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1.
Cell ; 173(5): 1244-1253.e10, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29681455

RESUMO

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.


Assuntos
Amiloide/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Alinhamento de Sequência
2.
Immunity ; 47(4): 635-647.e6, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29045898

RESUMO

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.


Assuntos
Amiloide/imunologia , Proteínas de Transporte/imunologia , Proteínas de Drosophila/imunologia , NF-kappa B/imunologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Amiloide/metabolismo , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Feminino , Expressão Gênica/imunologia , Masculino , Microscopia Confocal , Modelos Imunológicos , Mutação , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
3.
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
5.
Proc Natl Acad Sci U S A ; 107(25): 11295-300, 2010 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-20534509

RESUMO

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 A resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Citoplasma/metabolismo , Flagelina/química , Modelos Biológicos , Chaperonas Moleculares/química , Estrutura Terciária de Proteína , Transporte Proteico
6.
Proc Natl Acad Sci U S A ; 106(50): 21131-6, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19948960

RESUMO

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and alpha-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the alpha-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the alpha-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg(2+)- and ADP-Mg(2+)-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP.AlF(4)(-)-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361-366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Proteínas de Membrana/química , Trifosfato de Adenosina/metabolismo , Clostridium thermocellum/química , Ligação Proteica , Transporte Proteico
7.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 3): 295-303, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20179341

RESUMO

The signal recognition particle (SRP) is a conserved ribonucleoprotein (RNP) complex that co-translationally targets membrane and secretory proteins to membranes. The assembly of the particle depends on the proper folding of the SRP RNA, which in mammalia and archaea involves an induced-fit mechanism within helices 6 and 8 in the S domain of SRP. The two helices are juxtaposed and clamped together upon binding of the SRP19 protein to their apices. In the current assembly paradigm, archaeal SRP19 causes the asymmetric loop of helix 8 to bulge out and expose the binding platform for the key player SRP54. Based on a heterologous archaeal SRP19-human SRP RNA structure, mammalian SRP19 was thought not to be able to induce this change, thus explaining the different requirements of SRP19 for SRP54 recruitment. In contrast, the crystal structures of a crenarchaeal and the all-human SRP19-SRP RNA binary complexes presented here show that the asymmetric loop is bulged out in both binary complexes. Differences in SRP assembly between mammalia and archaea are therefore independent of SRP19 and are based on differences in SRP RNA itself. A new SRP-assembly scheme is presented.


Assuntos
Proteínas Arqueais/química , Partícula de Reconhecimento de Sinal/química , Sulfolobus solfataricus/química , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sequência de Bases , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , Alinhamento de Sequência , Partícula de Reconhecimento de Sinal/metabolismo , Sulfolobus solfataricus/metabolismo
8.
Sci Rep ; 8: 45912, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28374821

RESUMO

Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.


Assuntos
Aciltransferases/genética , Dissulfetos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Dissulfetos/química , Glicosilação , Glicosilfosfatidilinositóis/genética , Humanos , Insetos/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Domínios Proteicos/genética , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Trypanosoma/genética , Trypanosoma/patogenicidade
9.
Nat Med ; 21(5): 457-66, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25849135

RESUMO

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML-RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Leucêmica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Leucemia Promielocítica Aguda/metabolismo , Peptidilprolil Isomerase/genética , Tretinoína/metabolismo , Animais , Antineoplásicos/química , Neoplasias da Mama/genética , Catálise , Domínio Catalítico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/metabolismo , Células HEK293 , Humanos , Leucemia Promielocítica Aguda/genética , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Peptidilprolil Isomerase de Interação com NIMA , Transplante de Neoplasias , Fosfatos/química , Fosforilação , Prolina/química , Neoplasias de Mama Triplo Negativas/metabolismo
10.
Nat Protoc ; 8(9): 1787-99, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23989673

RESUMO

Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Parede Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Espectrometria de Massas
11.
Cell Rep ; 1(5): 461-71, 2012 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-22832272

RESUMO

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.


Assuntos
Linfócitos T CD8-Positivos/patologia , Epitopos/metabolismo , Glicoproteínas/metabolismo , Antígenos H-2/metabolismo , Infecções por Herpesviridae/fisiopatologia , Receptores de Antígenos de Linfócitos T/metabolismo , Rhadinovirus/metabolismo , Proteínas Virais/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/prevenção & controle , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Fenótipo , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/fisiopatologia , Infecções Tumorais por Vírus/prevenção & controle , Carga Viral/fisiologia
12.
FEBS Lett ; 584(8): 1509-14, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20206626

RESUMO

Tail-anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided-entry of tail-anchored proteins (Get) pathway. We present the 2A crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an alpha-solenoid fold with particular deviations from the regular pairwise arrangement of alpha-helices. A conserved hydrophobic groove accommodates the flexible C-terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein-protein interactions in the Get pathway.


Assuntos
Chaetomium/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína , Adaptação Fisiológica , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Proteínas Fúngicas/biossíntese , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica
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