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1.
Nucleic Acids Res ; 48(4): 2091-2106, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31875226

RESUMO

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the ß1-ß2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.


Assuntos
Fator 1 de Ribosilação do ADP/química , Proteínas do Citoesqueleto/química , Motivo de Ligação ao RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/química , Fator 1 de Ribosilação do ADP/genética , Sítios de Ligação/genética , Citoplasma/química , Citoplasma/genética , Proteínas do Citoesqueleto/genética , Humanos , Conformação Proteica , Estabilidade de RNA/genética , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética
2.
Biochemistry ; 58(10): 1423-1431, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30735034

RESUMO

Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers. Here, we report on the production of a functional human Arf1 membrane platform on nanodiscs for biophysical studies. Large scale bacterial production of highly pure, N-myristoylated human Arf1 has been achieved, including complex isotopic labeling for nuclear magnetic resonance (NMR) studies, and the myr-Arf1 can be readily assembled in small nanoscale lipid bilayers (nanodiscs, NDs). It is determined that myr-Arf1 requires a minimum binding surface in the NDs of ∼20 lipids. Fluorescence and NMR were used to establish nucleotide exchange and ArfGAP-stimulated GTP hydrolysis at the membrane, indicating that phophoinositide stimulation of the activity of the ArfGAP ASAP1 is ≥2000-fold. Differences in nonhydrolyzable GTP analogues are observed, and GMPPCP is found to be the most stable. Combined, these observations establish a functional environment for biophysical studies of Arf1 effectors and interactions at the membrane.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Bicamadas Lipídicas/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo , Ácido Mirístico/metabolismo
3.
FEBS J ; 285(11): 2004-2018, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29603662

RESUMO

The Arf family of small GTP-binding and -hydrolyzing proteins are some of the most important intracellular regulators of membrane dynamics. In this study, we identified the Golgi-localized Arf family G protein Arf1 in Candida albicans and confirmed its conserved function in regulating the secretory pathway. Interestingly, deletion of ARF1 resulted in intracellular reactive oxygen species (ROS) accumulation, and induced formation of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES). Moreover, N-acetylcysteine-mediated ROS scavenging in the arf1Δ/Δ strain attenuated ERMES formation, indicating that intracellular ROS accumulation resulting from ARF1 deletion facilitated ERMES formation. In addition, Arf1 regulated many key physiological processes in C. albicans, including cell cycle progression, morphogenesis and virulence. This study uncovers a role for Arf family G proteins in regulating ERMES formation and sheds new light on the crucial contribution of ROS to membrane dynamics.


Assuntos
Fator 1 de Ribosilação do ADP/genética , Candida albicans/genética , Retículo Endoplasmático/genética , Mitocôndrias/genética , Fator 1 de Ribosilação do ADP/química , Ciclo Celular/genética , Membrana Celular/química , Membrana Celular/genética , Retículo Endoplasmático/química , Complexo de Golgi/química , Complexo de Golgi/genética , Mitocôndrias/química , Morfogênese/genética , Espécies Reativas de Oxigênio/química , Transdução de Sinais , Virulência
4.
Sci Rep ; 7(1): 7148, 2017 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-28769048

RESUMO

AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular , Vesículas Revestidas por Clatrina/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Mutação , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/genética , Rede trans-Golgi/metabolismo
5.
Elife ; 62017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28621666

RESUMO

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of ßδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , Complexo I de Proteína do Envoltório/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Fator 1 de Ribosilação do ADP/química , Animais , Complexo I de Proteína do Envoltório/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Camundongos , Modelos Moleculares , Conformação Proteica
6.
Biochemistry ; 56(1): 271-280, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27936598

RESUMO

G proteins are part of the G-protein-coupled receptor (GPCR) signal transduction cascade in which they transfer a signal from the membrane-embedded GPCR to other proteins in the cell. In the case of the inhibitory G-protein heterotrimer, permanent N-terminal myristoylation can transiently localize the Gαi subunit at the membrane as well as crucially influence Gαi's function in the GTP-bound conformation. The attachment of lipids to proteins is known to be essential for membrane trafficking; however, our results suggest that lipidation is also important for protein-protein interactions during signal transduction. Here we investigate the effect of myristoylation on the structure and dynamics of soluble Gαi1 and its possible implication for signal transduction. A 2 µs classical molecular dynamics simulation of a myristoylated Gαi1-GTP complex suggests that the myristoyl-induced conformational changes of the switch II and alpha helical domains create new possibilities for protein-protein interactions and emphasize the importance of permanent lipid attachment for the conformation and functional tunability of signaling proteins.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/química , Ácido Mirístico/química , Conformação Proteica , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Ácido Mirístico/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Eletricidade Estática
7.
Chembiochem ; 18(3): 324-330, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27925692

RESUMO

Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motifs, coupled with bio-orthogonal chemistry, have been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP were measured and compared with the unmodified myristoylated Arf1. The ketone-modified myristoylated Arf1 could be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Ácido Mirístico/química , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrazinas/química , Hidrólise , Lipossomos/química , Lipossomos/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem
8.
Methods Mol Biol ; 1496: 41-53, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27632000

RESUMO

Protein-protein and protein-membrane interactions play a critical role in shaping biological membranes through direct physical contact with the membrane surface. This is particularly evident in many steps of membrane trafficking, in which proteins deform the membrane and induce fission to form transport carriers. The small GTPase Arf1 and related proteins have the ability to remodel membranes by insertion of an amphipathic helix into the membrane. Arf1 and the exomer cargo adaptor coordinate cargo sorting into subset of secretory vesicle carriers in the model organism Saccharomyces cerevisiae. Here, we detail the assays we used to explore the cooperative action of Arf1 and exomer to bind and remodel membranes. We expect these methods are broadly applicable to other small GTPase/effector systems where investigation of membrane binding and remodeling is of interest.


Assuntos
Fator 1 de Ribosilação do ADP , Membrana Celular/enzimologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Protein Cell ; 7(8): 586-600, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27472951

RESUMO

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.


Assuntos
Proteína Coatomer/química , Citosol/química , Membranas Artificiais , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Proteína Coatomer/metabolismo , Citosol/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Ratos
10.
Fish Shellfish Immunol ; 55: 123-30, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27231192

RESUMO

ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda.


Assuntos
Fator 1 de Ribosilação do ADP/genética , Amônia/toxicidade , Proteínas de Artrópodes/genética , Regulação da Expressão Gênica , Palaemonidae/genética , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Palaemonidae/efeitos dos fármacos , Palaemonidae/imunologia , Palaemonidae/microbiologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência
11.
Science ; 350(6259): aac5137, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26494761

RESUMO

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.


Assuntos
Fator 1 de Ribosilação do ADP/química , Complexo 1 de Proteínas Adaptadoras/química , Antígenos CD/química , Vesículas Revestidas por Clatrina/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Clatrina/química , Clatrina/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Proteínas Ligadas por GPI/química , Humanos , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Produtos do Gene nef do Vírus da Imunodeficiência Humana/ultraestrutura , Rede trans-Golgi/metabolismo
12.
Methods Cell Biol ; 130: 69-80, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26360029

RESUMO

Defining the interaction of Arf GAPs with specific Arfs is important for understanding their functions in the endocytic system. Cell-based approaches have been valuable for identifying Arfs and Arf GAPs active in the endocytic compartment; however, the cell-based assays have some limitations in establishing relationships among the Arfs and ArfGAPs. Here we describe a simple in vitro assay that will provide a means for comparing Arfs as substrates and serve to complement cell-based studies.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/biossíntese , Fator 1 de Ribosilação do ADP/isolamento & purificação , Ensaios Enzimáticos , Escherichia coli , Guanosina Trifosfato/química , Humanos , Hidrólise , Lipossomas Unilamelares
13.
Methods Cell Biol ; 130: 101-26, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26360031

RESUMO

The ADP ribosylation factor (Arf) family of small guanosine triphosphatases (GTPases) regulates vesicular transport at several locations within the cell, and is in turn regulated by guanine nucleotide exchange factors (GEFs) via a conserved catalytic domain, termed the Sec7 domain. The catalytic activity of the Sec7 domain is well characterized in the context of a few GEFs acting at the periphery of the cell. This chapter describes the techniques used to extend the biochemical analysis of activity to the much larger GEFs acting on the Arf family in the core secretory pathway, using the activity of Saccharomyces cerevisiae Sec7 on Arf1, regulating export from the trans-Golgi network, as a model. The complete methods for purification to near homogeneity of all proteins required, including several Sec7 constructs and multiple relevant small GTPases, are detailed. These are followed by methods for the quantification of the nucleotide exchange activity of Sec7 in a physiologically relevant context, including modifications required to dissect the signal integration functions of Sec7 as an effector of several other small GTPases, and methods for identifying stable Sec7-small GTPase interactions in the presence of membranes. These techniques may be extended to the analysis of similar members of the Sec7 GEF subfamily in other species and acting elsewhere in the secretory pathway.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fatores de Troca do Nucleotídeo Guanina/química , Fator 1 de Ribosilação do ADP/isolamento & purificação , Animais , Baculoviridae/genética , Linhagem Celular , Cromatografia de Afinidade , Cromatografia em Gel , Ensaios Enzimáticos , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Cinética , Lipossomos/química , Mariposas , Transporte Proteico , Saccharomyces cerevisiae/enzimologia
14.
Science ; 349(6244): 195-8, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-26160949

RESUMO

Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Complexo I de Proteína do Envoltório/química , Fator 1 de Ribosilação do ADP/química , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Proteínas Ativadoras de GTPase/química , Humanos , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química
15.
Anal Chem ; 87(14): 7022-9, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26134943

RESUMO

Hydrogen exchange (HX) mass spectrometry (MS) is valuable for providing conformational information for proteins/peptides that are very difficult to analyze with other methods such as peripheral membrane proteins and peptides that interact with membranes. We developed a new type of HX MS measurement that integrates Langmuir monolayers. A lipid monolayer was generated, a peptide or protein associated with it, and then the monolayer-associated peptide or protein was exposed to deuterium. The deuterated species was recovered from the monolayer, digested, and deuterium incorporation monitored by MS. Test peptides showed that deuterium recovery in an optimized protocol was equivalent to deuterium recovery in conventional solution HX MS. The reproducibility of the measurements was high, despite the requirement of generating a new monolayer for each deuterium labeling time. We validated that known conformational changes in the presence of a monolayer/membrane could be observed with the peptide melittin and the myristoylated protein Arf-1. Results in an accompanying paper show that the method can reveal details of conformational changes in a protein (HIV-1 Nef), which adopts a different conformation, depending on whether or not it is able to insert into the lipid layer. Overall, the HX MS Langmuir monolayer method provided new and meaningful conformational information for proteins that associate with lipid layers. The combination of HX MS results with neutron or X-ray reflection of the same proteins in Langmuir monolayers can be more informative than the isolated use of either method.


Assuntos
Fator 1 de Ribosilação do ADP/química , Hidrogênio/química , Espectrometria de Massas , Meliteno/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Medição da Troca de Deutério , Ácidos Graxos Monoinsaturados/química , HIV-1/metabolismo , Humanos , Meliteno/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo
16.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 5): 594-9, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25945714

RESUMO

Entamoeba histolytica is the etiological agent of amebiasis, a diarrheal disease which causes amoebic liver abscesses and amoebic colitis. Approximately 50 million people are infected worldwide with E. histolytica. With only 10% of infected people developing symptomatic amebiasis, there are still an estimated 100,000 deaths each year. Because of the emergence of resistant strains of the parasite, it is necessary to find a treatment which would be a proper response to this challenge. ADP-ribosylation factor (ARF) is a member of the ARF family of GTP-binding proteins. These proteins are ubiquitous in eukaryotic cells; they generally associate with cell membranes and regulate vesicular traffic and intracellular signalling. The crystal structure of ARF1 from E. histolytica has been determined bound to magnesium and GDP at 1.8 Å resolution. Comparison with other structures of eukaryotic ARF proteins shows a highly conserved structure and supports the interswitch toggle mechanism of communicating the conformational state to partner proteins.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Entamoeba histolytica/química , Entamoeba histolytica/metabolismo , Guanosina Difosfato/metabolismo , Magnésio/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Sequência de Aminoácidos , Cristalização , Entamoeba histolytica/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína
17.
Mol Cell ; 58(1): 110-22, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25773595

RESUMO

N-myristoylation is an essential fatty acid modification that governs the localization and activity of cell signaling enzymes, architectural proteins, and immune regulatory factors. Despite its importance in health and disease, there are currently no methods for reversing protein myristoylation in vivo. Recently, the Shigella flexneri protease IpaJ was found to cleave myristoylated glycine of eukaryotic proteins, yet the discriminatory mechanisms of substrate selection required for targeted demyristoylation have not yet been evaluated. Here, we performed global myristoylome profiling of cells treated with IpaJ under distinct physiological conditions. The protease is highly promiscuous among diverse N-myristoylated proteins in vitro but is remarkably specific to Golgi-associated ARF/ARL family GTPases during Shigella infection. Reconstitution studies revealed a mechanistic framework for substrate discrimination based on IpaJ's function as a GTPase "effector" of bacterial origin. We now propose a concerted model for IpaJ function that highlights its potential for programmable demyristoylation in vivo.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Antígenos de Bactérias/metabolismo , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Shigella flexneri/química , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ácido Mirístico/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shigella flexneri/enzimologia , Transdução de Sinais
18.
Curr Opin Struct Biol ; 29: 67-76, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25460270

RESUMO

Arf GTPases are major regulators of membrane traffic and organelle structure in eukaryotes where they recruit many different effectors, including components of vesicular coats, proteins that tether membranes, sort lipids or have diverse other functions in vesicular traffic, and bacterial proteins that divert Arf functions in host cells. A dozen of structures of unrelated effectors bound to Arf1, Arf6 or their close relative Arl1 are available, revealing that Arf GTPases do not recognize preferred structures in their effectors. In contrast, a trait common to many Arf/effector complexes is that they are juxtaposed to membranes by multiple protein/membrane contacts, yet of diverse sizes, shapes and physicochemistry. The common function of Arf GTPases thus appears to be their ability to assemble versatile, multivalent membrane-binding platforms, resulting in optimal orientation and allosteric regulation of their effectors leading to a plethora of membrane-localized functions.


Assuntos
Fator 1 de Ribosilação do ADP/química , Proteínas de Transporte/química , Complexos Multiproteicos/química , Conformação Proteica , Multimerização Proteica , Transporte Proteico
19.
Proc Natl Acad Sci U S A ; 111(34): 12378-83, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25114232

RESUMO

Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Substituição de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cricetinae , Ativação Enzimática , Retroalimentação Fisiológica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Lipossomos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismo
20.
Structure ; 22(3): 363-5, 2014 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-24607142

RESUMO

In this issue of Structure, Liu and colleagues describe an experimentally rigorous and innovative approach for understanding the role of membranes in the function and regulation of peripheral membrane proteins. This work is the beginning of a new era of experimental work that promises many advances relevant to molecular mechanisms and therapeutic targeting of this important class of proteins.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos
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