RESUMO
Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Assuntos
Regeneração Nervosa , Humanos , Neoplasias/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Transdução de Sinais/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Cardiotônicos/farmacologia , Animais , Biocatálise/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Compressão Nervosa , Proliferação de Células/efeitos dos fármacosRESUMO
ATF4 is a cellular stress induced bZIP transcription factor that is a hallmark effector of the integrated stress response. The integrated stress response is triggered by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 complex that can be carried out by the cellular stress responsive kinases; GCN2, PERK, PKR, and HRI. eIF2α phosphorylation downregulates mRNA translation initiation en masse, however ATF4 translation is upregulated. The integrated stress response can output two contradicting outcomes in cells; pro-survival or apoptosis. The mechanism for choice between these outcomes is unknown, however combinations of ATF4 heterodimerisation partners and post-translational modifications have been linked to this regulation. This semi-systematic review article covers ATF4 target genes, heterodimerisation partners and post-translational modifications. Together, this review aims to be a useful resource to elucidate the mechanisms controlling the effects of the integrated stress response. Additional putative roles of the ATF4 protein in cell division and synaptic plasticity are outlined.
RESUMO
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming part of the standard repertoire of techniques used by molecular biologists to investigate protein structure and dynamics. This is partly due to the increased use of automation in all stages of the technique and its versatility of application-many proteins that present challenges with techniques such as X-ray crystallography and cryoelectron microscopy are amenable to investigation with HDX-MS. The present review is aimed at scientists who are curious about the technique, and how it may aid their research. It describes the fundamental basis of solvent exchange, the basics of a standard HDX-MS experiment, as well as highlighting emerging novel experimental advances, which point to where the field is heading.
Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Espectrometria de Massas/métodos , Microscopia Crioeletrônica , Medição da Troca de Deutério/métodos , Proteínas/químicaRESUMO
GCN2 (general control nonderepessible 2) is an eIF2α kinase responsible for entirely rewiring the metabolism of cells when they are put under amino acid starvation stress. Recently, there has been renewed interest in GCN2 as a potential oncotarget, with several studies reporting the development of small molecule inhibitors. The foundation of this work is built upon biochemical and cellular data which suggest GCN2 may be aberrantly overexpressed and is responsible for keeping cells on 'life-support' while tumours undergo significant nutritional stress during tumorigenesis, allowing cancer stem cells to develop chemotherapeutic resistance. However, most studies which have investigated the role of GCN2 in cancer have been conducted in various cancer model systems, often under a specific set of stresses, mutational backgrounds and drug cocktails. This review aims to comprehensively summarise the biochemical, molecular and cellular literature associated with GCN2 and its role in various cancers and determine whether a consensus can be developed to discern under which circumstances we may wish to target GCN2.
Assuntos
Neoplasias , Proteínas Serina-Treonina Quinases , Aminoácidos/metabolismo , Humanos , Mutação , FosforilaçãoRESUMO
The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli , Regulação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intracelular/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Modelos Moleculares , Domínios PDZ , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Serina-Treonina Quinases TOR/genéticaRESUMO
The tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN) is a tightly regulated enzyme responsible for dephosphorylating the progrowth lipid messenger molecule phosphatidylinositol 3,4,5-trisphosphate (PIP3) on the plasma membrane. The carboxy-terminal tail (CTT) of PTEN is key for regulation of the enzyme. When phosphorylated, the unstructured CTT interacts with the phosphatase-C2 superdomain to inactivate the enzyme by preventing membrane association. PTEN mutations associated with cancer also inactivate the enzyme. Alternate translation-initiation sites generate extended isoforms of PTEN, such as PTEN-L that has multiple roles in cells. The extended amino-terminal region bears a signal sequence and a polyarginine sequence to facilitate exit from and entry into cells, respectively, and a membrane-binding helix that activates the enzyme. This amino-terminal region also facilitates mitochondrial and nucleolar localization. This review explores PTEN structure and its impact on localization and regulation.
Assuntos
PTEN Fosfo-Hidrolase/química , Sequência de Aminoácidos , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , FosforilaçãoRESUMO
The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Dimerização , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lisossomos/metabolismo , Espectrometria de Massas , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/sangue , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteína Regulatória Associada a mTOR/química , Proteínas de Saccharomyces cerevisiae/sangue , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Cells must be able to sense and adapt to their surroundings to thrive in a dynamic environment. Key to adapting to a low nutrient environment is the Integrated Stress Response (ISR), a short-lived pathway that allows cells to either regain cellular homeostasis or facilitate apoptosis during periods of stress. Central to the ISR is the protein kinase General Control Non-depressible 2 (GCN2), which is responsible for sensing starvation. Upon amino acid deficiency, GCN2 is activated and initiates the ISR by phosphorylating the translation initiation factor eIF2α, stalling protein translation, and activating the transcription factor ATF4, which in turn up-regulates autophagy and biosynthesis pathways. A key outstanding question is how GCN2 is activated from an autoinhibited state. Until recently, a model of activation focussed on the increase of deacylated tRNA associated with amino acid starvation, with deacylated tRNA binding directly to GCN2 and releasing autoinhibition. However, in vivo experiments have pointed towards an alternative, deacylated-tRNA-independent mechanism of activation. Here, we review the various factors that may facilitate GCN2 activation, including recent research showing that the P-stalk complex, a ribosome-associated heteropentameric protein complex, is a potent activator of GCN2.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Ativação Enzimática , Humanos , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , RNA de Transferência/metabolismoRESUMO
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de HidrogênioRESUMO
Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo. Here, we show that, essential for anti-bacterial autophagy, the cargo receptor NDP52 forms a trimeric complex with FIP200 and SINTBAD/NAP1, which are subunits of the autophagy-initiating ULK and the TBK1 kinase complex, respectively. FIP200 and SINTBAD/NAP1 are each recruited independently to bacteria via NDP52, as revealed by selective point mutations in their respective binding sites, but only in their combined presence does xenophagy proceed. Such recruitment of the upstream autophagy machinery by NDP52 reveals how detection of cargo-associated "eat me" signals, induction of autophagy, and juxtaposition of cargo and phagophores are integrated in higher eukaryotes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia , Sítios de Ligação/genética , Citoplasma/microbiologia , Citosol/microbiologia , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Mutação Puntual/genética , Ligação Proteica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidadeRESUMO
Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , Motivos de Aminoácidos , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Relação Estrutura-AtividadeRESUMO
Receptor tyrosine kinase FGFR3 is involved in many signaling networks and is frequently mutated in developmental disorders and cancer. The Hsp90/Cdc37 chaperone system is essential for function of normal and neoplastic cells. Here we uncover the mechanistic inter-relationships between these proteins by combining approaches including NMR, HDX-MS, and SAXS. We show that several disease-linked mutations convert FGFR3 to a stronger client, where the determinant underpinning client strength involves an allosteric network through the N-lobe and at the lobe interface. We determine the architecture of the client kinase/Cdc37 complex and demonstrate, together with site-specific information, that binding of Cdc37 to unrelated kinases induces a common, extensive conformational remodeling of the kinase N-lobe, beyond localized changes and interactions within the binary complex. As further shown for FGFR3, this processing by Cdc37 deactivates the kinase and presents it, in a specific orientation established in the complex, for direct recognition by Hsp90.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mutação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Sítio Alostérico , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Ativação Enzimática , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Fosfopeptídeos/metabolismo , Domínios ProteicosRESUMO
INTRODUCTION: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule interactions. The development of novel methodologies and technical advancements in mass spectrometers has greatly expanded the accessibility and acceptance of this technique within both academia and industry. Areas covered: This review examines the theoretical basis of how amide exchange occurs, how different mass spectrometer approaches can be used for HDX-MS experiments, as well as the use of HDX-MS in drug development, specifically focusing on how HDX-MS is used to characterize bio-therapeutics, and its use in examining protein-protein and protein small molecule interactions. Expert opinion: HDX-MS has been widely accepted within the pharmaceutical industry for the characterization of bio-therapeutics as well as in the mapping of antibody drug epitopes. However, there is room for this technique to be more widely used in the drug discovery process. This is particularly true in the use of HDX-MS as a complement to other high-resolution structural approaches, as well as in the development of small molecule therapeutics that can target both active-site and allosteric binding sites.
Assuntos
Medição da Troca de Deutério/métodos , Descoberta de Drogas/métodos , Espectrometria de Massas/métodos , Sítio Alostérico , Sítios de Ligação , Desenho de Fármacos , Indústria Farmacêutica/métodos , Humanos , Dobramento de Proteína , Proteínas/metabolismoRESUMO
Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.
Assuntos
Antineoplásicos/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Medição da Troca de Deutério , Avaliação Pré-Clínica de Medicamentos/métodos , Transporte de Elétrons , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Indazóis/química , Indazóis/metabolismo , Indazóis/farmacologia , Peso Molecular , Oligonucleotídeos/antagonistas & inibidores , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Conformação Proteica , Purinas/química , Purinas/metabolismo , Purinas/farmacologia , Piridazinas , Quinazolinonas/química , Quinazolinonas/metabolismo , Quinazolinonas/farmacologia , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Processamento de Sinais Assistido por Computador , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Espectrometria de Massas em Tandem , Triazinas/química , Triazinas/metabolismo , Triazinas/farmacologiaRESUMO
The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Proteínas Relacionadas à Autofagia/química , Cristalografia por Raios X , Medição da Troca de Deutério , Células HEK293 , Humanos , Espectrometria de Massas , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Despite its small size and deceptively simple domain organization, PTEN remains a challenging structural target due to its N- and C-terminal intrinsically disordered segments, and the conformational heterogeneity caused by phosphorylation of its C terminus. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS), it is possible to probe the conformational dynamics of the disordered termini, and also to determine how PTEN binds to lipid membranes. Here, we describe how to purify recombinant, homogenously dephosphorylated PTEN from a eukaryotic system for subsequent investigation with HDX-MS or crystallography.
Assuntos
Cristalografia por Raios X/métodos , Espectrometria de Massas/métodos , PTEN Fosfo-Hidrolase/química , Animais , Medição da Troca de Deutério/métodos , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismoRESUMO
The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIß) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIß in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIß-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and both activities are necessary for its role as a tumour suppressor. PTEN activity is controlled by phosphorylation of its intrinsically disordered C-terminal tail. A recently discovered variant of PTEN, PTEN-long (PTEN-L), has a 173-residue N-terminal extension that causes PTEN-L to exhibit unique behaviour, such as movement from one cell to another. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and biophysical assays, we show that both the N-terminal extension of PTEN-L and C-terminal tail of PTEN affect the phosphatase activity using unique mechanisms. Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN. Partially dephosphorylating PTEN on pThr(366)/pSer(370) results in sufficient exposure of the active site to allow a selective activation for soluble substrates. Using HDX-MS, we identified a membrane-binding element in the N-terminal extension of PTEN-L, termed the membrane-binding helix (MBH). The MBH radically alters the membrane binding mechanism of PTEN-L compared with PTEN, switching PTEN-L to a 'scooting' mode of catalysis from the 'hopping' mode that is characteristic of PTEN.
Assuntos
Membrana Celular/genética , Membrana Celular/metabolismo , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/fisiologia , Sequência de Aminoácidos , Animais , Insetos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Spodoptera , Especificidade por Substrato/fisiologiaRESUMO
Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.