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.

The X-linked retinitis pigmentosa protein RP2 facilitates G protein traffic.

The X-linked retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins are implicated in the traffic of proteins to the primary cilia. Here, we show that RP2 can facilitate the traffic of the Gß subunit of transducin (Gß1). Glutathione S-transferase (GST)-RP2 pulled down Gß from retinal lysates and the interaction was specific to Gß1, as Gß3 or Gß5L did not bind RP2. RP2 did not appear to interact with the Gß:Gγ heterodimer, in contrast Gγ1 competed with RP2 for Gß binding. Overexpression of Gß1 in SK-N-SH cells led to a cytoplasmic accumulation of Gß1, while co-expression of RP2 or Gγ1 with Gß1 restored membrane association of Gß1. Furthermore, RP2 small interfering RNA in ARPE19 cells resulted in a reduction in Gß1 membrane association that was rescued by Gγ1 overexpression. The interaction of RP2 with Gß1 required RP2 N-terminal myristolyation and the co-factor C (TBCC) homology domain. The interaction was also disrupted by the pathogenic mutation R118H, which blocks Arl3 GAP activity. Interestingly, Arl3-Q71L competed with Gß1 for RP2 binding, suggesting that Arl3-GTP binding by RP2 would release Gß1. RP2 also stimulated the association of Gß1 with Rab11 vesicles. Collectively, the data support a role for RP2 in facilitating the membrane association and traffic of Gß1, potentially prior to the formation of the obligate Gß:Gγ heterodimer. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in the cilia-associated traffic of G proteins.

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage.

OBJECTIVE: To investigate whether cartilage injury activates protein tyrosine kinases distinct from fibroblast growth factor (FGF)-related signaling, and whether they contribute to injury-induced gene responses. METHODS: Phosphokinases and protein tyrosine phosphorylation were assayed by Western blotting of cartilage lysates. Immunoprecipitation and Western blotting with 4G10 antibody and immunoprecipitation kinase assay were carried out. Tyrosine-phosphorylated proteins on silver-stained gels of injured cartilage lysates were identified by mass spectrometry. Messenger RNA induction in cartilage explants was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling. CONCLUSION: Injury to the surface of intact articular cartilage activates Src-like kinases as well as MAPKs and IKK (implying NF-κB activation). FGF-2 contributes to MAPK/IKK activation but not to Src-like signaling, suggesting that the latter is a parallel pathway that also regulates the injury-induced inflammatory gene response.

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

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated ß1-integrin-dependent cell migration.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human ß1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of ß1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from ß1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Tenascin-C fragments are endogenous inducers of cartilage matrix degradation.

Cartilage destruction is a hallmark of osteoarthritis (OA) and is characterized by increased protease activity resulting in the degradation of critical extracellular matrix (ECM) proteins essential for maintaining cartilage integrity. Tenascin-C (TN-C) is an ECM glycoprotein, and its expression is upregulated in OA cartilage. We aimed to investigate the presence of TN-C fragments in arthritic cartilage and establish whether they promote cartilage degradation. Expression of TN-C and its fragments was evaluated in cartilage from subjects undergoing joint replacement surgery for OA and RA compared with normal subjects by western blotting. The localization of TN-C in arthritic cartilage was also established by immunohistochemistry. Recombinant TN-C fragments were then tested to evaluate which regions of TN-C are responsible for cartilage-degrading activity in an ex vivo cartilage explant assay measuring glycosaminoglycan (GAG) release, aggrecanase and matrix metalloproteinase (MMP) activity. We found that specific TN-C fragments are highly upregulated in arthritic cartilage. Recombinant TN-C fragments containing the same regions as those identified from OA cartilage mediate cartilage degradation by the induction of aggrecanase activity. TN-C fragments mapping to the EGF-L and FN type III domains 3-8 of TN-C had the highest levels of aggrecan-degrading ability that was not observed either with full-length TN-C or with other domains of TN-C. TN-C fragments represent a novel mechanism for cartilage degradation in arthritis and may present new therapeutic targets for the inhibition of cartilage degradation.

The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response.

Following estrogenic activation, the estrogen receptor-alpha (ERalpha) directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) modulated by ERalpha have the potential to fine tune these regulatory systems and also provide an alternate mechanism that could impact on estrogen-dependent developmental and pathological systems. Through a microarray approach, we identify the subset of microRNAs (miRNAs) modulated by ERalpha, which include upregulation of miRNAs derived from the processing of the paralogous primary transcripts (pri-) mir-17-92 and mir-106a-363. Characterization of the mir-17-92 locus confirms that the ERalpha target protein c-MYC binds its promoter in an estrogen-dependent manner. We observe that levels of pri-mir-17-92 increase earlier than the mature miRNAs derived from it, implicating precursor cleavage modulation after transcription. Pri-mir-17-92 is immediately cleaved by DROSHA to pre-miR-18a, indicating that its regulation occurs during the formation of the mature molecule from the precursor. The clinical implications of this novel regulatory system were confirmed by demonstrating that pre-miR-18a was significantly upregulated in ERalpha-positive compared to ERalpha-negative breast cancers. Mechanistically, miRNAs derived from these paralogous pri-miRNAs (miR-18a, miR-19b, and miR-20b) target and downregulate ERalpha, while a subset of pri-miRNA-derived miRNAs inhibit protein translation of the ERalpha transcriptional p160 coactivator, AIB1. Therefore, different subsets of miRNAs identified act as part of a negative autoregulatory feedback loop. We propose that ERalpha, c-MYC, and miRNA transcriptional programs invoke a sophisticated network of interactions able to provide the wide range of coordinated cellular responses to estrogen.

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis.

BACKGROUND: Human T lymphotropic virus Type 1 (HTLV-1) causes a chronic inflammatory disease of the central nervous system known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM) which resembles chronic spinal forms of multiple sclerosis (MS). The pathogenesis of HAM remains uncertain. To aid in the differential diagnosis of HAM and to identify pathogenetic mechanisms, we analysed the plasma proteome in asymptomatic HTLV-1 carriers (ACs), patients with HAM, uninfected controls, and patients with MS. We used surface-enhanced laser desorption-ionization (SELDI) mass spectrometry to analyse the plasma proteome in 68 HTLV-1-infected individuals (in two non-overlapping sets, each comprising 17 patients with HAM and 17 ACs), 16 uninfected controls, and 11 patients with secondary progressive MS. Candidate biomarkers were identified by tandem Q-TOF mass spectrometry. RESULTS: The concentrations of three plasma proteins--high [ß2-microglobulin], high [Calgranulin B], and low [apolipoprotein A2]--were specifically associated with HAM, independently of proviral load. The plasma [ß2-microglobulin] was positively correlated with disease severity. CONCLUSIONS: The results indicate that monocytes are activated by contact with activated endothelium in HAM. Using ß2-microglobulin and Calgranulin B alone we derive a diagnostic algorithm that correctly classified the disease status (presence or absence of HAM) in 81% of HTLV-1-infected subjects in the cohort.

Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis.

OBJECTIVE: To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). METHODS: The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis-knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and α-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. RESULTS: Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or α-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. CONCLUSION: Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis-mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA.

Proteomic and metabolomic analysis of atrial profibrillatory remodelling in congestive heart failure.

Congestive heart failure (CHF) leads to atrial structural remodelling and increased susceptibility to atrial fibrillation. The underlying molecular mechanisms are poorly understood. We applied high-throughput proteomic and metabolomic analysis to left-atrial cardiomyocytes and tissues obtained from sham and ventricular-tachypaced (VTP, 240 bpm × 24 h and × 2 weeks) CHF dogs. Protein-extracts were subjected to two-dimensional gel electrophoresis using differential in-gel electrophoresis technology. Differentially expressed (P<0.05) proteins were identified by tandem mass-spectrometry. Cardiac metabolites were assayed with high-resolution NMR spectroscopy. Extensive changes occurred in structural proteins, particularly at 2-week VTP, with desmin and filamin fragmentation suggesting structural damage, which was confirmed by electron-microscopy. Oxidant stress was evidenced by decreased antioxidant proteins (superoxide dismutase and peroxiredoxin) at 2-week VTP. Extensive changes in cardioprotective heat shock proteins (HSPs) occurred, with several proteins increasing rapidly (HSP27, HSP60 and HSP70) and others showing a delayed rise (GRP78, α-B-crystallin, and HSP90). An evolving adaptive response to metabolic stress was suggested by early upregulation of malate dehydrogenase (DH), α-/ß-enolase and pyruvate dehydrogenase (α-subunit of E1 component) and delayed downregulation of a host of enzymes, along with extensive metabolomic changes. Early changes in metabolite expression that persisted as CHF developed included increased concentrations of glucose and alanine. ADP/ATP accumulation and alpha-ketoisovalerate depletion at 2-week VTP suggested a combination of metabolic stress and less effective energy utilization, as well as a shift from glycolysis to alpha-ketoacid metabolism. We conclude that VTP-induced CHF causes time-dependent changes in the atrial proteome and metabolome, providing insights into molecular mechanisms contributing to arrhythmogenic atrial remodelling.

Proteomic analysis of human low-density lipoprotein reveals the presence of prenylcysteine lyase, a hydrogen peroxide-generating enzyme.

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid-phase IEF has been used to resolve LDL proteins into well-defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL-associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol-binding protein, and paraoxonase-1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time-dependent generation of H(2)O(2) which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H(2)O(2) generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis.

Evolutionarily conserved antigens in autoimmune disease: implications for an infective aetiology.

The immune system has evolved to eliminate or inactivate infectious organisms. An inappropriate response against self-components (autoantigens) can result in autoimmune disease. Here we examine the hypothesis that some evolutionarily conserved proteins, present in pathogenic and commensal organisms and their hosts, provide the stimulus that initiates autoimmune disease in susceptible individuals. We focus on seven autoantigens, of which at least four, glutamate decarboxylase, pyruvate dehydrogenase, histidyl-tRNA synthetase and alpha enolase, have orthologs in bacteria. Citrullinated alpha-enolase, a target for autoantibodies in 40% of patients with rheumatoid arthritis, is our main example. The major epitope is highly conserved, with over 90% identity to human in some bacteria. We propose that this reactivity of autoantibodies to shared sequences provides a model of autoimmunity in rheumatoid arthritis, which may well extend to other autoimmune disease in humans.

Development and characterization of polyspecific anti-mitochondrion antibodies for proteomics studies on in toto tissue homogenates.

We describe the characterization of polyclonal antibodies directed against the whole mitochondrial subproteome, as obtained by hyperimmunization of rabbits with an organelle fraction purified from human skeletal muscle and lysed by sonication. After 2-DE separations with either blue native electrophoresis or IPG as first dimension and blotting, the polyspecific antibodies detect 113 proteins in human muscle mitochondria, representative of all major biochemical pathways and oxidative phosphorylation (OXPHOS) complexes, and cross-react with 28 proteins in rat heart mitochondria. Using as sample cryosections of human muscle biopsies lysed in urea/thiourea/CHAPS, the mitochondrial subproteome can be detected against the background of contractile proteins. When comparing with controls samples from mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes patients, immunoblotting shows in the latter a drastic reduction for the subunits of OXPHOS complex I as well as an increase of several enzymes, including ATP synthase. This finding is the first evidence at the proteomic level of massive up-regulation in a number of metabolic pathways by which the affected tissues try to compensate for the deficit in the OXPHOS machinery.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts.

The amylose extender (ae(-)) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein-protein interactions in amyloplasts of ae(-) mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein-protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae(-) amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae(-) endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae(-)), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.

Proteomic analysis of the lymphocyte plasma membrane using cell surface biotinylation and solution-phase isoelectric focusing.

Plasma membrane (PM) proteins are of particular interest to cell biologists because of their role in transducing information from the external environment to the cell interior, and because of their potential as therapeutic targets. The hydrophobicity and large size of these proteins renders their analysis by conventional proteomic approaches using 2D-electrophoresis problematic, limiting our ability to evaluate alterations of cell surface architecture as a function of varying physiological, pathological, or developmental state.In this chapter, we describe a simple method for enrichment and separation of plasma membrane proteins, prior to their identification by tandem mass spectrometry. Cell surface proteins are labeled with biotin using a reagent which does not enter the cell, purified by differential centrifugation and then affinity captured with streptavidin-agarose beads, before separation by a combination of solution-phase isoelectric focusing, and gradient gel electrophoresis, resulting in highly enriched membrane protein fractions suitable for characterization by mass spectrometry. We discuss the application of this protocol to the semiquantitative comparison of the plasma membrane proteins from resting and activated murine lymphocytes.

A proteomic reference map for pig serum proteins as a prerequisite for diagnostic applications.

A reference protein map for pig serum was set up using two-dimensional electrophoresis (2-DE). Thirty-nine protein chains or spots deriving from 26 different proteins were identified by immunological and mass spectrometric methods. Thus, the positions of most medium to higher abundance serum proteins could be determined on the 2-DE gels. The plasma protein fibrinogen was also included in our study. The overall pig protein pattern differs in some respect to serum/plasma maps of other mammalian species, e.g. in levels and properties of single proteins such as haptoglobin or IgM or in species-specific proteins like pig major acute phase protein. Serum protein maps are a useful tool to get an overview on expressed proteins, and to monitor changes in concentration as well as isotype distribution of the identified proteins. As a consequence, more detailed knowledge on protein pattern changes may give deeper insights into the metabolic development of some pathologic conditions and may lead to putative biomarkers for further investigation. Selected examples for protein pattern changes in pigs infected by a viral (porcine circovirus type 2) and a bacterial (Actinobacillus pleuropneumoniae) pathogen illustrate the usefulness of the method.

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia.

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 alpha (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre-hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.

Proteomic analysis of proteins regulated by TRPS1 transcription factor in DU145 prostate cancer cells.

The aim of the present study was to identify proteins differentially regulated by TRPS1 in human prostate cancer cells in order to better understand the role of TRPS1 in prostate cancer development. The proteomes of androgen-independent DU145 prostate cancer cells, that do not express TRPS1 and of genetically engineered DU145 cells that stable and inducible express recombinant TRPS1 protein, were compared. Using two-dimensional electrophoresis followed by mass spectrometric analysis, 13 proteins that were differentially expressed between these two cell lines were identified. These proteins represent a dominant reduction of expression of antioxidant proteins, including superoxide dismutase, protein disulfide isomerase A3 precursor, endoplasmin precursor and annexin A2. Furthermore, regulation was observed for mitochondrion-associated proteins, glycolytic enzymes, a cytoskeleton-associated protein, a nuclear protein and proteins involved in apoptosis. Our data indicate that overexpression of TRPS1 protein is correlated with reduced protein expression of certain antioxidants. This suggests a possible involvement of TRPS1 in oxidative stress, and possibly in apoptosis in androgen-independent DU145 prostate cancer cells.

Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae--involvement of the mitochondrial FAD transporter, Flx1p.

The mitochondrial FAD transporter, Flx1p, is a member of the mitochondrial carrier family responsible for FAD transport in Saccharomyces cerevisiae. It has also been suggested that it has a role in maintaining the normal activity of mitochondrial FAD-binding enzymes, including lipoamide dehydrogenase and succinate dehydrogenase flavoprotein subunit Sdh1p. A decrease in the amount of Sdh1p in the flx1Delta mutant strain has been determined here to be due to a post-transcriptional control that involves regulatory sequences located upstream of the SDH1 coding sequence. The SDH1 coding sequence and the regulatory sequences located downstream of the SDH1 coding region, as well as protein import and cofactor attachment, seem to be not involved in the decrease in the amount of protein.

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction.

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.