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1.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , Proteínas de Ligação a RNA/ultraestrutura , RNA Polimerase Dependente de RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
2.
Nature ; 572(7769): 382-386, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31330532

RESUMO

The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease1-3. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown4-6. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages4,5. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca2+ concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.


Assuntos
Proteínas de Bactérias/metabolismo , Calmodulina/metabolismo , Ácido Glutâmico/metabolismo , Legionella pneumophila/enzimologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina/metabolismo , Fatores de Virulência/metabolismo , ADP-Ribosilação , Apoproteínas/metabolismo , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Calmodulina/farmacologia , Catálise , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HEK293 , Humanos , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Virulência/agonistas , Fatores de Virulência/química
3.
Nat Commun ; 10(1): 2370, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147549

RESUMO

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Forma das Organelas/genética , Autofagia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico/genética
4.
Nature ; 557(7707): 734-738, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29795347

RESUMO

Conventional ubiquitination regulates key cellular processes by catalysing the ATP-dependent formation of an isopeptide bond between ubiquitin (Ub) and primary amines in substrate proteins 1 . Recently, the SidE family of bacterial effector proteins (SdeA, SdeB, SdeC and SidE) from pathogenic Legionella pneumophila were shown to use NAD+ to mediate phosphoribosyl-linked ubiquitination of serine residues in host proteins2, 3. However, the molecular architecture of the catalytic platform that enables this complex multistep process remains unknown. Here we describe the structure of the catalytic core of SdeA, comprising mono-ADP-ribosyltransferase (mART) and phosphodiesterase (PDE) domains, and shed light on the activity of two distinct catalytic sites for serine ubiquitination. The mART catalytic site is composed of an α-helical lobe (AHL) that, together with the mART core, creates a chamber for NAD+ binding and ADP-ribosylation of ubiquitin. The catalytic site in the PDE domain cleaves ADP-ribosylated ubiquitin to phosphoribosyl ubiquitin (PR-Ub) and mediates a two-step PR-Ub transfer reaction: first to a catalytic histidine 277 (forming a transient SdeA H277-PR-Ub intermediate) and subsequently to a serine residue in host proteins. Structural analysis revealed a substrate binding cleft in the PDE domain, juxtaposed with the catalytic site, that is essential for positioning serines for ubiquitination. Using degenerate substrate peptides and newly identified ubiquitination sites in RTN4B, we show that disordered polypeptides with hydrophobic residues surrounding the target serine residues are preferred substrates for SdeA ubiquitination. Infection studies with L. pneumophila expressing substrate-binding mutants of SdeA revealed that substrate ubiquitination, rather than modification of the cellular ubiquitin pool, determines the pathophysiological effect of SdeA during acute bacterial infection.


Assuntos
Biocatálise , Legionella pneumophila/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Serina/metabolismo , Ubiquitinação , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Proteínas de Membrana/genética , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Estrutura Secundária de Proteína , Especificidade por Substrato , Ubiquitina/metabolismo
5.
Nat Commun ; 8: 14004, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28084320

RESUMO

The hallmark of Salmonella Typhimurium infection is an acute intestinal inflammatory response, which is mediated through the action of secreted bacterial effector proteins. The pro-inflammatory Salmonella effector SopA is a HECT-like E3 ligase, which was previously proposed to activate host RING ligases TRIM56 and TRIM65. Here we elucidate an inhibitory mechanism of TRIM56 and TRIM65 targeting by SopA. We present the crystal structure of SopA in complex with the RING domain of human TRIM56, revealing the atomic details of their interaction and the basis for SopA selectivity towards TRIM56 and TRIM65. Structure-guided biochemical analysis shows that SopA inhibits TRIM56 E3 ligase activity by occluding the E2-interacting surface of TRIM56. We further demonstrate that SopA ubiquitinates TRIM56 and TRIM65, resulting in their proteasomal degradation during infection. Our results provide the basis for how a bacterial HECT ligase blocks host RING ligases and exemplifies the multivalent power of bacterial effectors during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Salmonella/enzimologia , Salmonella typhimurium/enzimologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno , Humanos , Proteólise , Infecções por Salmonella/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/química , Salmonella typhimurium/genética , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
6.
Cell ; 167(6): 1636-1649.e13, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27912065

RESUMO

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.


Assuntos
Legionella pneumophila/fisiologia , Doença dos Legionários/microbiologia , Ubiquitinação , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias , Enzimas Reparadoras do DNA , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Doença dos Legionários/metabolismo , Proteínas de Membrana/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirofosfatases/metabolismo , Saccharomyces cerevisiae , Serina/metabolismo , Ubiquitina/metabolismo
7.
Elife ; 52016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27852435

RESUMO

Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)- a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.


Assuntos
Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Proteínas de Saccharomyces cerevisiae/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mamíferos , Mutação
8.
Proc Natl Acad Sci U S A ; 110(43): 17296-301, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24101465

RESUMO

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na(+) symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na(+)-dependent. The transport activity of R262 mutants increased by 30-40% in the presence of a membrane potential, indicating substrate/Na(+) cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1' helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na(+)-coupled substrate symporters. We propose that a similar mechanism is operative in other Na(+)/H(+)-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.


Assuntos
Antiporters/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Proteus mirabilis/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Antiporters/química , Antiporters/genética , Arginina/química , Arginina/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Transporte Biológico/genética , Carnitina/química , Carnitina/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteus mirabilis/química , Proteus mirabilis/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
9.
J Biol Chem ; 284(14): 9115-23, 2009 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-19201753

RESUMO

Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3'-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg(2+) due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.


Assuntos
Sequência Conservada , Ligases/química , Ligases/metabolismo , Motivos de Aminoácidos , Biocatálise , Difosfatos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Ligases/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Nucleotídeos/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por Substrato
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