEK inhibitors, suggesting that the obstruction of differentiation was dependent on activation of ERK. A dominant interfering H-Ras mutant (Ras N17) did not inhibit differentiation, but led to increased expression of genes important for energy dissipation in brown fat cells, including UCP1.These data suggest that the intensity of Ras signalling is important for differentiation and UCP1 expression in models of brown adipogenesis.
Keywords: Abbreviations; AEBP1; AE-binding protein 1; C/EBP; CCAAT/enhancer-binding protein; CS; citrate synthase; DMEM; Dulbecco's Modified Eagle; '; s Medium; DMSO; dimethyl sulfoxide; ERK; extracellular signal-regulated kinase; FABP4; fatty acid-binding protein 4; FBS; foetal bovine serum; IGF-1; insulin-like growth factor-1; MEFs; mouse embryo fibroblasts; MEK; mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PGC-1α; PPARγ co-activator-1α; PI3K; phosphatidylinositol 3-kinase; PKB; protein kinase B; PPARγ; peroxisome proliferator-activated receptor γ; PRDM16; PR domain containing 16; Pref-1; pre-adipocyte factor-1; Rb; retinoblastoma gene; RT-qPCR; reverse transcription-quantitative polymerase chain reaction; SV40 TAg; simian virus 40 large T antigen; TBP; TATA-binding protein; TFIIB; transcription factor IIB; UCP1; uncoupling protein 1Brown fat; UCP1; Adipogenesis; Ras; ERK; MEK
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