Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Differential Contributions of mSWI/SNF Chromatin Remodeler Sub-Families to Myoblast Differentiation.

Mammalian SWI/SNF (mSWI/SNF) complexes are ATP-dependent chromatin remodeling enzymes that are critical for normal cellular functions. mSWI/SNF enzymes are classified into three sub-families based on the presence of specific subunit proteins. The sub-families are Brm- or Brg1-associated factor (BAF), ncBAF (non-canonical BAF), and polybromo-associated BAF (PBAF). The biological roles for the different enzyme sub-families are poorly described. We knocked down the expression of genes encoding unique subunit proteins for each sub-family, Baf250A, Brd9, and Baf180, which mark the BAF, ncBAF, and PBAF sub-families, respectively, and examined the requirement for each in myoblast differentiation. We found that Baf250A and the BAF complex were required to drive lineage-specific gene expression. KD of Brd9 delayed differentiation. However, while the Baf250A-dependent gene expression profile included myogenic genes, the Brd9-dependent gene expression profile did not, suggesting Brd9 and the ncBAF complex indirectly contributed to differentiation. Baf180 was dispensable for myoblast differentiation. The results distinguish between the roles of the mSWI/SNF enzyme sub-families during myoblast differentiation.

Oxidative Reactions Catalyzed by Hydrogen Peroxide Produced by Streptococcus pneumoniae and Other Streptococci Cause the Release and Degradation of Heme from Hemoglobin.

Streptococcus pneumoniae (Spn) strains cause pneumonia that kills millions every year worldwide. Spn produces Ply, a hemolysin that lyses erythrocytes releasing hemoglobin, and also produces the pro-oxidant hydrogen peroxide (Spn-H2O2) during growth. The hallmark of the pathophysiology of hemolytic diseases is the oxidation of hemoglobin, but oxidative reactions catalyzed by Spn-H2O2 have been poorly studied. We characterized the oxidation of hemoglobin by Spn-H2O2. We prepared a series of single-mutant (ΔspxB or ΔlctO), double-mutant (ΔspxB ΔlctO), and complemented strains in TIGR4, D39, and EF3030. We then utilized an in vitro model with oxyhemoglobin to demonstrate that oxyhemoglobin was oxidized rapidly, within 30 min of incubation, by Spn-H2O2 to methemoglobin and that the main source of Spn-H2O2 was pyruvate oxidase (SpxB). Moreover, extended incubation caused the release and the degradation of heme. We then assessed oxidation of hemoglobin and heme degradation by other bacterial inhabitants of the respiratory tract. All hydrogen peroxide-producing streptococci tested caused the oxidation of hemoglobin and heme degradation, whereas bacterial species that produce <1 µM H2O2 neither oxidized hemoglobin nor degraded heme. An ex vivo bacteremia model confirmed that oxidation of hemoglobin and heme degradation occurred concurrently with hemoglobin that was released from erythrocytes by Ply. Finally, gene expression studies demonstrated that heme, but not red blood cells or hemoglobin, induced upregulated transcription of the spxB gene. Oxidation of hemoglobin may be important for pathogenesis and for the symbiosis of hydrogen peroxide-producing bacteria with other species by providing nutrients such as iron.

The molecular and cellular basis of copper dysregulation and its relationship with human pathologies.

Copper (Cu) is an essential micronutrient required for the activity of redox-active enzymes involved in critical metabolic reactions, signaling pathways, and biological functions. Transporters and chaperones control Cu ion levels and bioavailability to ensure proper subcellular and systemic Cu distribution. Intensive research has focused on understanding how mammalian cells maintain Cu homeostasis, and how molecular signals coordinate Cu acquisition and storage within organs. In humans, mutations of genes that regulate Cu homeostasis or facilitate interactions with Cu ions lead to numerous pathologic conditions. Malfunctions of the Cu+ -transporting ATPases ATP7A and ATP7B cause Menkes disease and Wilson disease, respectively. Additionally, defects in the mitochondrial and cellular distributions and homeostasis of Cu lead to severe neurodegenerative conditions, mitochondrial myopathies, and metabolic diseases. Cu has a dual nature in carcinogenesis as a promotor of tumor growth and an inducer of redox stress in cancer cells. Cu also plays role in cancer treatment as a component of drugs and a regulator of drug sensitivity and uptake. In this review, we provide an overview of the current knowledge of Cu metabolism and transport and its relation to various human pathologies.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

The Oncopig as an Emerging Model to Investigate Copper Regulation in Cancer.

Emerging evidence points to several fundamental contributions that copper (Cu) has to promote the development of human pathologies such as cancer. These recent and increasing identification of the roles of Cu in cancer biology highlights a promising field in the development of novel strategies against cancer. Cu and its network of regulatory proteins are involved in many different contextual aspects of cancer from driving cell signaling, modulating cell cycle progression, establishing the epithelial-mesenchymal transition, and promoting tumor growth and metastasis. Human cancer research in general requires refined models to bridge the gap between basic science research and meaningful clinical trials. Classic studies in cultured cancer cell lines and animal models such as mice and rats often present caveats when extended to humans due to inherent genetic and physiological differences. However, larger animal models such as pigs are emerging as more appropriate tools for translational research as they present more similarities with humans in terms of genetics, anatomical structures, organ sizes, and pathological manifestations of diseases like cancer. These similarities make porcine models well-suited for addressing long standing questions in cancer biology as well as in the arena of novel drug and therapeutic development against human cancers. With the emergent roles of Cu in human health and pathology, the pig presents an emerging and valuable model to further investigate the contributions of this metal to human cancers. The Oncopig Cancer Model is a transgenic swine model that recapitulates human cancer through development of site and cell specific tumors. In this review, we briefly outline the relationship between Cu and cancer, and how the novel Oncopig Cancer Model may be used to provide a better understanding of the mechanisms and causal relationships between Cu and molecular targets involved in cancer.

Loss of the Metastasis Suppressor NME1, But Not of Its Highly Related Isoform NME2, Induces a Hybrid Epithelial-Mesenchymal State in Cancer Cells.

Epithelial-mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive 'triple-negative' phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell-cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.

The classic metal-sensing transcription factor MTF1 promotes myogenesis in response to copper.

Metal-regulatory transcription factor 1 (MTF1) is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements. MTF1 responds to both metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. To examine the role for MTF1 in cell differentiation, we use multiple experimental strategies [including gene knockdown (KD) mediated by small hairpin RNA and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), immunofluorescence, chromatin immunopreciptation sequencing, subcellular fractionation, and atomic absorbance spectroscopy] and report a previously unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, both MTF1 expression and nuclear localization increased. Mtf1 KD impaired differentiation, whereas addition of nontoxic concentrations of Cu+-enhanced MTF1 expression and promoted myogenesis. Furthermore, we observed that Cu+ binds stoichiometrically to a C terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes, and Cu addition stimulated this binding. Of note, MTF1 formed a complex with myogenic differentiation (MYOD)1, the master transcriptional regulator of the myogenic lineage, at myogenic promoters. These findings uncover unexpected mechanisms by which Cu and MTF1 regulate gene expression during myoblast differentiation.-Tavera-Montañez, C., Hainer, S. J., Cangussu, D., Gordon, S. J. V., Xiao, Y., Reyes-Gutierrez, P., Imbalzano, A. N., Navea, J. G., Fazzio, T. G., Padilla-Benavides, T. The classic metal-sensing transcription factor MTF1 promotes myogenesis in response to copper.

CK2-Dependent Phosphorylation of the Brg1 Chromatin Remodeling Enzyme Occurs during Mitosis.

Brg1 (Brahma-related gene 1) is one of two mutually exclusive ATPases that can act as the catalytic subunit of mammalian SWI/SNF (mSWI/SfigureNF) chromatin remodeling enzymes that facilitate utilization of the DNA in eukaryotic cells. Brg1 is a phospho-protein, and its activity is regulated by specific kinases and phosphatases. Previously, we showed that Brg1 interacts with and is phosphorylated by casein kinase 2 (CK2) in a manner that regulates myoblast proliferation. Here, we use biochemical and cell and molecular biology approaches to demonstrate that the Brg1-CK2 interaction occurred during mitosis in embryonic mouse somites and in primary myoblasts derived from satellite cells isolated from mouse skeletal muscle tissue. The interaction of CK2 with Brg1 and the incorporation of a number of other subunits into the mSWI/SNF enzyme complex were independent of CK2 enzymatic activity. CK2-mediated hyperphosphorylation of Brg1 was observed in mitotic cells derived from multiple cell types and organisms, suggesting functional conservation across tissues and species. The mitotically hyperphosphorylated form of Brg1 was localized with soluble chromatin, demonstrating that CK2-mediated phosphorylation of Brg1 is associated with specific partitioning of Brg1 within subcellular compartments. Thus, CK2 acts as a mitotic kinase that regulates Brg1 phosphorylation and subcellular localization.

Ouabain Enhances Cell-Cell Adhesion Mediated by ß1 Subunits of the Na+,K+-ATPase in CHO Fibroblasts.

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.

Extracellular-Signal Regulated Kinase: A Central Molecule Driving Epithelial-Mesenchymal Transition in Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible cellular process, characterized by changes in gene expression and activation of proteins, favoring the trans-differentiation of the epithelial phenotype to a mesenchymal phenotype. This process increases cell migration and invasion of tumor cells, progression of the cell cycle, and resistance to apoptosis and chemotherapy, all of which support tumor progression. One of the signaling pathways involved in tumor progression is the MAPK pathway. Within this family, the ERK subfamily of proteins is known for its contributions to EMT. The ERK subfamily is divided into typical (ERK 1/2/5), and atypical (ERK 3/4/7/8) members. These kinases are overexpressed and hyperactive in various types of cancer. They regulate diverse cellular processes such as proliferation, migration, metastasis, resistance to chemotherapy, and EMT. In this context, in vitro and in vivo assays, as well as studies in human patients, have shown that ERK favors the expression, function, and subcellular relocalization of various proteins that regulate EMT, thus promoting tumor progression. In this review, we discuss the mechanistic roles of the ERK subfamily members in EMT and tumor progression in diverse biological systems.

Casein kinase 2-mediated phosphorylation of Brahma-related gene 1 controls myoblast proliferation and contributes to SWI/SNF complex composition.

Transcriptional regulation is modulated in part by chromatin-remodeling enzymes that control gene accessibility by altering chromatin compaction or nucleosome positioning. Brahma-related gene 1 (Brg1), a catalytic subunit of the mammalian SWI/SNF chromatin-remodeling enzymes, is required for both myoblast proliferation and differentiation, and the control of Brg1 phosphorylation by calcineurin, PKCß1, and p38 regulates the transition to differentiation. However, we hypothesized that Brg1 activity might be regulated by additional kinases. Here, we report that Brg1 is also a target of casein kinase 2 (CK2), a serine/threonine kinase, in proliferating myoblasts. We found that CK2 interacts with Brg1, and mutation of putative phosphorylation sites to non-phosphorylatable (Ser to Ala, SA) or phosphomimetic residues (Ser to Glu, SE) reduced Brg1 phosphorylation by CK2. Although BRG1-deleted myoblasts that ectopically express the SA-Brg1 mutant proliferated similarly to the parental cells or cells ectopically expressing wild-type (WT) Brg1, ectopic expression of the SE-Brg1 mutant reduced proliferation and increased cell death, similar to observations from cells lacking Brg1. Moreover, pharmacological inhibition of CK2 increased myoblast proliferation. Furthermore, the Pax7 promoter, which controls expression of a key transcription factor required for myoblast proliferation, was in an inaccessible chromatin state in the SE-Brg1 mutant, suggesting that hyperphosphorylated Brg1 cannot remodel chromatin. WT-, SA-, and SE-Brg1 exhibited distinct differences in interacting with and affecting expression of the SWI/SNF subunits Baf155 and Baf170 and displayed differential sub-nuclear localization. Our results indicate that CK2-mediated phosphorylation of Brg1 regulates myoblast proliferation and provides insight into one mechanism by which composition of the mammalian SWI/SNF enzyme complex is regulated.

Signaling Pathways Induced by Leptin during Epithelial⁻Mesenchymal Transition in Breast Cancer.

Leptin is an adipokine that is overexpressed in obese and overweight people. Interestingly, women with breast cancer present high levels of leptin and of its receptor ObR. Leptin plays an important role in breast cancer progression due to the biological processes it participates in, such as epithelial⁻mesenchymal transition (EMT). EMT consists of a series of orchestrated events in which cell⁻cell and cell⁻extracellular matrix interactions are altered and lead to the release of epithelial cells from the surrounding tissue. The cytoskeleton is also re-arranged, allowing the three-dimensional movement of epithelial cells into the extracellular matrix. This transition provides cells with the ability to migrate and invade adjacent or distal tissues, which is a classic feature of invasive or metastatic carcinoma cells. In recent years, the number of cases of breast cancer has increased, making this disease a public health problem worldwide and the leading cause of death due to cancer in women. In this review, we focus on recent advances that establish: (1) leptin as a risk factor for the development of breast cancer, and (2) leptin as an inducer of EMT, an event that promotes tumor progression.

Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis.

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu(+) import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu(+)-delivery mechanism and provides a unique snapshot into the evolution of a Cu(+) distribution pathway. We also provide evidence for an interaction between PAA2, the Cu(+)-ATPase in thylakoids, and the Cu(+)-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu(+) network that has evolved to fine-tune Cu(+) distribution.

Lipogenic Enzymes Complexes and Cytoplasmic Lipid Droplet Formation During Adipogenesis.

Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc.

Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF.

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu(2+/+). In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu(+) to target transporters or metalloenzymes. Transmembrane Cu(+)-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). Cytosolic Cu(+) chaperones (CopZ) interact with a structural platform in Cu(+)-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu(+) chaperone that supplies Cu(+) to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu(+) transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu(+)-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu(+) transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu(+) transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu(+) transfer was minimally affected. These results show how soluble chaperones obtain Cu(+) from transmembrane transporters. Furthermore, by explaining the movement of Cu(+) from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu(+) can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.

Brg1 Controls the Expression of Pax7 to Promote Viability and Proliferation of Mouse Primary Myoblasts.

Brg1 (Brahma-related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP-dependent chromatin remodeling enzymes that disrupt histone-DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1-deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1-mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well.

Differential roles for the Co(2+) /Ni(2+) transporting ATPases, CtpD and CtpJ, in Mycobacterium tuberculosis virulence.

The genome of Mycobacterium tuberculosis encodes two paralogous P1 B 4 -ATPases, CtpD (Rv1469) and CtpJ (Rv3743). Both proteins showed ATPase activation by Co(2+) and Ni(2+) , and both appear to be required for metal efflux from the cell. However, using a combination of biochemical and genetic studies we found that these proteins play non-redundant roles in virulence and metal efflux. CtpJ expression is induced by Co(2+) and this protein possesses a relatively high turnover rate. A ctpJ deletion mutant accumulated Co(2+) , indicating that this ATPase controls cytoplasmic metal levels. In contrast, CtpD expression is induced by redox stressors and this protein displays a relatively low turnover rate. A ctpD mutant failed to accumulate metal, suggesting an alternative cellular function. ctpD is cotranscribed with two thioredoxin genes trxA (Rv1470), trxB (Rv1471), and an enoyl-coA hydratase (Rv1472), indicating a possible role for CtpD in the metallation of these redox-active proteins. Supporting this, in vitro metal binding assays showed that TrxA binds Co(2+) and Ni(2+) . Mutation of ctpD, but not ctpJ, reduced bacterial fitness in the mouse lung, suggesting that redox maintenance, but not Co(2+) accumulation, is important for growth in vivo.

A novel antimycobacterial compound acts as an intracellular iron chelator.

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.

The mechanism of Cu+ transport ATPases: interaction with CU+ chaperones and the role of transient metal-binding sites.

Cu(+)-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu(+), delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu(+)-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59-64) shows that a kinked transmembrane segment forms a "platform" exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the "entrance" of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu(+)-ATPase CopA do not affect ATPase activity in the presence of Cu(+) free in solution. However, Cu(+) bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu(+) to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu(+) transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu(+) chaperone. This docking likely places the Cu(+) proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu(+) with the pump is transient because Cu(+) is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation.