Functionally specified protein signatures distinctive for each of the different blue copper proteins

BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences

Long-Chain Acyl Coenzyme A Synthetase 1 Overexpression in Primary Cultured Schwann Cells Prevents Long Chain Fatty Acid-Induced Oxidative Stress and Mitochondrial Dysfunction

Aims: High circulating long chain fatty acids (LCFAs) are implicated in diabetic neuropathy (DN) development. Expression of the long-chain acyl-CoA synthetase 1 (Acsl1) gene, a gene required for LCFA metabolic activation, is altered in human and mouse diabetic peripheral nerve. We assessed the significance of Acsl1 upregulation in primary cultured Schwann cells. Results: Acsl1 overexpression prevented oxidative stress (nitrotyrosine; hydroxyoctadecadienoic acids [HODEs]) and attenuated cellular injury (TUNEL) in Schwann cells following 12?h exposure to LCFAs (palmitate, linoleate, and oleate, 100??M). Acsl1 overexpression potentiated the observed increase in medium to long-chain acyl-carnitines following 12?h LCFA exposure. Data are consistent with increased mitochondrial LCFA uptake, largely directed to incomplete beta-oxidation. LCFAs uncoupled mitochondrial oxygen consumption from ATP production. Acsl1 overexpression corrected mitochondrial dysfunction, increasing coupling efficiency and decreasing proton leak. Innovation: Schwann cell mitochondrial function is critical for peripheral nerve function, but research on Schwann cell mitochondrial dysfunction in response to hyperlipidemia is minimal. We demonstrate that high levels of a physiologically relevant mixture of LCFAs induce Schwann cell injury, but that improved mitochondrial uptake and metabolism attenuate this lipotoxicity. Conclusion: Acsl1 overexpression improves Schwann cell function and survival following high LCFA exposure in vitro; however, the observed endogenous Acsl1 upregulation in peripheral nerve in response to diabetes is not sufficient to prevent the development of DN in murine models of DN. Therefore, targeted improvement in Schwann cell metabolic disposal of LCFAs may improve DN phenotypes. Antioxid. Redox Signal. 21, 588?600.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140243/1/ars.2013.5248.pd

Type I interferons modulate vascular function, repair, thrombosis, and plaque progression in murine models of lupus and atherosclerosis

Objective Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. Methods Lupus‐prone New Zealand mixed 2328 (NZM) mice and atherosclerosis‐prone apolipoprotein E– knockout (apoE −/− ) mice were compared to mice lacking type I IFN receptor (INZM and apoE −/− IFNAR −/− mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE −/− mice exposed to an IFNα‐containing or empty adenovirus. Results Loss of type I IFN receptor signaling improved endothelium‐dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus‐prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus‐prone and non–lupus‐prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE −/− IFNAR −/− mice, compared to apoE −/− mice, while NZM and apoE −/− mice exposed to IFNα developed accelerated thrombosis and platelet activation. Conclusion These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93543/1/34504_ftp.pd

Neutrophil Extracellular Trap–Derived Enzymes Oxidize High‐Density Lipoprotein: An Additional Proatherogenic Mechanism in Systemic Lupus Erythematosus

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108351/1/art38703.pd

Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Objective Citrullinated proteins are immunogenic in rheumatoid arthritis (RA), particularly in patients who carry shared epitope (SE)–coding HLA–DRB1 alleles. The mechanism underlying this association is unknown. We have previously identified the SE as a ligand that interacts with cell surface calreticulin (CRT) and activates immune dysregulation. This study was undertaken to determine the effect of CRT citrullination on SE signaling. Methods CRT–SE binding affinity was measured by surface plasmon resonance. The role of individual CRT arginine residues was determined by site‐directed mutagenesis, and nitric oxide levels were measured using a fluorochrome‐based assay. CRT citrullination in synovial tissue samples and cell cultures was determined by 2‐dimensional gel electrophoresis, immunoblotting, and mass spectrometry techniques. Results Synovial tissue and fibroblast‐like synoviocytes from RA patients were found to express a higher abundance of citrullinated CRT than samples from osteoarthritis patients. Citrullinated CRT showed more robust interaction with the SE ligand, and transduced SE signaling at a 10,000‐fold higher potency, compared to noncitrullinated CRT. Site‐directed mutation analysis identified Arg 205 , which is spatially adjacent to the SE binding site in the CRT P‐domain, as a dominant inhibitor of SE–CRT interaction and signaling, while a more remote arginine residue, Arg 261 , was found to enhance these SE functions. Conclusion Our findings indicate that citrullinated CRT is overabundant in the RA synovium and potentiates SE‐activated signaling in vitro. These findings could introduce a new mechanistic model of gene–environment interaction in RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96655/1/37814_ftp.pd

Endothelial Acyl-CoA Synthetase 1 Is Not Required for Inflammatory and Apoptotic Effects of a Saturated Fatty Acid-Rich Environment

Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells (ECs) and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSL). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating EC responses to a saturated fatty acid-rich environment in vitro and in vivo

Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1

Author contributions: J.E.K., L.B., C.N.S., and K.E.B. designed research; J.E.K., F.K., S.B., M.M.A., A.V.-G., T.V., L.O.L., L.B., K.R.B., and S.P.-P. performed research; W.Y., A.C., S.S., T.N.W., J.W.H., and R.A.C. contributed new reagents/analytic tools; J.E.K., L.B., S.S., S.P., and K.E.B. analyzed data; and J.E.K. and K.E.B. wrote the paper

Metabolomic Profiling Reveals a Role for Androgen in Activating Amino Acid Metabolism and Methylation in Prostate Cancer Cells

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer