Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria

The Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria translocates effector proteins via a specialized type III secretion (TTS) system into the host cell cytosol. The efficient secretion of many effector proteins depends on the global TTS chaperone HpaB. Here, we identified a novel export control protein, HpaC, which significantly contributes to bacterial pathogenicity. Deletion of hpaC leads to a severe reduction in secretion of effector proteins and the putative type III translocon proteins HrpF and XopA. By contrast, secretion of the TTS pilus protein HrpE is not affected. We provide experimental evidence that HpaC differentiates between two classes of effector proteins. Using an in vivo reporter assay, we found that HpaC specifically promotes the translocation of the effector proteins XopJ and XopF1 into the plant cell, whereas AvrBs3 and XopC are efficiently translocated even in the absence of HpaC. Similar findings were obtained for HpaB. Inhibition of protein synthesis suggests that HpaB is involved in the secretion of stored effector proteins. Furthermore, protein-protein interaction studies revealed that HpaB and HpaC form an oligomeric protein complex and that they interact with members of both effector protein classes and the conserved TTS system component HrcV. Taken together, our data indicate that HpaB and HpaC play a central role in recruiting TTS substrates to the secretion apparatus.     

Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.     

Factor VIIa inhibitors: chemical optimization, preclinical pharmacokinetics, pharmacodynamics, and efficacy in an arterial baboon thrombosis model

Highly selective and potent factor VIIa-tissue factor (fVIIa.TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentration-response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenox) in the baboon model are also presented.     

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.     

Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state

ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.     

The proteoglycan osteoglycin/mimecan is correlated with arteriogenesis

Arteriogenesis or collateral growth is able to compensate for the stenosis of major arteries. Using differential display RT-PCR on growing and quiescent collateral arteries in a rabbit femoral artery ligation model, we cloned the rabbit full-length cDNA of osteoglycin/mimecan. Osteoglycin was present in the adventitia of collateral arteries as a glycosylated protein without keratan sulfate side chains, mainly produced by smooth muscle cells (SMCs) and perivascular fibroblasts. Northern blot, Western blot, and immunohistochemistry confirmed a collateral artery-specific downregulation of osteoglycin from 6 h to 3 weeks after the onset of arteriogenesis. Treatment of primary SMCs with the arteriogenic protein fibroblast growth factor-2 (FGF-2) resulted in a similar reduction of osteoglycin expression as observed in vivo. Application of the FGF-2 inhibitor polyanethole sulfonic acid (PAS) blocked the downregulation of osteoglycin and interfered with arteriogenesis. From our study we conclude that downregulation of osteoglycin is a fundamental requirement for proper arteriogenesis.     

Improving derivatization efficiency of BMAA utilizing AccQ-Tag<Superscript>®</Superscript> in a complex cyanobacterial matrix

Two different assays have been developed and used in order to investigate the optimal conditions for derivatization and detection of acid beta-N-methyl-amino-L-alanine (BMAA) in a cyanobacterial sample. BMAA was extracted from cyanobacterial cultures both from the cytosolic ("free") fraction and in the precipitated ("protein") fraction using a newly developed extraction scheme and the sample matrix was standardized according to protein concentration to ensure the highest possible derivative yield. A rapid and sensitive HPLC method for fluorescence detection of the non-protein amino acid BMAA in cyanobacteria, utilizing the Waters AccQ-Tag chemistry and Chromolith Performance RP-18e columns was developed. Using this new method and utilizing a different buffer system and column than that recommended by Waters, we decreased the time between injections by 75%. The limit of quantification was determined to be 12 nmol and limit of detection as 120 fmol. The linear range was in the range of 8.5 nmol-84 pmol. Accuracy and precision were well within FDA guidelines for bioanalysis.     

ADAM12 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity

ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.