A Proteomic Investigation of Isolated Soy Proteins with Variable Effects in Experimental and Clinical Studies

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Proteome analysis of serovars Typhimurium and Pullorum of Salmonella enterica subspecies I

BACKGROUND: Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella. RESULTS: In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars. CONCLUSION: Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods

Identification of citrullinated α-enolase as a candidate autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA

Changes in male rat urinary protein profile during puberty: a pilot study

BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, alpha2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n = 3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease. RESULTS: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (alpha2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins. CONCLUSIONS: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models

Antiandrogens Act as Selective Androgen Receptor Modulators at the Proteome Level in Prostate Cancer Cells*

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context

Smooth muscle cells in human atherosclerosis: proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease.

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Fibronectin III 13-14 Domains Induce Joint Damage via Toll-Like Receptor 4 Activation and Synergize with Interleukin-1 and Tumour Necrosis Factor

Cartilage loss is a feature of chronic arthritis. It results from degradation of the extracellular matrix which is composed predominantly of aggrecan and type II collagen. Extracellular matrix degradation is mediated by aggrecanases and matrix metalloproteinases (MMPs). Recently, a number of endogenous matrix molecules, including fibronectin (FN), have been implicated in mediating cartilage degradation. We were interested in studying the C-terminal heparin-binding region of FN since it mediates aggrecan and type II collagen breakdown in cartilage, but the specific FN domains responsible for proteolytic enzyme activity and their receptors in cartilage are unknown. In this study, the ability of recombinant FN domains to induce cartilage breakdown was tested. We found that the FN III 13-14 domains in the C-terminal heparin-binding region of FN are potent inducers of aggrecanase activity in articular cartilage. In murine studies, the FN III 13-14-induced aggrecanase activity was inhibited in Toll-like receptor 4 (TLR4) knockout mice but not wild-type mice. FN III 13-14 domains also synergized with the known catabolic cytokines interleukin-1α and tumour necrosis factor and induced secretion of MMP-1, MMP-3, gp38 and serum amyloid-like protein A in chondrocytes. Our studies provide a mechanistic link between the innate immune receptor TLR4 and sterile arthritis induced by the FN III 13-14 domains of the endogenous matrix molecule FN