Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time-and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against S. aureus at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin, S. aureus strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by S. aureus contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional nonactive site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E 0 ) of the Trx1 active site (؊230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an ␣ helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the nonactive site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.Thioredoxin (Trx1) 1 is a ubiquitous 12-kDa protein that functions as a reductant for ribonucleotide reductase, peroxiredoxins, and transcription factors (e.g. Fos, Jun, NF-B, p53), controlling key aspects of cell proliferation and survival (1-4). The active site of Trx1, WCGPC, is conserved among species from cyanobacteria to humans (5). The active site cysteines are readily accessible on the surface of the protein and become oxidized to a disulfide upon reduction of a target protein. This disulfide is cycled back to the dithiol by Trx reductase (6).Unlike Trxs from lower species, mammalian Trx1 contains additional conserved cysteine residues (at positions 62, 69, and 73 of human Trx1; See Fig. 1). Whether these non-active site Cys residues have biologic function is unknown. Cys-73 was present as an intermolecular disulfide bond (Trx1 homodimer) in x-ray crystal studies (7), suggesting a possible function for Cys-73. However, a mutant Trx1 bearing a serine at this position still appeared as a homodimer in the crystal structure, suggesting that Cys-73 was not essential for dimerization (7). More recently, S-glutathionylation of Trx1 at Cys-73 has been found during oxidative stress (8). In addition, S-nitrosylation of Cys-69 has recently been described (9).The midpoint potential (E 0 ) for the active site dithiol of Trx is available for several lower species (10 -14) but not for mammals. Equilibrium with NADPH in the presence of a catalytic amount of Trx reductase, where it is assumed that each mole of NADPH consumed translates into ...

The redox potential of the plasma cysteine/cystine couple (E h CySS) is oxidized in association with risk factors for cardiovascular disease (CVD), including age, smoking, type 2 diabetes, obesity, and alcohol abuse. Previous in vitro findings support a cause-effect relationship for extracellular E h CySS in cell signaling pathways associated with CVD, including those controlling monocyte adhesion to endothelial cells. In this study, we provide evidence that mitochondria are a major source of reactive oxygen species (ROS) in the signaling response to a more oxidized extracellular E h CySS. This increase in ROS was blocked by overexpression of mitochondrial thioredoxin-2 (Trx2) in endothelial cells from Trx2-transgenic mice, suggesting that mitochondrial thiol antioxidant status plays a key role in this redox signaling mechanism. Mass spectrometry-based redox proteomics showed that several classes of plasma membrane and cytoskeletal proteins involved in inflammation responded to this redox switch, including vascular cell adhesion molecule, integrins, actin, and several Ras family GTPases. Together, the data show that the proinflammatory effects of oxidized plasma E h CySS are due to a mitochondrial signaling pathway that is mediated through redox control of downstream effector proteins.

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.