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1.
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.

2.
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.

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 ; 4(1): 291, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674710

RESUMO

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome-lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended "shepherd's staff" architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome-lysosome fusion. The hEPG5-GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5's overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Catarata/genética , Catarata/metabolismo , Predisposição Genética para Doença , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/genética
6.
Blood ; 137(26): 3641-3655, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33786587

RESUMO

The abundance of genetic abnormalities and phenotypic heterogeneities in acute myeloid leukemia (AML) poses significant challenges to the development of improved treatments. Here, we demonstrated that a key growth arrest-specific gene 6/AXL axis is highly activated in cells from patients with AML, particularly in stem/progenitor cells. We developed a potent selective AXL inhibitor that has favorable pharmaceutical properties and efficacy against preclinical patient-derived xenotransplantation (PDX) models of AML. Importantly, inhibition of AXL sensitized AML stem/progenitor cells to venetoclax treatment, with strong synergistic effects in vitro and in PDX models. Mechanistically, single-cell RNA-sequencing and functional validation studies uncovered that AXL inhibition, alone or in combination with venetoclax, potentially targets intrinsic metabolic vulnerabilities of AML stem/progenitor cells and shows a distinct transcriptomic profile and inhibits mitochondrial oxidative phosphorylation. Inhibition of AXL or BCL-2 also differentially targets key signaling proteins to synergize in leukemic cell killing. These findings have a direct translational impact on the treatment of AML and other cancers with high AXL activity.

7.
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
8.
Int J Mol Sci ; 21(21)2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33126766

RESUMO

Macroautophagy (also known as autophagy) is a major pathway for selective degradation of misfolded/aggregated proteins and damaged organelles and non-selective degradation of cytoplasmic constituents for the generation of power during nutrient deprivation. The multi-step degradation process, from sequestering cytoplasmic cargo into the double-membrane vesicle termed autophagosome to the delivery of the autophagosome to the lysosome or lytic vacuole for breakdown, is mediated by the core autophagy machinery composed of multiple Atg proteins, as well as the divergent sequence family of selective autophagy receptors. Single-particle electron microscopy (EM) is a molecular imaging approach that has become an increasingly important tool in the structural characterization of proteins and macromolecular complexes. This article summarizes the contributions single-particle EM have made in advancing our understanding of the core autophagy machinery and selective autophagy receptors. We also discuss current technical challenges and roadblocks, as well as look into the future of single-particle EM in autophagy research.


Assuntos
Autofagossomos , Autofagia , Microscopia Eletrônica/métodos , Imagem Individual de Molécula/métodos , Animais , Humanos , Vacúolos
9.
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
10.
J Phys Chem Lett ; 9(17): 4901-4909, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30102541

RESUMO

Mapping the nanomechanical properties of amyloids can provide valuable insights into structure and assembly mechanisms of protein aggregates that underlie the development of various human diseases. Although it is well-known that amyloids exhibit an intrinsic stiffness comparable to that of silk (1-10 GPa), a detailed understanding of the directional dependence (anisotropy) of the stiffness of amyloids and how it relates to structural features in these protein aggregates is missing. Here we used steered molecular dynamics (SMD) simulations and amplitude modulation-frequency modulation (AM-FM) atomic force microscopy to measure the directional variation in stiffness of GNNQQNY amyloid crystals. We reveal that individual crystals display significant mechanical anisotropy and relate this anisotropy to subtle but mechanically important differences in interactions between interfaces that define the crystal architecture. Our results provide detailed insights into the structure-mechanics relationship of amyloid that may help in designing amyloid-based nanomaterials with tailored mechanical properties.

11.
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
12.
Appl Environ Microbiol ; 84(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29625982

RESUMO

Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the RhodobacteralesIMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.


Assuntos
Proteínas de Bactérias/genética , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Rhodobacter capsulatus/enzimologia , Rhodobacter capsulatus/genética , Endopeptidase Clp/metabolismo , Transferência Genética Horizontal , Fosforilação , Domínios Proteicos
13.
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
14.
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
15.
Autophagy ; 13(12): 2018-2027, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28976798

RESUMO

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.


Assuntos
Sequência Conservada , Complexos Multiproteicos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/ultraestrutura
16.
Biochim Biophys Acta Proteins Proteom ; 1865(11 Pt B): 1613-1622, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28652207

RESUMO

Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This organization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated factors. These chromatin modifications are often catalyzed by multi-subunit enzyme complexes, whose large size, sophisticated composition, and inherent conformational flexibility pose significant technical challenges to their biochemical and structural characterization. Multiple structural approaches including nuclear magnetic resonance spectroscopy, X-ray crystallography, single-particle electron microscopy, and crosslinking coupled to mass spectrometry are often used synergistically to probe the overall architecture, subunit organization, and catalytic mechanisms of these macromolecular assemblies. In this review, we highlight several recent chromatin-modifying complexes studies that embodies this multipronged structural approach, and explore common themes amongst them. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Proteínas de Ligação a DNA/química , DNA/química , Complexos Multiproteicos/química , Animais , Cromatina/metabolismo , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Ressonância Magnética Nuclear Biomolecular
17.
RNA ; 23(6): 952-967, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28325844

RESUMO

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.


Assuntos
Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Rodófitas/genética , Rodófitas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional/métodos , Imunoprecipitação , Modelos Moleculares , Conformação de Ácido Nucleico , Filogenia , Ligação Proteica , Conformação Proteica , Transporte Proteico , Precursores de RNA/química , Estabilidade de RNA , RNA Mensageiro/química , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA/química , Espectrometria de Massas em Tandem
18.
Biophys J ; 112(4): 584-594, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28256219

RESUMO

Amyloids are fibrillar nanostructures of proteins that are assembled in several physiological processes in human cells (e.g., hormone storage) but also during the course of infectious (prion) and noninfectious (nonprion) diseases such as Creutzfeldt-Jakob and Alzheimer's diseases, respectively. How the amyloid state, a state accessible to all proteins and peptides, can be exploited for functional purposes but also have detrimental effects remains to be determined. Here, we measure the nanomechanical properties of different amyloids and link them to features found in their structure models. Specifically, we use shape fluctuation analysis and sonication-induced scission in combination with full-atom molecular dynamics simulations to reveal that the amyloid fibrils of the mammalian prion protein PrP are mechanically unstable, most likely due to a very low hydrogen bond density in the fibril structure. Interestingly, amyloid fibrils formed by HET-s, a fungal protein that can confer functional prion behavior, have a much higher Young's modulus and tensile strength than those of PrP, i.e., they are much stiffer and stronger due to a tighter packing in the fibril structure. By contrast, amyloids of the proteins RIP1/RIP3 that have been shown to be of functional use in human cells are significantly stiffer than PrP fibrils but have comparable tensile strength. Our study demonstrates that amyloids are biomaterials with a broad range of nanomechanical properties, and we provide further support for the strong link between nanomechanics and ß-sheet characteristics in the amyloid core.


Assuntos
Amiloide/química , Fenômenos Mecânicos , Multimerização Proteica , Fenômenos Biomecânicos , Humanos , Ligação de Hidrogênio , Insulina/química , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
19.
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
20.
Nat Struct Mol Biol ; 23(8): 758-60, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27428773

RESUMO

The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Complexos Multiproteicos/ultraestrutura , Estrutura Quaternária de Proteína
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