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1.
Traffic ; 25(1): e12927, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38272446

RESUMO

Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.


Assuntos
Dobramento de Proteína , Doenças Raras , Animais , Humanos , Doenças Raras/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Retículo Endoplasmático/metabolismo , Mutação , Mamíferos/metabolismo , Glucosiltransferases/metabolismo
2.
J Biol Chem ; 300(2): 105651, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38237679

RESUMO

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.


Assuntos
Proteínas 14-3-3 , Proteínas Proto-Oncogênicas c-mdm2 , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo
3.
Proteins ; 91(12): 1571-1599, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37493353

RESUMO

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.


Assuntos
Biologia Computacional , Proteínas , Conformação Proteica , Modelos Moleculares , Biologia Computacional/métodos , Proteínas/química
4.
Molecules ; 26(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063908

RESUMO

The experimental electron density distribution (EDD) of 1-methyluracil (1-MUR) was obtained by single crystal X-ray diffraction (XRD) experiments at 23 K. Four different structural models fitting an extensive set of XRD data to a resolution of (sinθ/λ)max = 1.143 Å-1 are compared. Two of the models include anharmonic temperature factors, whose inclusion is supported by the Hamilton test at a 99.95% level of confidence. Positive Fourier residuals up to 0.5 eÅ-3 in magnitude were found close to the methyl group and in the region of hydrogen bonds. Residual density analysis (RDA) and molecular dynamics simulations in the solid-state demonstrate that these residuals can be likely attributed to unresolved disorder, possibly dynamical and long-range in nature. Atomic volumes and charges, molecular moments up to hexadecapoles, as well as maps of the molecular electrostatic potential were obtained from distributed multipole analysis of the EDD. The derived electrostatic properties neither depend on the details of the multipole model, nor are significantly affected by the explicit inclusion of anharmonicity in the least-squares model. The distribution of atomic charges in 1-MUR is not affected by the crystal environment in a significant way. The quality of experimental findings is discussed in light of in-crystal and gas-phase quantum simulations.

5.
Nat Immunol ; 9(7): 753-60, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18536718

RESUMO

To provide insight into the structural and functional properties of human complement component 5 (C5), we determined its crystal structure at a resolution of 3.1 A. The core of C5 adopted a structure resembling that of C3, with the domain arrangement at the position corresponding to the C3 thioester being very well conserved. However, in contrast to C3, the convertase cleavage site in C5 was ordered and the C345C domain flexibly attached to the core of C5. Binding of the tick C5 inhibitor OmCI to C5 resulted in stabilization of the global conformation of C5 but did not block the convertase cleavage site. The structure of C5 may render possible a structure-based approach for the design of new selective complement inhibitors.


Assuntos
Complemento C5/química , Complemento C5/metabolismo , Proteínas de Insetos/metabolismo , Estrutura Quaternária de Proteína , Animais , Proteínas de Artrópodes , Sítios de Ligação , Proteínas de Transporte , Complemento C3 , Cristalografia por Raios X , Humanos , Proteínas de Insetos/química , Ressonância de Plasmônio de Superfície
6.
Proc Natl Acad Sci U S A ; 114(32): 8544-8549, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28739903

RESUMO

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two ß-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first ß-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.


Assuntos
Glucosiltransferases/química , Glucosiltransferases/fisiologia , Animais , Chaetomium/genética , Chaetomium/metabolismo , Cristalografia por Raios X/métodos , Retículo Endoplasmático/metabolismo , Eucariotos/metabolismo , Células Eucarióticas/metabolismo , Glucosiltransferases/metabolismo , Glicoproteínas/metabolismo , Conformação Molecular , Domínios Proteicos/fisiologia , Dobramento de Proteína , Transporte Proteico/fisiologia , Especificidade por Substrato
7.
Proc Natl Acad Sci U S A ; 113(32): E4630-8, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27462106

RESUMO

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined small-angle X-ray scattering (SAXS) and crystal structures of the main ERQC enzyme, ER α-glucosidase II (α-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the +1 and +2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of d-mannose at the +1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the α-GluII +1 and +2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.


Assuntos
Antivirais/farmacologia , Retículo Endoplasmático/enzimologia , Inibidores de Glicosídeo Hidrolases/farmacologia , alfa-Glucosidases/química , Animais , Catálise , Cristalografia por Raios X , Camundongos , Conformação Proteica , Subunidades Proteicas , Espalhamento a Baixo Ângulo , Especificidade por Substrato
9.
Biochim Biophys Acta Gen Subj ; 1862(9): 1948-1955, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29908816

RESUMO

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.


Assuntos
Hidroximetilbilano Sintase/química , Hidroximetilbilano Sintase/metabolismo , Pirróis/química , Pirróis/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Polimerização , Porfobilinogênio/química , Porfobilinogênio/metabolismo , Conformação Proteica , Uroporfirinogênios/química , Uroporfirinogênios/metabolismo
10.
Nature ; 492(7428): 210-4, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23201679

RESUMO

The twin-arginine translocation (Tat) pathway is one of two general protein transport systems found in the prokaryotic cytoplasmic membrane and is conserved in the thylakoid membrane of plant chloroplasts. The defining, and highly unusual, property of the Tat pathway is that it transports folded proteins, a task that must be achieved without allowing appreciable ion leakage across the membrane. The integral membrane TatC protein is the central component of the Tat pathway. TatC captures substrate proteins by binding their signal peptides. TatC then recruits TatA family proteins to form the active translocation complex. Here we report the crystal structure of TatC from the hyperthermophilic bacterium Aquifex aeolicus. This structure provides a molecular description of the core of the Tat translocation system and a framework for understanding the unique Tat transport mechanism.


Assuntos
Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Sítios de Ligação , Escherichia coli/genética , Bactérias Gram-Negativas/genética , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
11.
Adv Exp Med Biol ; 1062: 265-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29845539

RESUMO

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and ß-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.


Assuntos
Retículo Endoplasmático/enzimologia , Viroses/enzimologia , Viroses/imunologia , Fenômenos Fisiológicos Virais , alfa-Glucosidases/química , alfa-Glucosidases/imunologia , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/virologia , Interações Hospedeiro-Patógeno , Humanos , Viroses/genética , Viroses/virologia , Vírus/genética , alfa-Glucosidases/genética
12.
Int J Mol Sci ; 19(7)2018 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-30041423

RESUMO

Small molecule modulators of the Endoplasmic Reticulum glycoprotein folding quality control (ERQC) machinery have broad-spectrum antiviral activity against a number of enveloped viruses and have the potential to rescue secretion of misfolded but active glycoproteins in rare diseases. In vivo assays of candidate inhibitors in mammals are expensive and cannot be afforded at the preliminary stages of drug development programs. The strong conservation of the ERQC machinery across eukaryotes makes transgenic plants an attractive system for low-cost, easy and fast proof-of-concept screening of candidate ERQC inhibitors. The Arabidopsis thaliana immune response is mediated by glycoproteins, the folding of which is controlled by ERQC. We have used the plant response to bacterial peptides as a means of assaying an ERQC inhibitor in vivo. We show that the treatment of the plant with the iminosugar NB-DNJ, which is a known ER α-glucosidase inhibitor in mammals, influences the immune response of the plant to the bacterial peptide elf18 but not to the flagellin-derived flg22 peptide. In the NB-DNJ-treated plant, the responses to elf18 and flg22 treatments closely follow the ones observed for the ER α-glucosidase II impaired plant, At psl5-1. We propose Arabidopsis thaliana as a promising platform for the development of low-cost proof-of-concept in vivo ERQC modulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Inibidores de Glicosídeo Hidrolases/química , Proteínas Recombinantes/metabolismo , alfa-Glucosidases/metabolismo
13.
Mol Microbiol ; 92(1): 153-63, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24673795

RESUMO

It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Ferroquelatase/química , Heme/análogos & derivados , Paracoccus pantotrophus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/biossíntese , Histidina/genética , Modelos Moleculares , Mutação , Paracoccus pantotrophus/genética , Estrutura Terciária de Proteína
14.
Nature ; 458(7240): 890-3, 2009 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-19225461

RESUMO

The complement system is an essential component of the innate and acquired immune system, and consists of a series of proteolytic cascades that are initiated by the presence of microorganisms. In health, activation of complement is precisely controlled through membrane-bound and soluble plasma-regulatory proteins including complement factor H (fH; ref. 2), a 155 kDa protein composed of 20 domains (termed complement control protein repeats). Many pathogens have evolved the ability to avoid immune-killing by recruiting host complement regulators and several pathogens have adapted to avoid complement-mediated killing by sequestering fH to their surface. Here we present the structure of a complement regulator in complex with its pathogen surface-protein ligand. This reveals how the important human pathogen Neisseria meningitidis subverts immune responses by mimicking the host, using protein instead of charged-carbohydrate chemistry to recruit the host complement regulator, fH. The structure also indicates the molecular basis of the host-specificity of the interaction between fH and the meningococcus, and informs attempts to develop novel therapeutics and vaccines.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboidratos/química , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Mimetismo Molecular , Neisseria meningitidis/metabolismo , Sítios de Ligação , Fator H do Complemento/imunologia , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Neisseria meningitidis/química , Neisseria meningitidis/imunologia , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
15.
J Biol Chem ; 288(26): 18789-802, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23625922

RESUMO

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB4 may be useful for treatment of human immune complex-dependent diseases.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Complexo Antígeno-Anticorpo/farmacologia , Proteínas de Artrópodes/farmacologia , Proteínas de Transporte/farmacologia , Lipocalinas/farmacologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/terapia , Animais , Proteínas de Artrópodes/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia Gasosa , Complemento C5/metabolismo , Eicosanoides/metabolismo , Ácidos Graxos/metabolismo , Técnicas Imunoenzimáticas , Leucotrieno B4/metabolismo , Lipocalinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/metabolismo , Ovinos , Ressonância de Plasmônio de Superfície , Trombina/metabolismo
16.
Eur J Clin Invest ; 44(1): 93-102, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24164255

RESUMO

BACKGROUND: Numerous epidemiologic studies have linked the presence of chronic obstructive pulmonary disease (COPD) to coronary artery disease (CAD). However, prevalence, pathological processes, clinical manifestations and therapy are still debated, as progress towards uncovering the link between these two disorders has been hindered by the complex nature of multimorbidity. METHODS: Articles targeting CAD in patients with COPD were identified from the searches of MEDLINE and EMBASE databases in July 2013. Three authors reviewed available evidence, focusing on the latest development on disease prevalence, pathogenesis, clinical manifestations and therapeutic strategies. Both clinical trial and previous reviews have been included in this work. RESULTS: The most accredited hypothesis asserts that the main common risk factors, that is, cigarette smoke and ageing, elicit a chronic low-grade systemic inflammatory response, which affects both cardiovascular endothelial cells and airways/lung parenchyma. The development of CAD in patients with COPD potentiates the morbidity of COPD, leading to increased hospitalizations, mortality and health costs. Moreover, correct diagnosis is challenging and therapies are not clearly defined. CONCLUSIONS: Evidence from recently published articles highlights the importance of multimorbidity in patient management and future research. Moreover, many authors emphasize the importance of low-grade systemic inflammation as a common pathological mechanism and a possible future therapeutic target.


Assuntos
Doença da Artéria Coronariana/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Fatores Etários , Doença da Artéria Coronariana/imunologia , Progressão da Doença , Humanos , Inflamação , Doença Pulmonar Obstrutiva Crônica/imunologia , Fatores de Risco , Fumar
17.
Proc Natl Acad Sci U S A ; 108(36): 14879-84, 2011 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-21873236

RESUMO

Initiation of the innate immune response requires agonist recognition by pathogen-recognition receptors such as the Toll-like receptors (TLRs). Toll/interleukin-1 receptor (TIR) domain-containing adaptors are critical in orchestrating the signal transduction pathways after TLR and interleukin-1 receptor activation. Myeloid differentiation primary response gene 88 (MyD88) adaptor-like (MAL)/TIR domain-containing adaptor protein (TIRAP) is involved in bridging MyD88 to TLR2 and TLR4 in response to bacterial infection. Genetic studies have associated a number of unique single-nucleotide polymorphisms in MAL with protection against invasive microbial infection, but a molecular understanding has been hampered by a lack of structural information. The present study describes the crystal structure of MAL TIR domain. Significant structural differences exist in the overall fold of MAL compared with other TIR domain structures: A sequence motif comprising a ß-strand in other TIR domains instead corresponds to a long loop, placing the functionally important "BB loop" proline motif in a unique surface position in MAL. The structure suggests possible dimerization and MyD88-interacting interfaces, and we confirm the key interface residues by coimmunoprecipitation using site-directed mutants. Jointly, our results provide a molecular and structural basis for the role of MAL in TLR signaling and disease protection.


Assuntos
Imunidade Inata/fisiologia , Glicoproteínas de Membrana/química , Receptores de Interleucina-1/química , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Humanos , Infecções , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Multimerização Proteica/imunologia , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Relação Estrutura-Atividade , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
18.
Proc Natl Acad Sci U S A ; 108(31): 12839-44, 2011 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-21768352

RESUMO

The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors.


Assuntos
Fator I do Complemento/química , Fator I do Complemento/genética , Polimorfismo Genético , Estrutura Terciária de Proteína , Regulação Alostérica , Sítios de Ligação/genética , Domínio Catalítico , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4b/química , Complemento C4b/metabolismo , Fator I do Complemento/metabolismo , Cristalização , Cristalografia por Raios X , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Predisposição Genética para Doença/genética , Glicosilação , Humanos , Modelos Moleculares , Mutação , Ligação Proteica
19.
J Biol Chem ; 287(39): 32913-21, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22854970

RESUMO

The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Šlong. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9.


Assuntos
Lectina de Ligação a Manose da Via do Complemento/fisiologia , Glicoproteínas , Lectinas , Lectina de Ligação a Manose , Serina Proteases Associadas a Proteína de Ligação a Manose , Multimerização Proteica/fisiologia , Processamento Alternativo/fisiologia , Animais , Células CHO , Complemento C3/química , Complemento C3/metabolismo , Complemento C9/química , Complemento C9/metabolismo , Cricetinae , Cricetulus , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Lectinas/química , Lectinas/metabolismo , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/química , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína
20.
J Exp Med ; 204(10): 2277-83, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17893204

RESUMO

Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.


Assuntos
Envelhecimento/fisiologia , Fator H do Complemento/química , Fator H do Complemento/metabolismo , Sítios de Ligação , Fator H do Complemento/genética , Cristalografia por Raios X , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Ligantes , Modelos Moleculares , Mutação/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sacarose/análogos & derivados , Sacarose/química , Sacarose/metabolismo , Propriedades de Superfície
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