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1.
Structure ; 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34348129

RESUMO

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

2.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

3.
J Mol Biol ; 433(18): 167145, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34229011

RESUMO

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.


Assuntos
Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Mamíferos/genética , Conformação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética
4.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
5.
Commun Biol ; 3(1): 735, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277614

RESUMO

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Insetos , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas , Especificidade por Substrato , Espectrometria de Massas em Tandem , Proteínas rab de Ligação ao GTP/genética
6.
Sci Rep ; 8(1): 17353, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478341

RESUMO

Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped viruses (e.g. hepatitis A virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket paralysis virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.


Assuntos
Dicistroviridae/patogenicidade , Drosophila melanogaster/virologia , Vesículas Extracelulares/virologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Virais/metabolismo , Animais , Dicistroviridae/genética , Dicistroviridae/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Exossomos/virologia , RNA Viral/metabolismo , Proteínas Virais/análise
7.
J Mol Biol ; 430(18 Pt B): 3129-3142, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30031006

RESUMO

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP2 and PIP3. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , Regulação Alostérica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas/química , Proteínas Recombinantes
8.
Mol Cell Biol ; 38(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29463645

RESUMO

Conserved from yeast to humans, the NuA4 histone acetyltransferase is a large multisubunit complex essential for cell viability through the regulation of gene expression, genome maintenance, metabolism, and cell fate during development and stress. How the different NuA4 subunits work in concert with one another to perform these diverse functions remains unclear, and addressing this central question requires a comprehensive understanding of NuA4's molecular architecture and subunit organization. We have determined the structure of fully assembled native yeast NuA4 by single-particle electron microscopy. Our data revealed that NuA4 adopts a trilobal overall architecture, with each of the three lobes constituted by one or two functional modules. By performing cross-linking coupled to mass spectrometry analysis and in vitro protein interaction studies, we further mapped novel intermolecular interfaces within NuA4. Finally, we combined these new data with other known structural information of NuA4 subunits and subassemblies to construct a multiscale model to illustrate how the different NuA4 subunits and modules are spatially arranged. This model shows that the multiple chromatin reader domains are clustered together around the catalytic core, suggesting that NuA4's multimodular architecture enables it to engage in multivalent interactions with its nucleosome substrate.


Assuntos
Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Acetilação , Sequência de Aminoácidos , Histona Acetiltransferases/genética , Histonas , Microscopia Eletrônica/métodos , Nucleossomos/fisiologia , Subunidades Proteicas/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
9.
Cell Mol Life Sci ; 75(9): 1613-1622, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29332244

RESUMO

Conserved from yeast to humans, Elongator is a protein complex implicated in multiple processes including transcription regulation, α-tubulin acetylation, and tRNA modification, and its defects have been shown to cause human diseases such as familial dysautonomia. Elongator consists of two copies of six core subunits (Elp1, Elp2, Elp3, Elp4, Elp5, and Elp6) that are organized into two subcomplexes: Elp1/2/3 and Elp4/5/6 and form a stable assembly of ~ 850 kDa in size. Although the catalytic subunit of Elongator is Elp3, which contains a radical S-adenosyl-L-methionine (SAM) domain and a putative histone acetyltransferase domain, the Elp4/5/6 subcomplex also possesses ATP-modulated tRNA binding activity. How at the molecular level, Elongator performs its multiple functions and how the different subunits regulate Elongator's activities remains poorly understood. Here, we provide an overview of the proposed functions of Elongator and describe how recent structural studies provide new insights into the mechanism of action of this multifunctional complex.


Assuntos
Fatores de Alongamento de Peptídeos/metabolismo , Animais , Histona Acetiltransferases/metabolismo , Humanos , Ligação Proteica/fisiologia , Subunidades Proteicas/metabolismo , RNA de Transferência/metabolismo , Transcrição Genética/fisiologia
10.
EMBO Rep ; 18(2): 280-291, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27872205

RESUMO

Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.


Assuntos
Proteínas Fúngicas/química , Histona Acetiltransferases/química , Modelos Moleculares , Complexos Multiproteicos/química , Subunidades Proteicas/química , Sequência Conservada , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Espectrometria de Massas , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade
11.
Infect Immun ; 85(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27795363

RESUMO

Enteropathogenic Escherichia coli (EPEC)-induced diarrhea is often associated with disruption of intestinal epithelial tight junctions. Although studies have shown alterations in the expression and localization of bicellular tight junction proteins during EPEC infections, little is known about whether tricellular tight junction proteins (tTJs) are affected. Using Caco-2 cell monolayers, we investigated if EPEC is capable of targeting the tTJ protein tricellulin. Our results demonstrated that at 4 h postinfection, EPEC induced a significant reduction in tricellulin levels, accompanied by a significant loss of transepithelial resistance (TEER) and a corresponding increase in paracellular permeability. Conversely, cells overexpressing tricellulin were highly resistant to EPEC-induced barrier disruption. Confocal microscopy revealed the distribution of tricellulin into the plasma membrane of infected epithelial cells and confirmed the localization of EPEC aggregates in close proximity to tTJs. Moreover, infections with EPEC strains lacking genes encoding specific type III secreted effector proteins demonstrated a crucial role for the effector EspG1 in modulating tricellulin expression. Complementation studies suggest that the EspG-induced depletion of tricellulin is microtubule dependent. Overall, our results show that EPEC-induced epithelial barrier dysfunction is mediated in part by EspG1-induced microtubule-dependent depletion of tricellulin.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteína 2 com Domínio MARVEL/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Junções Íntimas/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Diarreia/metabolismo , Diarreia/microbiologia , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Microtúbulos/metabolismo , Microtúbulos/microbiologia , Permeabilidade , Junções Íntimas/microbiologia
12.
Infect Immun ; 82(9): 3753-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24958710

RESUMO

Intestinal epithelial cells (IECs), including secretory goblet cells, form essential physiochemical barriers that separate luminal bacteria from underlying immune cells in the intestinal mucosa. IECs are common targets for enteric bacterial pathogens, with hosts responding to these microbes through innate toll-like receptors that predominantly signal through the MyD88 adaptor protein. In fact, MyD88 signaling confers protection against several enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Since IECs are considered innately hyporesponsive, it is unclear whether MyD88 signaling within IECs contributes to this protection. We infected mice lacking MyD88 solely in their IECs (IEC-Myd88(-/-)) with S. Typhimurium. Compared to wild-type (WT) mice, infected IEC-Myd88(-/-) mice suffered accelerated tissue damage, exaggerated barrier disruption, and impaired goblet cell responses (Muc2 and RELMß). Immunostaining revealed S. Typhimurium penetrated the IECs of IEC-Myd88(-/-) mice, unlike in WT mice, where they were sequestered to the lumen. When isolated crypts were assayed for their antimicrobial actions, crypts from IEC-Myd88(-/-) mice were severely impaired in their antimicrobial activity against S. Typhimurium. We also examined whether MyD88 signaling in IECs impacted host defense against C. rodentium, with IEC-Myd88(-/-) mice again suffering exaggerated tissue damage, impaired goblet cell responses, and reduced antimicrobial activity against C. rodentium. These results demonstrate that MyD88 signaling within IECs plays an important protective role at early stages of infection, influencing host susceptibility to infection by controlling the ability of the pathogen to reach and survive at the intestinal mucosal surface.


Assuntos
Anti-Infecciosos/imunologia , Colite/imunologia , Células Caliciformes/imunologia , Mucosa Intestinal/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/imunologia , Animais , Citrobacter rodentium/imunologia , Colite/microbiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Gastroenterite/imunologia , Gastroenterite/microbiologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Células Caliciformes/microbiologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia
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