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Serratia marcescens is a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein ‘secretome’ comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the ‘chitinolytic machinery’, which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, as normally only 1% of the population expresses the chitinolytic machinery. In this study, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60 %. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens , and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.

Salmonella enterica serovar Typhimurium has the ability to use molecular hydrogen as a respiratory electron donor. This is facilitated by three [NiFe]‐hydrogenases termed Hyd‐1, Hyd‐2, and Hyd‐5. Hyd‐1 and Hyd‐5 are homologous oxygen‐tolerant [NiFe]‐hydrogenases. A critical step in the biosynthesis of a [NiFe]‐hydrogenase is the proteolytic processing of the catalytic subunit. In this work, the role of the maturation protease encoded within the Hyd‐5 operon, HydD, was found to be partially complemented by the maturation protease encoded in the Hyd‐1 operon, HyaD. In addition, both maturation proteases were shown to form stable complexes, in vivo and in vitro, with the catalytic subunit of Hyd‐5. The protein–protein interactions were not detectable in a strain that could not make the enzyme metallocofactor.

Serratia marcescensis a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein ‘secretome’ comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the ‘chitinolytic machinery’, which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon and nitrogen source. Secretion of the chitinases across the outer membrane is governed by thechiWXYZoperon encoding a holin/endopeptidase pair. Expression of thechiWXYZoperon is co-ordinated with the chitinase genes and is also bimodal, since normally only 1% of the population expresses the chitinolytic machinery. In this work, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation ofchiWXYZwith bothchiAandchiC. Moreover, overexpression ofchiRalone was able to increase the proportion of the cell population expressing chitinase genes to >60%. In addition, quantitative label-free proteomic analysis of cells overexpressingchiRestablished that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression inS. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.IMPORTANCEThe opportunistic pathogenSerratia marcescenssecretes chitinases through the action of thechiWXYZoperon, which encodes a holin/endopeptidase pair. Expression ofchiWXYZis normally bimodal, with only 1% of the population transcribing these genes. In this work, it is demonstrated that overexpression ofchiRinduces exquisitely co-ordinated holin/endopeptidase and chitinase gene expression in the majority of the population of cells, establishing that ChiR is a key player in biomodal gene expression. This work also reveals for the first time that co-operating pathways are induced by ChiR, including enzymes involved in ammonia, nitrite and nitrate metabolism. This work expands knowledge of basic bacterial physiology and could have applications in the biomedical and biotechnological research fields.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues.Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored. Refereed/Peer-reviewed

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.

The twin arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. Here we use disulfide crosslinking and molecular modelling to show there are two binding sites for TatA/B proteins on TatC. TatA and TatB are each able to occupy both sites if they are the only TatA/B protein present. However, under resting conditions the sites are differentially occupied with TatB occupying the ‘polar cluster’ site while TatA binds adjacently at the TatC transmembrane helix 6 binding site. When the Tat system is activated by the overproduction of a substrate, TatA and TatB switch their binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

The vast majority of polytopic membrane proteins are inserted into the cytoplasmic membrane of prokaryotes by the general secretory (Sec) pathway. However, a subset of monotopic proteins that contain non-covalently-bound redox cofactors depend on the twin-arginine translocase (Tat) machinery for membrane integration. Recently actinobacterial Rieske iron-sulfur cluster-containing proteins were identified as an unusual class of membrane proteins that require both the Sec and Tat pathways for the insertion of their three transmembrane domains (TMDs). The Sec pathway inserts the first two TMDs of these proteins co-translationally, but releases the polypeptide prior to the integration of TMD3 to allow folding of the cofactor-containing domain and its translocation by Tat. Here we have investigated features of theStreptomyces coelicolorRieske polypeptide that modulate its interaction with the Sec and Tat machineries. Mutagenesis of a highly conserved loop region between Sec-dependent TMD2 and Tat-dependent TMD3 shows that it plays no significant role in coordinating the activities of the two translocases, but that a minimum loop length of approximately eight amino acids is required for the Tat machinery to recognise TMD3. Instead we show that a combination of relatively low hydrophobicity of TMD3, coupled with the presence of C-terminal positively-charged amino acids, results in abortive insertion of TMD3 by the Sec pathway and its release at the cytoplasmic side of the membrane. Bioinformatic analysis identified two further families of polytopic membrane proteins that share features of dual Sec-Tat-targeted membrane proteins. A predicted heme-molybdenum cofactor-containing protein with five TMDs, and a polyferredoxin also with five predicted TMDs, are encoded across bacterial and archaeal genomes. We demonstrate that membrane insertion of representatives of each of these newly-identified protein families is dependent on more than one protein translocase, with the Tat machinery recognising TMD5. Importantly, the combination of low hydrophobicity of the final TMD and the presence of multiple C-terminal positive charges that serve as critical Sec-release features for the actinobacterial Rieske protein also dictate Sec release in these further protein families. Therefore we conclude that a simple unifying mechanism governs the assembly of dual targeted membrane proteins.

Prostate cancer development and progression is the result of complex interactions between epithelia cells and fibroblasts/myofibroblasts, in a series of dynamic process amenable to regulation by hormones. Whilst androgen action through the androgen receptor (AR) is a well-established component of prostate cancer biology, it has been becoming increasingly apparent that changes in AR signalling in the surrounding stroma can dramatically influence tumour cell behavior. This is reflected in the consistent finding of a strong association between stromal AR expression and patient outcomes. In this review, we explore the relationship between AR signalling in fibroblasts/myofibroblasts and prostate cancer cells in the primary site, and detail the known functions, actions, and mechanisms of fibroblast AR signaling. We conclude with an evidence-based summary of how androgen action in stroma dramatically influences disease progression.

Prostate cancer is hormone-dependent and regulated by androgens as well as oestrogens. The tumour microenvironment also provides regulatory control, but the balance and interplay between androgens and oestrogens at the human prostate tumour interface is unknown. This study reveals a central and dominant role for oestrogen in the microenvironment, fuelling a pro-tumourigenic loop of inflammatory cytokines involving recruitment of mast cells by carcinoma-associated fibroblasts (CAFs). Mast cell numbers were increased in human PCa clinical specimens, specifically within the peritumoural stroma. Human mast cells were also shown to express ERα and ERβ, with oestradiol directly stimulating mast cell proliferation and migration as well as altered cytokine/chemokine expression. There was a significant shift in the oestrogen:androgen balance in CAFs versus normal prostatic fibroblasts (NPFs), with a profound increase to ER:AR expression. Androgen signalling is also reduced in CAFs, while ERα and ERβ transcriptional activity is not, allowing oestrogen to dictate hormone action in the tumour microenvironment. Gene microarray analyses identified CXCL12 as a major oestrogen-driven target gene in CAFs, and CAFs recruit mast cells via CXCL12 in a CXCR4-dependent manner. Collectively, these data reveal multicellular oestrogen action in the tumour microenvironment and show dominant oestrogen, rather than androgen, signalling at the prostatic tumour interface.

BACKGROUND Contribution of stromal Hedgehog (Hh) signaling is evident in the prostate gland in mice, but needs translation to human tissues if Hh therapeutics are to be used effectively. Our goal was to determine if primary human prostate fibroblasts contain cilia, and respond to prostate Hh signaling. METHODS Primary human prostate cancer-associated (CAFs), and adjacent non-malignant (NPFs) fibroblasts isolated from human tissue specimens were analyzed using immunofluorescence, real-time PCR, and available array data. Cell culture and tissue recombination were used to determine responsiveness of human fibroblasts to Hh pathway manipulation and the paracrine effects of stromal Hh signaling, respectively. RESULTS Prostatic fibroblasts were capable of forming primary cilia, with the capacity for active Hh signaling as seen by Smo co-localization to the tip of the primary cilium. Expression of genes known to represent a signature of active Hh signaling in the prostate (especially Fgf5 and Igfbp6) were increased in CAFs compared to NPFs. The level of canonical Hh genes and prostate Hh signature genes were rarely synchronous; with lower doses of Purmorphamine/BMS-833923 regulating canonical transcription factors, and higher doses effecting prostate Hh signature genes. Grafts consisting of NPFs with constitutively active Hh signaling induced increased proliferation and dedifferentiation of adjacent non-malignant BPH-1 epithelial cells. CONCLUSIONS These data show that human prostatic fibroblasts have the capacity for Hh signaling and manipulation. Increased expression of a signature of prostatic Hh genes in the prostate tumor microenvironment suggests a role in the epithelial transformations driving prostate cancer (PCa). Prostate 73:1810–1823, 2013. © 2013 Wiley Periodicals, Inc.

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The essential components of the Tat pathway are the membrane proteins TatA, TatB, and TatC. TatA is thought to form the protein translocating element of the Tat system. Current models for Tat transport make predictions about the oligomeric state of TatA and whether, and how, this state changes during the transport cycle. We determined the oligomeric state of TatA directly at native levels of expression in living cells by photophysical analysis of individual yellow fluorescent protein-labeled TatA complexes. TatA forms complexes exhibiting a broad range of stoichiometries with an average of approximately 25 TatA subunits per complex. Fourier analysis of the stoichiometry distribution suggests the complexes are assembled from tetramer units. Modeling the diffusion behavior of the complexes suggests that TatA protomers associate as a ring and not a bundle. Each cell contains approximately 15 mobile TatA complexes and a pool of approximately 100 TatA molecules in a more disperse state in the membrane. Dissipation of the protonmotive force that drives Tat transport has no affect on TatA complex stoichiometry. TatA complexes do not form in cells lacking TatBC, suggesting that TatBC controls the oligomeric state of TatA. Our data support the TatA polymerization model for the mechanism of Tat transport.

Androgen receptor (AR) signalling in fibroblasts is important in prostate development and carcinogenesis, and is inversely related to prostate cancer mortality. However, the molecular mechanisms of AR action in fibroblasts and other non-epithelial cell types are largely unknown. The genome-wide DNA binding profile of AR in human prostate fibroblasts was identified by chromatin immunoprecipitation sequencing (ChIP-Seq), and found to be common to other fibroblast lines but disparate from AR cistromes of prostate cancer cells and tissue. Although AR binding sites specific to fibroblasts were less well conserved evolutionarily than those shared with cancer epithelia, they were likewise correlated with androgen regulation of fibroblast gene expression. Whereas FOXA1 is the key pioneer factor of AR in cancer epithelia, our data indicated that AP-1 likely plays a more important role in the AR cistrome in fibroblasts. The specificity of AP-1 and FOXA1 to binding in these cells is demonstrated using immunoblot and immunohistochemistry. Importantly, we find the fibroblast cistrome is represented in whole tissue/in vivo ChIP-seq studies at both genomic and resulting protein levels, highlighting the importance of the stroma in whole tissue -omic studies. This is the first nuclear receptor ChIP-seq study in prostatic fibroblasts, and provides novel insight into the action of fibroblast AR in prostate cancer.

The cellular response to circulating sex steroids is more than the sum of individual hormone actions, instead representing an interplay between activities of the evolutionarily related steroid hormone receptors. An example of this interaction is in breast cancer, where the risk of dying from estrogen receptor-α (ERα)-positive disease decreases approximately 4-fold when androgen receptor (AR) expression is high. In this study, we used chromatin immunoprecipitation sequencing (ChIP-seq) and microarray expression profiling to investigate the genomic and transcriptional cross talk between AR and ERα signaling in a luminal breast cancer cell line model, ZR-75-1. Expression profiling demonstrated reciprocal interference between 5α-dihydrotestosterone (DHT)- and 17β-estradiol (E(2))-induced transcriptional programs. Specifically, regulation of 26% of E(2) and 15% of DHT target genes was significantly affected by cotreatment with the other hormone, in the majority of cases (78-83%) antagonistically. Pathway analysis suggested that DHT cotreatment, for example, depleted E(2)-regulated pathways in cell survival and proliferation. ChIP-seq identified substantial overlap between the steroid receptor cistromes in ZR-75-1 cells, with 10-13% of AR- and ERα-binding sites located within 10 kb of the other receptor. Enrichment of androgen response elements in ERα-binding sites and vice versa was revealed by motif analysis, and AR-binding sites were enriched about E(2)-responsive genes affected by DHT cotreatment. Targeted ChIP and expression analysis revealed locus-specific outcomes when AR and ERα bind to the same DNA region. This work provides the first cistrome data for two steroid receptors in the same cell, insight into the antagonistic interplay between estrogens and androgens in luminal breast cancer, and an important resource for future work aimed at evaluating interrelated steroid receptors in different cellular systems.

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins. © 2012 Blackwell Publishing Ltd.

Karen Chiam, Natalie K. Ryan, Carmela Ricciardelli,Tanya K. Day, Grant Buchanan, Aleksandra M. Ochnik, Krisna Murti, Luke A. Selth, Australian Prostate Cancer BioResource,Lisa M. Butler, Wayne D. Tilley, and Tina Bianco-Miotto

Ligand-dependent activity of steroid receptors is affected by tetratricopeptide repeat (TPR)-containing co-chaperones, such as small glutamine-rich tetratricopeptide repeat-containing alpha (SGTA). However, the precise mechanisms by which the predominantly cytoplasmic TPR proteins affect downstream transcriptional outcomes of steroid signaling remain unclear. In this study, we assessed how SGTA affects ligand sensitivity and action of the androgen receptor (AR) using a transactivation profiling approach. Deletion mapping coupled with structural prediction, transcriptional assays, andin vivoregulation of AR-responsive promoters were used to assess the role of SGTA domains in AR responses. At subsaturating ligand concentrations of ≤0.1 nM 5α-dihydrotestosterone, SGTA overexpression constricted AR activity by an average of 32% (Pin vivo. The strength of the SGTA effect was associated with the presence or absence of bioinformatically predicated transcription factor motifs at each site. Homodimerizaion of SGTA, which is thought to be necessary for chaperone complex formation, was found to be dependent on the structural integrity of amino acids 1–80, and a core evolutionary conserved peptide within this region (amino acids 21–40) necessary for an effect of SGTA on the activity of both exogenous and endogenous AR. This study provides new insights into the subdomain structure of SGTA and how SGTA acts as a regulator of AR ligand sensitivity. A change in AR:SGTA ratio will impact the cellular and molecular response of prostate cancer cells to maintain androgenic signals, which may influence tumor progression.

Mutations in the androgen receptor (AR) have been detected in experimental and clinical prostate tumors. Mice with enforced prostate-specific expression of one such receptor variant, AR-E231G, invariably develop prostatic intraepithelial neoplasia by 12 weeks and metastatic prostate cancer by 52 weeks. The aim of this study was to identify genes with altered expression in the prostates of AR-E231G mice at an early stage of disease that may act as drivers of AR-mediated tumorigenesis. The gene expression profile of AR-E231G prostate tissue from 12-week-old mice was compared to an equivalent profile from mice expressing the AR-T857A receptor variant (analogous to the AR-T877A variant in LNCaP cells), which do not develop prostate tumors. One hundred and thirty-two genes were differentially expressed in AR-E231G prostates. Classification of these genes revealed enrichment for cellular pathways known to be involved in prostate cancer, including cell cycle and lipid metabolism. Suppression of two genes upregulated in the AR-E231G model, ADM and CITED1, increased cell death and reduced proliferation of human prostate cancer cells. Many genes differentially expressed in AR-E231G prostates are also deregulated in human tumors. Three of these genes, ID4, NR2F1 and PTGDS, which were expressed at consistently lower levels in clinical prostate cancer compared to nonmalignant tissues, formed a signature that predicted biochemical relapse (hazard ratio 2.2, p = 0.038). We believe that our findings support the value of this novel mouse model of prostate cancer to identify candidate therapeutic targets and/or biomarkers of human disease.

The response of prostate cells to androgens reflects a combination of androgen receptor (AR) transactivation and transrepression, but how these two processes differ mechanistically and influence prostate cancer risk and disease outcome remain elusive. Given recent interest in targeting AR transrepressive processes, a better understanding of AR/corepressor interaction and responses is warranted. Here, we used transactivation and interaction assays with wild-type and mutant ARs, and deletion AR fragments, to dissect the relationship between AR and the corepressor, silencing mediator for retinoic acid and thyroid hormone receptors (SMRT). We additionally tested how these processes are influenced by AR agonist and antagonist ligands, as well as by variation in the polyglutamine tract in the AR amino terminal domain (NTD), which is encoded by a polymorphic CAG repeat in the gene. SMRT was recruited to the AR ligand binding domain by agonist ligand, and as determined by the effect of strategic mutations in activation function 2 (AF-2), requires a precise conformation of that domain. A distinct region of SMRT also mediated interaction with the AR-NTD via the transactivation unit 5 (TAU5; residues 315–538) region. The degree to which SMRT was able to repress AR increased from 17% to 56% as the AR polyglutamine repeat length was increased from 9 to 42 residues, but critically this effect could be abolished by increasing the SMRT:AR molar ratio. These data suggest that the the extent to which the CAG encoded polyglutamine repeat influences AR activity represents a balance between corepressor and coactivator occupancy of the same ligand-dependent and independent AR interaction surfaces. Changes in the homeostatic relationship of AR to these molecules, including SMRT, may explain the variable penetrance of the CAG repeat and the loss of AR signalling flexibility in prostate cancer progression.

The twin arginine protein transport (Tat) system transports folded proteins across the cytoplasmic membranes of prokaryotes and the thylakoid membranes of plant chloroplasts. In Escherichia coli, the TatB and TatC components form a multivalent receptor complex that binds Tat substrates. Here, we have used a genetic fusion approach to construct covalent TatC oligomers in order to probe the organisation of TatC. A fused dimer of TatC supported Tat transport activity and was fully stable in vivo. Inactivating point mutations in one or other of the TatC units in the fused TatC dimer did not inactivate TatC function, indicating that only one TatC protomer in the TatC fused dimer needs to be active. Larger covalent fusions of TatC also supported Tat transport activity but were degraded in vivo to release smaller TatC forms. Taken together, these results strongly suggest that TatC forms a functional dimer, and support the idea that there is an even number of TatC protomers in the TatBC complex.

James Baddiley was a biochemist who used the methods and insight of the organic chemist to answer important questions in biology, notably coenzyme structure and the structure and function of bacterial cell walls. A graduate of Manchester University, he moved to Cambridge in 1944 with A. R. Todd, where he synthesized adenosine triphosphate, the nucleotide concerned with essential energy transformations in all forms of life. As an independent researcher at the Lister Institute in London he elucidated the structure of coenzyme A and other coenzymes. He was appointed Professor of Organic Chemistry in Newcastle, where the exploration of the structures of two cytidine nucleotides led to the discovery of the teichoic acids, major components of the cell walls and membranes of Gram-positive bacteria. These discoveries were extended to cover the structures, biosynthesis, function and immunology of the teichoic acids. Baddiley became Professor of Chemical Microbiology in 1977. Moving to Cambridge after his retirement, he was able to continue his researches in the Department of Biochemistry. He was elected a Fellow of Pembroke College and as an elder statesman undertook extensive committee work, often as chairman, both in Cambridge University and nationally. He was knighted in 1977.

Summary Objective To assess if a cell-based readout of androgen action in serum demonstrates a closer association with recognized classical parameters of androgen action in men than current measures of serum testosterone (T). Design To develop, validate and utilize a mammalian cell-based assay to measure specifically bioactive T and determine if this measure is a physiologically relevant fraction of serum T. Measurements and participants We have developed a specific serum T bioassay using human prostate cancer cells. A rapid 5-min exposure to 100% serum followed by serum withdrawal confers specificity of the assay to serum T and provides sufficient sensitivity to measure T in male serum samples. Matrix effects were experimentally discounted as a confounding issue. A total of 960 male serum samples from the Florey Adelaide Male Ageing Study (FAMAS) with previous comprehensive cohort data and serum measurements were utilized. Results Bioassay T measurement in the 960 FAMAS serum samples returned a median of 10AE7 nmol/l (1AE7–45AE4), and was most closely related to immunoassayed total T, but not immunoassayed bioavailable T or calculated free T. Immunoassayed total T demonstrated a positive association with isometric grip-strength (R 2 =0 AE127, P

The yjeE , yeaZ , and ygjD genes are highly conserved in the genomes of eubacteria, and ygjD orthologs are also found throughout the Archaea and eukaryotes. In this study, we have constructed conditional expression strains for each of these genes in the model organism Escherichia coli K12. We show that each gene is essential for the viability of E. coli under laboratory growth conditions. Growth of the conditional strains under nonpermissive conditions results in dramatic changes in cell ultrastructure. Deliberate repression of the expression of yeaZ results in cells with highly condensed nucleoids, while repression of yjeE and ygjD expression results in at least a proportion of very enlarged cells with an unusual peripheral distribution of DNA. Each of the three conditional expression strains can be complemented by multicopy clones harboring the rstA gene, which encodes a two-component-system response regulator, strongly suggesting that these proteins are involved in the same essential cellular pathway. The results of bacterial two-hybrid experiments show that YeaZ can interact with both YjeE and YgjD but that YgjD is the preferred interaction partner. The results of in vitro experiments indicate that YeaZ mediates the proteolysis of YgjD, suggesting that YeaZ and YjeE act as regulators to control the activity of this protein. Our results are consistent with these proteins forming a link between DNA metabolism and cell division.

Runx2 and androgen receptor (AR) are master transcription factors with pivotal roles in bone metabolism and prostate cancer (PCa). We dissected AR-mediated repression of Runx2 in dihydrotestosterone (DHT)-treated osteoblastic and PCa cells using reporter assays and endogenous Runx2 target genes. Repression required DHT, but not AR’s transactivation function, and was associated with nuclear colocalization of the two proteins. Runx2 and AR coimmunoprecipitated and interacted directly in glutathione-S-transferase pull-down assays. Interaction was ionic in nature. Intact AR DNA-binding domain (DBD) was necessary and sufficient for both interaction with Runx2 and its repression. Runx2 sequences required for interaction were the C-terminal 132 amino acid residues together with the Runt DBD. Runx2 DNA binding was abrogated by endogenous AR in chromatin immunoprecipitation assays and by recombinant AR-DBD in gel shift assays. Furthermore, AR caused increased nuclear mobility of Runx2 as indicated by faster fluorescence recovery after photobleaching. Thus, AR binds Runx2 and abrogates its binding to DNA and possibly to other nuclear components. Clinical relevance of our results was suggested by an inverse correlation between expression of AR-responsive prostate-specific antigen and osteocalcin genes in PCa biopsies. Given the tumor suppressor properties of Runx2, its repression by AR may constitute a mechanism of hormone carcinogenesis. Attenuation of Runx2 by AR in osteoblasts may play a role in skeletal metabolism: the bone-sparing effect of androgens is attributable, in part, to keeping Runx2 activity in check and preventing high-turnover bone disease such as seen after castration and in transgenic mice overexpressing Runx2 in osteoblasts.

The Tat (twin arginine translocation) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The integral membrane proteins TatA, TatB, and TatC are essential components of the Tat pathway. TatA forms high order oligomers and is thought to constitute the protein-translocating unit of the Tat system. Cysteine scanning mutagenesis was used to systematically investigate the functional importance of residues in the essential N-terminal transmembrane and amphipathic helices of Escherichia coli TatA. Cysteine substitutions of most residues in the amphipathic helix, including all the residues on the hydrophobic face of the helix, severely compromise Tat function. Glutamine 8 was identified as the only residue in the transmembrane helix that is critical for TatA function. The cysteine variants in the transmembrane helix were used in disulfide mapping experiments to probe the oligomeric arrangement of TatA protomers within the larger TatA complex. Residues in the center of the transmembrane helix (including residues 10-16) show a distinct pattern of cross-linking indicating that this region of the protein forms well defined interactions with other protomers. At least two interacting faces were detected. The results of our TatA studies are compared with analogous data for the homologous, but functionally distinct, TatB protein. This comparison reveals that it is only in TatA that the amphipathic helix is sensitive to amino acid substitutions. The TatA amphipathic helix may play a role in forming and controlling the path of substrate movement across the membrane.

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.

usc Refereed/Peer-reviewed Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of ∼ 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.

There is increasing evidence that sensitization of the androgen receptor (AR) signaling pathway contributes to the failure of androgen ablation therapy for prostate cancer, and that direct targeting of the AR may be a useful therapeutic approach. To better understand how AR function could be abrogated in prostate cancer cells, we have developed a series of putative dominant-negative variants of the human AR, containing deletions or mutations in activation functions AF-1, AF-5, and/or AF-2. One construct, AR inhibitor (ARi)-410, containing a deletion of AF-1 and part of AF-5 of the AR, had no intrinsic transactivation activity but inhibited wild-type AR (wtAR) in a ligand-dependent manner by at least 95% when transfected at a 4:1 molar ratio. ARi-410 was an equally potent inhibitor of gain-of-function AR variants. Ectopic expression of ARi-410 inhibited the proliferation of AR-positive LNCaP cells, but not AR-negative PC-3 cells. Whereas ARi-410 also marginally inhibited progesterone receptor activity, this was far less pronounced than the effect on AR (50% vs. 95% maximal inhibition, respectively), and there was no inhibition of either vitamin D or estrogen receptor activity. In the presence of ligand, ARi-410 interacted with wtAR, and both receptors translocated into the nucleus. Whereas the amino-carboxy terminal interaction was not necessary for optimal dominant-negative activity, disruption of dimerization through the ligand binding domain reduced the efficacy of ARi-410. In addition, although inhibition of AR function by ARi-410 was not dependent on DNA binding, the DNA binding domain was required for dominant-negative activity. Taken together, our results suggest that interaction between ARi-410 and the endogenous AR in prostate cancer cells, potentially through the DNA binding and ligand binding domains, results in a functionally significant reduction in AR signaling and AR-dependent cell growth.

Previously, we reported that androgen receptor (AR), but not estrogen receptor (ER) or progesterone receptor (PR), is predictive of response to the synthetic progestin, medroxyprogesterone acetate (MPA), in a cohort of 83 patients with metastatic breast cancer. To further investigate the role of AR in determining response to MPA in this cohort, we analyzed AR levels by immunohistochemistry with two discrete antisera directed at either the NH2 or the COOH termini of the receptor. Compared with tumors that responded to MPA (n = 31), there was a significant decrease in the intensity and extent of AR immunoreactivity with both AR antisera in tumors from nonresponders (n = 52). Whereas only a single AR immunostaining pattern was detected in responders to MPA, reflecting concordance of immunoreactivity with the two AR antisera, tumors from nonresponders exhibited four distinct AR immunostaining patterns: (a) concordance with the two antibodies (31%), (b) staining only with the COOH-terminal antibody (33%), (c) staining only with the NH2-terminal antibody (22%), or (d) no immunoreactivity with either NH2- or COOH-terminal antibody (14%). DNA sequencing and functional analysis identified inactivating missense gene mutations in the ligand-binding domain of the AR in tumors from two of nine nonresponders positive with the NH2-terminal AR antisera but negative for COOH-terminal immunoreactivity and lacking specific, high-affinity dihydrotestosterone binding in tumor cytosol fractions. Tumors with more AR than the median level (37 fmol/mg protein) had significantly lower levels of PR (30 fmol/mg protein) than tumors with low AR (PR; 127 fmol/mg protein) despite comparable levels of ER. Ligand-dependent activation of the AR in human T47D and MCF-7 breast cancer cells resulted in inhibition of estradiol-stimulated cell proliferation and a reduction in the capacity of the ER to induce expression of the PR. These effects could be reversed using a specific AR antisense oligonucleotide. Increasing the ratio of AR to ER resulted in a greater androgen-dependent inhibition of ER function. Collectively, these data suggest that reduced levels of AR or impaired AR function contribute to the failure of MPA therapy potentially due to abrogation of the inhibitory effect of AR on ER signaling.

The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial ‘proofreading' or ‘quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

The categorization of prostate cancers that are progressing after castration as ‘hormone-refractory’ evolved from the clinical observation that surgical or medical castration (i.e. androgen ablation therapy; AAT) is not curative and, despite an initial response, virtually all tumors eventually regrow. Successful AAT is contingent on the dependence of prostate cancer cells for androgen signaling through an intracellular mediator, the androgen receptor (AR) for survival. Current preclinical and clinical data imply that the AR is expressed and continues to mediate androgen signaling after failure of AAT. As AAT does not completely eliminate circulating androgens, sufficient concentrations of dihydrotestosterone may accumulate in tumor cells to maintain AR signaling, especially in the context of upregulated receptor levels or increased sensitivity of the AR for activation. In addition, ligands of non-testicular origin or ligand-independent activation can contribute to continued AR signaling. In many cases, therefore, from the perspective of the AR, a ‘hormone-refractory’ classification after failure of AAT is inappropriate. Classifying prostate tumors that progress after AAT as ‘castration-resistant’ may be more relevant. Clinical responses to second- and third-line hormonal therapies suggest that the mechanisms of AR activation are in part a function of previously administered AAT. Accordingly, the increasing trend to utilize AAT earlier in the course of the clinical disease may have a greater influence on the genotype and phenotype of the resistant tumor. In this article, we detail strategies to inhibit the growth of prostate cancer cells that specifically target the AR in addition to those practiced traditionally that indirectly target the receptor by reducing the amount of circulating ligand. We propose that treatment regimes combining AAT with direct AR targeting strategies may provide a more complete blockade of androgen signaling, thereby preventing or significantly delaying the emergence of treatment-resistant disease.

Summary The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.

Functional analysis of androgen receptor (AR) gene mutations isolated from prostate cancer has led to the identification of residues that play important roles in the structure and function of the receptor. Here we report the characteristics of a novel AR mutation A748T located in helix 5 of the ligand-binding domain, which was identified in metastatic prostate cancer. Despite a normal hormone-binding affinity, A748T causes hormone concentration-dependent defects in nuclear accumulation and transcriptional activation. Moreover, when equivalent amounts of DNA are transfected, the mutant is expressed at much lower levels than the wild-type AR (ARWT). Treatment with geldanamycin to disrupt receptor-heat shock protein complexes rapidly decreases the levels of ARWT but not A748T, suggesting that the lower expression and rapid degradation rate of A748T is due to weaker interactions with heat shock proteins. Further analysis revealed that hormone dissociates from A748T five times faster than from ARWT. Loss of the ability to form stable amino/carboxyl-terminal interactions causes accelerated dissociation rates in some AR mutants. However, A748T exhibits normal amino/carboxyl-terminal interactions at high hormone concentrations, suggesting that the mutation alters interactions with ligand. Consistent with this conclusion, our structural model predicts that A748T disrupts crucial contact points with ligand, thereby altering the conformation of the ligand-binding domain.

The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A ‘polar cluster’ site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase.

International audience; We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine- and heavy arginine-labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.

A holin (ChiW) and an endopeptidase (ChiX) operate in tandem as components of a protein secretion system used by the gram-negative bacterium Serratia marcescens to secrete the chitinolytic machinery., Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

The cytochrome bc 1 -cytochrome aa 3 complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc 1 complex shows a number of unusual features in comparison to other cytochrome bc 1 complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc 1 complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a Δ tatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc 1 complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc 1 complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mossbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Although prostate cancer is heterogeneous in its etiology and progression, androgen signaling through the androgen receptor (AR) appears to be involved in all aspects of the disease, from initiation to development of treatment resistance. Lifetime exposure to a constitutively more active AR, encoded by AR alleles as defined by two translated polymorphic microsatellites (CAG and GGC), results in a significant increase in prostate cancer risk. The AR gene is amplified or a target for somatic gain-of-function mutations in metastatic prostate cancer. Gain-of-function AR gene mutations may result in inappropriate activation of the AR, thereby contributing to the failure of conventional androgen-ablation treatments. In cases where no genetically altered receptors are observed, altered signaling through the AR, achieved by cross-talk with other signaling pathways (e.g. kinase-mediated pathways) and/or inappropriate expression of coregulatory proteins, may contribute to disease progression. Thus, the AR-signaling axis contributes to many aspects of prostate cancer, including initiation, progression and resistance to current forms of therapy. This recognition represents a paradigm shift in our understanding of the molecular mechanisms involved in progression of prostate cancer, and provides insight into novel AR-targeted therapies which ultimately may be more effective than current forms of androgen ablation.

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment.

Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of 5α-dihydrotestosterone (DHT) target genes was affected by SGTA knockdown, with gene-specific effects on basal or DHT-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100β levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.

Free to read Recent evidence indicates that the estrogen receptor-a-negative, androgen receptor (AR)- positive molecular apocrine subtype of breast cancer is driven by AR signaling. The MDA-MB-453 cell line is the prototypical model of this breast cancer subtype; its proliferation is stimulated by androgens such as 5a-dihydrotestosterone (DHT) but inhibited by the progestin medroxyprogesterone acetate (MPA) via AR-mediated mechanisms. We report here that the AR gene in MDAMB- 453 cells contains a G-T transversion in exon 7, resulting in a receptor variant with a glutamine to histidine substitution at amino acid 865 (Q865H) in the ligand binding domain. Compared with wild-type AR, the Q865H variant exhibited reduced sensitivity to DHT and MPA in transactivation assays in MDA-MB-453 and PC-3 cells but did not respond to non-androgenic ligands or receptor antagonists. Ligand binding, molecular modeling, mammalian two-hybrid and immunoblot assays revealed effects of the Q865H mutation on ligand dissociation, AR intramolecular interactions, and receptor stability. Microarray expression profiling demonstrated that DHT and MPA regulate distinct transcriptional programs in MDA-MB-453 cells. Gene Set Enrichment Analysis revealed that DHT- but not MPA-regulated genes were associated with estrogen-responsive transcriptomes from MCF-7 cells and the Wnt signaling pathway. These findings suggest that the divergent proliferative responses of MDA-MB-453 cells to DHT and MPA result from the different genetic programs elicited by these two ligands through the AR-Q865H variant. This work highlights the necessity to characterize additional models of molecular apocrine breast cancer to determine the precise role of AR signaling in this breast cancer subtype. Endocrine-Related Cancer (2012) 19 599–613

Free to read on publisher website The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.

l-Type amino acid transporters such as LAT1 and LAT3 mediate the uptake of essential amino acids. Here, we report that prostate cancer cells coordinate the expression of LAT1 and LAT3 to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth. Inhibiting LAT function was sufficient to decrease cell growth and mTORC1 signaling in prostate cancer cells. These cells maintained levels of amino acid influx through androgen receptor–mediated regulation of LAT3 expression and ATF4 regulation of LAT1 expression after amino acid deprivation. These responses remained intact in primary prostate cancer, as indicated by high levels of LAT3 in primary disease, and by increased levels of LAT1 after hormone ablation and in metastatic lesions. Taken together, our results show how prostate cancer cells respond to demands for increased essential amino acids by coordinately activating amino acid transporter pathways vital for tumor outgrowth. Cancer Res; 71(24); 7525–36. ©2011 AACR.