RESUMEN
There is a common misconception that the United States is suffering from a "STEM shortage," a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes.
Asunto(s)
Investigación Biomédica , Selección de Profesión , Investigadores , Ciencia , Estudiantes , Humanos , Estados UnidosRESUMEN
Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR), an essential post-translational modification whose function is important in many cellular processes including DNA damage signaling, cell death, and inflammation. All known PAR biology is intracellular, but we suspected it might also play a role in cell-to-cell communication during inflammation. We found that PAR activated cytokine release in human and mouse macrophages, a hallmark of innate immune activation, and determined structure-activity relationships. PAR was rapidly internalized by murine macrophages, while the monomer, ADP-ribose, was not. Inhibitors of Toll-like receptor 2 (TLR2) and TLR4 signaling blocked macrophage responses to PAR, and PAR induced TLR2 and TLR4 signaling in reporter cell lines suggesting it was recognized by these TLRs, much like bacterial pathogens. We propose that PAR acts as an extracellular damage associated molecular pattern that drives inflammatory signaling.
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Poli Adenosina Difosfato Ribosa/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Línea Celular , Citocinas/metabolismo , Dimerización , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Microscopía Confocal , Poli Adenosina Difosfato Ribosa/química , Relación Estructura-Actividad , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important posttranslational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed an assay for measuring PARP activity in cell lysates and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high- and low-activity lines. A discovered complex containing the NuA4 chromatin-remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA damage response.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Humanos , Células MCF-7 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Unión Proteica , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 (nd) and 3 (rd), 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers' synthesis of the outcomes.
RESUMEN
MOTIVATION: Structural characterization of protein interactions is necessary for understanding and modulating biological processes. On one hand, X-ray crystallography or NMR spectroscopy provide atomic resolution structures but the data collection process is typically long and the success rate is low. On the other hand, computational methods for modeling assembly structures from individual components frequently suffer from high false-positive rate, rarely resulting in a unique solution. RESULTS: Here, we present a combined approach that computationally integrates data from a variety of fast and accessible experimental techniques for rapid and accurate structure determination of protein-protein complexes. The integrative method uses atomistic models of two interacting proteins and one or more datasets from five accessible experimental techniques: a small-angle X-ray scattering (SAXS) profile, 2D class average images from negative-stain electron microscopy micrographs (EM), a 3D density map from single-particle negative-stain EM, residue type content of the protein-protein interface from NMR spectroscopy and chemical cross-linking detected by mass spectrometry. The method is tested on a docking benchmark consisting of 176 known complex structures and simulated experimental data. The near-native model is the top scoring one for up to 61% of benchmark cases depending on the included experimental datasets; in comparison to 10% for standard computational docking. We also collected SAXS, 2D class average images and 3D density map from negative-stain EM to model the PCSK9 antigen-J16 Fab antibody complex, followed by validation of the model by a subsequently available X-ray crystallographic structure.
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Simulación del Acoplamiento Molecular/métodos , Complejos Multiproteicos/química , Complejo Antígeno-Anticuerpo/química , Cristalografía por Rayos X , Microscopía Electrónica , Dispersión del Ángulo Pequeño , Programas Informáticos , Difracción de Rayos XRESUMEN
Hsp90, a dimeric ATP-dependent molecular chaperone, is required for the folding and activation of numerous essential substrate "client" proteins including nuclear receptors, cell cycle kinases, and telomerase. Fundamental to its mechanism is an ensemble of dramatically different conformational states that result from nucleotide binding and hydrolysis and distinct sets of interdomain interactions. Previous structural and biochemical work identified a conserved arginine residue (R380 in yeast) in the Hsp90 middle domain (MD) that is required for wild type hydrolysis activity in yeast, and hence proposed to be a catalytic residue. As part of our investigations on the origins of species-specific differences in Hsp90 conformational dynamics we probed the role of this MD arginine in bacterial, yeast, and human Hsp90s using a combination of structural and functional approaches. While the R380A mutation compromised ATPase activity in all three homologs, the impact on ATPase activity was both variable and much more modest (2-7 fold) than the mutation of an active site glutamate (40 fold) known to be required for hydrolysis. Single particle electron microscopy and small-angle X-ray scattering revealed that, for all Hsp90s, mutation of this arginine abrogated the ability to form the closed "ATP" conformational state in response to AMPPNP binding. Taken together with previous mutagenesis data exploring intra- and intermonomer interactions, these new data suggest that R380 does not directly participate in the hydrolysis reaction as a catalytic residue, but instead acts as an ATP-sensor to stabilize an NTD-MD conformation required for efficient ATP hydrolysis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Arginina/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Arginina/química , Bacterias/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas HSP90 de Choque Térmico/química , Humanos , Hidrólisis , Simulación de Dinámica Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Levaduras/química , Levaduras/metabolismoRESUMEN
Poly(ADP-ribose) (pADPr) is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD⺠by poly(ADP-ribose) polymerases (PARPs) and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. pADPr is involved in many cellular processes and exerts biological function through covalent modification and noncovalent binding to specific proteins. Very little is known about molecular recognition and structure-activity relationships for noncovalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on a large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at the hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.
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Bioquímica/métodos , Biopolímeros/biosíntesis , Poli Adenosina Difosfato Ribosa/biosíntesis , Animales , Biocatálisis , Biopolímeros/química , Calibración , Bovinos , Fraccionamiento Químico , Cromatografía Líquida de Alta Presión , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Humanos , Poli Adenosina Difosfato Ribosa/química , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Polimerizacion , Estructura Terciaria de Proteína , Tanquirasas/química , Tanquirasas/aislamiento & purificación , Tanquirasas/metabolismoRESUMEN
The ubiquitous molecular chaperone Hsp90 makes up 1-2% of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo-ATP-ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90, as well as the roles that nucleotide, co-chaperones, post-translational modification and substrates play. This view of Hsp90's conformational dynamics was enabled by the use of multiple complementary structural methods including, crystallography, small-angle X-ray scattering (SAXS), electron microscopy, Förster resonance energy transfer (FRET) and NMR. Finally, we discuss the effects of Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.
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Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Levaduras/metabolismoRESUMEN
Osmolytes are small molecules that play a central role in cellular homeostasis and the stress response by maintaining protein thermodynamic stability at controlled levels. The underlying physical chemistry that describes how different osmolytes impact folding free energy is well understood, however little is known about their influence on other crucial aspects of protein behavior, such as native-state conformational changes. Here we investigate this issue with the Hsp90 molecular chaperone, a large dimeric protein that populates a complex conformational equilibrium. Using small angle X-ray scattering we observe dramatic osmolyte-dependent structural changes within the native ensemble. The degree to which different osmolytes affect the Hsp90 conformation strongly correlates with thermodynamic metrics of their influence on stability. This observation suggests that the well-established osmolyte principles that govern stability also apply to large-scale conformational changes, a proposition that is corroborated by structure-based fitting of the scattering data, surface area comparisons and m-value analysis. This approach shows how osmolytes affect a highly cooperative open/closed structural transition between two conformations that differ by a domain-domain interaction. Hsp90 adopts an additional ligand-specific conformation in the presence of ATP and we find that osmolytes do not significantly affect this conformational change. Together, these results extend the scope of osmolytes by suggesting that they can maintain protein conformational heterogeneity at controlled levels using similar underlying principles that allow them to maintain protein stability; however the relative impact of osmolytes on different structural states can vary significantly.
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Adenilil Imidodifosfato/química , Proteínas de Escherichia coli/química , Proteínas HSP90 de Choque Térmico/química , Adenilil Imidodifosfato/farmacología , Betaína/química , Proteínas de Escherichia coli/metabolismo , Glicerol/química , Proteínas HSP90 de Choque Térmico/metabolismo , Metilaminas/química , Modelos Moleculares , Ósmosis , Conformación Proteica , Sarcosina/química , Dispersión del Ángulo Pequeño , Urea/química , Difracción de Rayos XRESUMEN
The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide-dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species-specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP-bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X-ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair-like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs.
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Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/química , Proteínas de la Membrana/química , Animales , Apoproteínas/química , Cristalografía por Rayos X , Citosol/química , Retículo Endoplásmico/química , Humanos , Ratones , Modelos Moleculares , Nucleótidos/química , Conformación Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Dispersión del Ángulo PequeñoRESUMEN
The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5'-adenylyl-beta,gamma-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Citrato (si)-Sintasa/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/ultraestructura , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
A major challenge in structural biology is to determine the configuration of domains and proteins in multidomain proteins and assemblies, respectively. All available data should be considered to maximize the accuracy and precision of these models. Small-angle X-ray scattering (SAXS) efficiently provides low-resolution experimental data about the shapes of proteins and their assemblies. Thus, we integrated SAXS profiles into our software for modeling proteins and their assemblies by satisfaction of spatial restraints. Specifically, we modeled the quaternary structures of multidomain proteins with structurally defined rigid domains as well as quaternary structures of binary complexes of structurally defined rigid proteins. In addition to SAXS profiles and the component structures, we used stereochemical restraints and an atomic distance-dependent statistical potential. The scoring function is optimized by a biased Monte Carlo protocol, including quasi-Newton and simulated annealing schemes. The final prediction corresponds to the best scoring solution in the largest cluster of many independently calculated solutions. To quantify how well the quaternary structures are determined based on their SAXS profiles, we used a benchmark of 12 simulated examples as well as an experimental SAXS profile of the homotetramer D-xylose isomerase. Optimization of the SAXS-dependent scoring function generally results in accurate models if sufficiently precise approximations for the constituent rigid bodies are available; otherwise, the best scoring models can have significant errors. Thus, SAXS profiles can play a useful role in the structural characterization of proteins and assemblies if they are combined with additional data and used judiciously. Our integration of a SAXS profile into modeling by satisfaction of spatial restraints will facilitate further integration of different kinds of data for structure determination of proteins and their assemblies.
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Modelos Moleculares , Conformación Proteica , Proteínas/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Biología Computacional , Simulación por Computador , Toxina Diftérica/química , Programas InformáticosRESUMEN
Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the gamma-phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.
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Adenosina Trifosfato/química , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Adenosina Trifosfatasas/química , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Escherichia coli/metabolismo , Hidrólisis , Modelos Biológicos , Conformación Molecular , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.