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1.
Nature ; 591(7850): 482-487, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33503651

RESUMO

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.


Assuntos
Anticorpos Antivirais/análise , Técnicas Biossensoriais/métodos , Vírus da Hepatite B/imunologia , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/análise , Troponina I/análise , Anticorpos Antivirais/imunologia , Técnicas Biossensoriais/normas , Toxinas Botulínicas/análise , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Limite de Detecção , Luminescência , Fosfoproteínas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Receptor ErbB-2/análise , Sensibilidade e Especificidade , Proteínas da Matriz Viral/imunologia
2.
Mol Cell ; 67(2): 334-347.e5, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28689660

RESUMO

Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase "head" and a "hinge" dimerization domain connected by a 49 nm coiled-coil "arm." The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Cisteína , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Relação Estrutura-Atividade
3.
Proc Natl Acad Sci U S A ; 119(23): e2122872119, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35653564

RESUMO

Adenosine diphosphate (ADP) ribosylation is a reversible posttranslational modification involved in the regulation of numerous cellular processes. Prototype ADP ribosyltransferases (ARTs) from many pathogenic bacteria are known to function as toxins, while other bacterial ARTs have just recently emerged. Recent studies have shown that bacteria also possess enzymes that function as poly-ADP ribose (ADPr) glycohydrolases (PARGs), which reverse poly-ADP ribosylation. However, how bacteria manipulate host target proteins by coordinated reactions of ARTs and ADPr hydrolases (ARHs) remains elusive. The intracellular bacterial pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, transports a large array of effector proteins via the Dot/Icm type IV secretion system to host cells. The effector proteins, which mostly function as enzymes, modulate host cellular processes for the bacteria's benefit. In this study, we identified a pair of L. pneumophila effector proteins, Lpg0080 and Lpg0081, which function as an ART and an ARH, respectively. The two proteins were shown to coordinately modulate mitochondrial ADP/adenosine triphosphate (ATP) translocases (ANTs) by their enzymatic activities to conjugate ADPr to, and remove it from, a key arginine residue. The crystal structures of Lpg0081 and the Lpg0081:ADPr complex indicated that Lpg0081 is a macroD-type ARH with a noncanonical macrodomain, whose folding topology is strikingly distinct from that of the canonical macrodomain that is ubiquitously found in eukaryotic PARGs and ARHs. Our results illustrate that L. pneumophila has acquired an effector pair that coordinately manipulate mitochondrial activity via reversible chemical modification of ANTs.


Assuntos
Legionella pneumophila , Legionella , Difosfato de Adenosina , Trifosfato de Adenosina , Proteínas de Bactérias , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP
4.
Cell ; 136(1): 85-96, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-19135891

RESUMO

Condensins are key mediators of chromosome condensation across organisms. Like other condensins, the bacterial MukBEF condensin complex consists of an SMC family protein dimer containing two ATPase head domains, MukB, and two interacting subunits, MukE and MukF. We report complete structural views of the intersubunit interactions of this condensin along with ensuing studies that reveal a role for the ATPase activity of MukB. MukE and MukF together form an elongated dimeric frame, and MukF's C-terminal winged-helix domains (C-WHDs) bind MukB heads to constitute closed ring-like structures. Surprisingly, one of the two bound C-WHDs is forced to detach upon ATP-mediated engagement of MukB heads. This detachment reaction depends on the linker segment preceding the C-WHD, and mutations on the linker restrict cell growth. Thus ATP-dependent transient disruption of the MukB-MukF interaction, which creates openings in condensin ring structures, is likely to be a critical feature of the functional mechanism of condensins.


Assuntos
Adenosina Trifosfatases/química , Bactérias/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Estrutura Terciária de Proteína
5.
Mol Cell ; 62(4): 507-19, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27203177

RESUMO

UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.


Assuntos
Autofagia/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Melanoma Experimental/enzimologia , Neoplasias Cutâneas/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/efeitos da radiação , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patologia , Proteólise , Interferência de RNA , Retina/enzimologia , Retina/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
6.
Mol Cell ; 57(2): 290-303, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25557547

RESUMO

SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive "hinge" dimerization domain with an ATP-regulated "head" dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB's association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc's dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.


Assuntos
Proteínas de Bactérias/ultraestrutura , Proteínas de Ciclo Celular/ultraestrutura , DNA Bacteriano/química , Pyrococcus furiosus , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína
7.
Biochem Biophys Res Commun ; 586: 49-54, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34826700

RESUMO

Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Glucose/química , Glicosídeo Hidrolases/química , Polissacarídeos/química , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Modelos Moleculares , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
8.
Nat Methods ; 16(11): 1095-1100, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611691

RESUMO

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and ß2-adrenergic receptor (ß2AR) optobodies suppressed endogenous gelsolin activity and ß2AR signaling, respectively.


Assuntos
Anticorpos/fisiologia , Gelsolina/fisiologia , Optogenética , Receptores Adrenérgicos beta 2/fisiologia , Animais , Células Cultivadas , Humanos
9.
PLoS Biol ; 17(7): e3000367, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31323018

RESUMO

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.


Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Neoplasias do Colo do Útero/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência de Aminoácidos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
10.
Proteins ; 89(4): 468-472, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33236809

RESUMO

We report the crystal structure of PYCH_01220, a hypothetical protein in Pyrococcus yayanosii CH1. This protein is composed of two domains, named Domain A and Domain B. While Domain B is not significantly homologous to known protein structures, Domain A is structurally analogous to the C-terminal ribonuclease domain of Escherichia coli colicin D. Domain A has a positively charged surface patch rendered by 13 basic residues, eight arginine or lysine residues of which are evolutionarily conserved. Electrophoretic mobility shift assays showed that PYCH_01220 binds to DNA, and charge-inversion mutations on this patch negatively affect the DNA binding, suggesting that the function of PYCH_01220 might involve nucleic acid-binding via the positively charged patch.


Assuntos
Proteínas Arqueais , DNA , Pyrococcus/química , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
11.
Nat Immunol ; 10(9): 949-57, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19668222

RESUMO

All metazoan guts are in permanent contact with the microbial realm. However, understanding of the exact mechanisms by which the strength of gut immune responses is regulated to achieve gut-microbe mutualism is far from complete. Here we identify a signaling network composed of complex positive and negative mechanisms that controlled the expression and activity of dual oxidase (DUOX), which 'fine tuned' the production of microbicidal reactive oxygen species depending on whether the gut encountered infectious or commensal microbes. Genetic analyses demonstrated that negative and positive regulation of DUOX was required for normal host survival in response to colonization with commensal and infectious microbes, respectively. Thus, the coordinated regulation of DUOX enables the host to achieve gut-microbe homeostasis by efficiently combating infection while tolerating commensal microbes.


Assuntos
Drosophila/imunologia , NADPH Oxidases/fisiologia , Fator 2 Ativador da Transcrição/fisiologia , Animais , Células CACO-2 , Calcineurina/fisiologia , Proteínas de Transporte/fisiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Intestinos/imunologia , Intestinos/microbiologia , MAP Quinase Quinase 3/fisiologia , MAP Quinase Quinase Quinase 1/fisiologia , NADPH Oxidases/genética , Fosfolipase C beta/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
12.
Nat Chem Biol ; 15(6): 607-614, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101917

RESUMO

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.


Assuntos
Dano ao DNA , DNA Bacteriano/metabolismo , Mycobacterium smegmatis/enzimologia , Uracila-DNA Glicosidase/metabolismo , Uracila/metabolismo , Sítios de Ligação , Biocatálise , Modelos Moleculares , Conformação Proteica , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/genética
13.
J Biol Chem ; 293(47): 18110-18122, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30282804

RESUMO

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site-coordinating core ß-sheet-containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.


Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sepse/enzimologia , Sepse/microbiologia , Vibrio vulnificus/enzimologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas , Endopeptidases/química , Endopeptidases/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Domínios Proteicos , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Sepse/genética , Vibrio vulnificus/química , Vibrio vulnificus/genética , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/genética
14.
EMBO Rep ; 18(5): 826-840, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28364023

RESUMO

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1121334151, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.


Assuntos
Sistemas CRISPR-Cas , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/metabolismo , RNA/metabolismo , Proteínas Arqueais/metabolismo , Thermococcus/genética
15.
Nucleic Acids Res ; 43(7): 3841-56, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25740648

RESUMO

In meiotic DNA recombination, the Hop2-Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2-Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2-Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasion.


Assuntos
Proteínas Cromossômicas não Histona/química , Meiose/fisiologia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Cromossômicas não Histona/fisiologia , Cristalografia por Raios X , Primers do DNA , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos
16.
J Virol ; 89(10): 5308-17, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25740994

RESUMO

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298-5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE: The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.


Assuntos
Herpesvirus Humano 8/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas Virais/fisiologia , Replicação Viral/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose , Autofagia , Sequência de Bases , Linhagem Celular , Replicação do DNA , DNA Viral/genética , Expressão Gênica , Técnicas de Inativação de Genes , Genoma Viral , Células HEK293 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Replicação Viral/genética
17.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 2): 535-43, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24531487

RESUMO

A multi-subunit ribonucleoprotein complex termed the Cmr RNA-silencing complex recognizes and destroys viral RNA in the CRISPR-mediated immune defence mechanism in many prokaryotes using an as yet unclear mechanism. In Archaeoglobus fulgidus, this complex consists of six subunits, Cmr1-Cmr6. Here, the crystal structure of Cmr1 from A. fulgidus is reported, revealing that the protein is composed of two tightly associated ferredoxin-like domains. The domain located at the N-terminus is structurally most similar to the N-terminal ferredoxin-like domain of the CRISPR RNA-processing enzyme Cas6 from Pyrococcus furiosus. An ensuing mutational analysis identified a highly conserved basic surface patch that binds single-stranded nucleic acids specifically, including the mature CRISPR RNA, but in a sequence-independent manner. In addition, this subunit was found to cleave single-stranded RNA. Together, these studies elucidate the structure and the catalytic activity of the Cmr1 subunit.


Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Subunidades Proteicas/química , RNA Viral/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Archaeoglobus fulgidus/imunologia , Archaeoglobus fulgidus/virologia , Sítios de Ligação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Interações Hospedeiro-Patógeno , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Pyrococcus furiosus/química , Pyrococcus furiosus/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato
18.
EMBO J ; 29(2): 496-504, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-19942850

RESUMO

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lipossomos/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico
19.
J Virol ; 87(22): 12499-503, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24027317

RESUMO

Autophagy is an important innate safeguard mechanism for protecting an organism against invasion by pathogens. We have previously discovered that Kaposi's sarcoma-associated herpesvirus (KSHV) evades this host defense through tight suppression of autophagy by targeting multiple steps of autophagy signal transduction. Here, we report that KSHV K7 protein interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step by blocking Vps34 enzymatic activity, further highlighting how KSHV deregulates autophagy-mediated host immunity for its life cycle.


Assuntos
Autofagia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Fagossomos/metabolismo , Sarcoma de Kaposi/patologia , Proteínas Virais/metabolismo , Proteínas Relacionadas à Autofagia , Células HeLa , Humanos , Immunoblotting , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Transdução de Sinais
20.
PLoS Pathog ; 8(12): e1003082, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23271971

RESUMO

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Legionella pneumophila/metabolismo , Legionelose/metabolismo , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Endossomos/genética , Endossomos/microbiologia , Endossomos/patologia , Células HeLa , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Legionelose/genética , Legionelose/patologia , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/microbiologia , Lisossomos/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/genética
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