RESUMO
Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Células CHO , Diferenciação Celular/efeitos dos fármacos , Cricetulus , Feminino , Fatores de Crescimento de Fibroblastos/classificação , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cultura Primária de CélulasRESUMO
Secreted proteins and their cognate plasma membrane receptors regulate human physiology by transducing signals from the extracellular environment into cells resulting in different cellular phenotypes. Systematic use of secretome proteins in assays enables discovery of novel biology and signaling pathways. Several secretome-based phenotypic screening platforms have been described in the literature and shown to facilitate target identification in drug discovery. In this review, we summarize the current status of secretome-based screening. This includes annotation, production, quality control, and sample management of secretome libraries, as well as how secretome libraries have been applied to discover novel target biology using different disease-relevant cell-based assays. A workflow for secretome-based screening is shared based on the AstraZeneca experience. The secretome library offers several advantages compared with other libraries used for target discovery: (1) screening using a secretome library directly identifies the active protein and, in many cases, its cognate receptor, enabling a rapid understanding of the disease pathway and subsequent formation of target hypotheses for drug discovery; (2) the secretome library covers significant areas of biological signaling space, although the size of this library is small; (3) secretome proteins can be added directly to cells without additional manipulation. These factors make the secretome library ideal for testing in physiologically relevant cell types, and therefore it represents an attractive approach to phenotypic target discovery.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Proteoma/genética , Microambiente Celular/genética , Biblioteca Gênica , HumanosRESUMO
The peroxisome proliferator-activated receptor gamma (PPARγ) is the target for the thiazolidinedione class of potent insulin-sensitizing drugs, which includes rosiglitazone and pioglitazone. However, their usage has been restricted due to severe side effects. Recent data have shown that specifically inhibiting the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser273 may lead to novel insulin sensitizers with fewer side effects. Here we describe a novel enzyme-linked immunosorbent assay (ELISA) in the 384-well format, which enables screening for PPARγ ligands that inhibit phosphorylation at Ser273 by Cdk5. The assay is robust with a Z-factor > 0.6, demonstrating its suitability for high-throughput screening. We demonstrate the suitability of this assay for profiling of published PPARγ ligands and identification of novel compounds that prevent the Cdk5-mediated phosphorylation of PPARγ at Ser273 in a 622 compound pilot study. Our assay enables the discovery and development of novel therapeutic agents for use in type-2 diabetes. Furthermore, our results in combination with structural analysis of reported PPARγ ligand binding domain X-ray structures give a molecular rationale for the Cdk5-mediated phosphorylation of PPARγ at Ser273.
Assuntos
Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , PPAR gama/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologiaRESUMO
Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.
Assuntos
Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Guanosina Pentafosfato/biossíntese , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , GTP Pirofosfoquinase/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.
Assuntos
RNA Helicases DEAD-box/química , Família Multigênica , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA/metabolismo , Alinhamento de SequênciaRESUMO
UNLABELLED: Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 A. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Assuntos
Metaloendopeptidases/química , ATPases Associadas a Diversas Atividades Celulares , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Impairment of the formation or action of hydrogen sulfide (H(2)S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine beta-synthase and cystathionine gamma-lyase (CSE) are two pyridoxal-5'-phosphate (PLP)-dependent enzymes largely responsible for the production of H(2)S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP.PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H(2)S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE.PLP.PAG complex highlights the particular importance of Tyr(114) in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.
Assuntos
Cistationina gama-Liase/química , Sulfeto de Hidrogênio/metabolismo , Biofísica , Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/química , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Modelos Moleculares , Poli(ADP-Ribose) Polimerases/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.
Assuntos
Desenho de Fármacos , Glutamato-Amônia Ligase/química , Glutamato-Amônia Ligase/metabolismo , Herbicidas/síntese química , Preparações Farmacêuticas/síntese química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Apoenzimas/química , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Cães , Interações Medicamentosas , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/isolamento & purificação , Herbicidas/química , Humanos , Ligação de Hidrogênio , Cinética , Ligantes , Magnésio/metabolismo , Magnésio/farmacologia , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Preparações Farmacêuticas/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
Argininosuccinate synthetase catalyzes the transformation of citrulline and aspartate into argininosuccinate and pyrophosphate using the hydrolysis of ATP to AMP and pyrophosphate. This enzymatic process constitutes the rate-limiting step in both the urea and arginine-citrulline cycles. Previous studies have investigated the crystal structures of argininosuccinate synthetase from bacterial species. In this work, the first crystal structure of human argininosuccinate synthetase in complex with the substrates citrulline and aspartate is presented. The human enzyme is compared with structures of argininosuccinate synthetase from bacteria. In addition, the structure also provides new insights into the function of the numerous clinical mutations identified in patients with type I citrullinaemia (also known as classic citrullinaemia).
Assuntos
Argininossuccinato Sintase/química , Modelos Moleculares , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Argininossuccinato Sintase/genética , Sítios de Ligação , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência de Proteína , Especificidade por SubstratoRESUMO
Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.
Assuntos
Domínio Catalítico , Tanquirases/química , Zinco/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Dados de Sequência Molecular , Estrutura MolecularRESUMO
Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helper protein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work we used lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery at different steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reacted with Ala-tmRNA*EF-Tu*GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was probed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we also analyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. We observed some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNA conformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA is in complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.