Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Int J Mol Sci ; 25(12)2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38928469

RESUMO

The SPRY domain-containing SOCS box proteins SPSB1, SPSB2, and SPSB4 utilize their SPRY/B30.2 domain to interact with a short region in the N-terminus of inducible nitric oxide synthase (iNOS), and recruit an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in the proteasomal degradation of iNOS. Inhibitors that can disrupt the endogenous SPSB-iNOS interactions could be used to augment cellular NO production, and may have antimicrobial and anticancer activities. We previously reported the rational design of a cyclic peptide inhibitor, cR8, cyclo(RGDINNNV), which bound to SPSB2 with moderate affinity. We, therefore, sought to develop SPSB inhibitors with higher affinity. Here, we show that cyclic peptides cR7, cyclo(RGDINNN), and cR9, cyclo(RGDINNNVE), have ~6.5-fold and ~2-fold, respectively, higher SPSB2-bindng affinities than cR8. We determined high-resolution crystal structures of the SPSB2-cR7 and SPSB2-cR9 complexes, which enabled a good understanding of the structure-activity relationships for these cyclic peptide inhibitors. Moreover, we show that these cyclic peptides displace full-length iNOS from SPSB2, SPSB1, and SPSB4, and that their inhibitory potencies correlate well with their SPSB2-binding affinities. The strongest inhibition was observed for cR7 against all three iNOS-binding SPSB proteins.


Assuntos
Peptídeos Cíclicos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Humanos , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Relação Estrutura-Atividade
2.
Biochem Biophys Res Commun ; 670: 73-78, 2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37285720

RESUMO

The second step in the de novo sphingolipid biosynthesis is the reduction of 3-ketodihydrosphingosine by 3-ketodihydrosphingosine reductase (KDSR) to produce dihydrosphingosine (sphinganine). Fungal TSC10 and mammalian KDSR (also named FVT-1) proteins are the enzymes responsible for this process and they belong to the short-chain dehydrogenase/reductase (SDR) superfamily. Albeit that both fungal and mammalian 3-ketodihydrosphingosine reductases were identified more than a decade ago, no structure of these enzymes from any species has been experimentally determined. Here we report the crystal structure of the catalytic domain of TSC10 from Cryptococcus neoformans in complex with NADPH. cnTSC10 adopts a Rossmann fold with a central seven-stranded ß-sheet flanked by α-helices on both sides. Several regions are disordered that include the segment connecting the serine and tyrosine residues of the catalytic triad, the so-called 'substrate loop', and the C-terminal region that often participates in homo-tetramerization in other SDRs. In addition, the cofactor NADPH is not fully ordered. These structural features indicate that the catalytic site of cnTSC10 possesses significant flexibility. cnTSC10 is predominantly dimeric in solution while a minor portion of the protein forms homo-tetramer. The crystal structure reveals that the homo-dimer interface involves both hydrophobic and hydrophilic interactions mediated by helices α4 and α5, as well as the loop connecting strand ß4 and helix α4. Because residues forming hydrogen bonds and salt bridges in the dimer interface are not conserved between fungal TSC10 and mammalian KDSR proteins, it might be possible to develop inhibitors that selectively target fungal TSC10 dimerization.


Assuntos
Cryptococcus neoformans , Sequência de Aminoácidos , Sítios de Ligação , Cryptococcus neoformans/metabolismo , Cristalografia por Raios X , Modelos Moleculares , NADP/metabolismo , Oxirredutases/metabolismo
3.
Nitric Oxide ; 113-114: 1-6, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33862200

RESUMO

Relatively high concentration of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in response to a variety of stimuli is a source of reactive nitrogen species, an important weapon of host innate immune defense. The SPRY domain-containing SOCS box protein 2 (SPSB2) is an E3 ubiquitin ligase that regulates the lifetime of iNOS. SPSB2 interacts with the N-terminal region of iNOS via a binding site on the SPRY domain of SPSB2, and recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, leading to its proteasomal degradation. Although critical residues for the SPSB2-iNOS interaction have been identified, structural basis for the interaction remains to be explicitly determined. In this study, we have determined a crystal structure of the N-terminal region of iNOS in complex with the SPRY domain of SPSB2 at 1.24 Å resolution. We have resolved the roles of some flanking residues, whose contribution to the SPSB2-iNOS interaction was structurally unclear previously. Furthermore, we have evaluated the effects of SPSB2 inhibitors on NO production using transient transfection and cell-penetrating peptide approaches, and found that such inhibitors can elevate NO production in RAW264.7 macrophages. These results thus provide a useful basis for the development of potent SPSB2 inhibitors as well as recruiting ligands for proteolysis targeting chimera (PROTAC) design.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Domínio B30.2-SPRY/efeitos dos fármacos , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/química , Peptídeos/farmacologia , Células RAW 264.7 , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/química
4.
Biochem Biophys Res Commun ; 531(3): 350-356, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32800543

RESUMO

The SPRY/B30.2 domain is one of the most abundant protein domains found in eukaryotes. Vast majority of the SPRY domain-containing proteins are multi-domain proteins. The SPRY domain-containing protein 7 (SPRY7, also named C13orf1, and named chronic lymphocytic leukemia deletion region gene 6 protein, CCLD6, encoded by the spryd7 gene) is the smallest SPRY domain protein in human that does not contain other accessory domains. Here we have determined the crystal structure of human SPRY7 at a resolution of 1.62 Å and found that SPRY7 has some unique structural features that are not present in other previously reported SRPY domain structures. Overall, SPRY7 may represent an evolutionary early version of the SPRY domain, and subsequent loop insertions and expansions, residue substitutions, as well as domain combinations have rendered the SPRY domain versatile binding specificities and broad biological functions. These results serve as a useful basis for a profound characterization of the molecular interactions of SPRY7.


Assuntos
Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/química , Sequência de Aminoácidos , Domínio B30.2-SPRY , Humanos , Modelos Moleculares , Eletricidade Estática , Homologia Estrutural de Proteína
5.
Biochem Biophys Res Commun ; 489(3): 346-352, 2017 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-28549582

RESUMO

SPRY domain-containing SOCS box protein 2 (SPSB2) is a negative regulator of inducible nitric oxide synthase (iNOS) that modulates the lifetime of iNOS and thus the levels of nitric oxide (NO) production. Inhibitors that can disrupt the endogenous SPSB2-iNOS interaction and augment NO production have potential as novel antimicrobial and anticancer drugs. In this study, we have designed a cyclic peptide (cR8), containing an RGD motif and the SPSB2 binding motif (DINNNV). ITC and chemical shift perturbation showed that cR8 binds to the iNOS binding site on SPSB2 with a Kd of 671 nM, and saturation transfer difference NMR showed that cR8 binds to αvß3 integrin-expressing cells. Moreover, we determined the crystal structure of SPSB2 in complex with cR8, at a resolution of 1.34 Å. cR8 forms extensive hydrogen bonding with SPSB2 residues, but loss of an intramolecular hydrogen bond that is present in SPSB2-bound iNOS peptide may destabilize the bound conformation of cR8 and lead to a gentle reduction in SPSB2 binding affinity. These results serve as a useful basis for designing site-directed SPSB2 inhibitors in the future.


Assuntos
Desenho de Fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Cristalização , Cristalografia , Humanos , Modelos Moleculares , Conformação Molecular , Oligopeptídeos/química , Peptídeos Cíclicos/síntese química , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade
6.
J Immunol ; 187(7): 3798-805, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21876038

RESUMO

The mammalian innate immune system has evolved to recognize foreign molecules derived from pathogens via the TLRs. TLR3 and TLR4 can signal via the TIR domain-containing adapter inducing IFN-ß (TRIF), which results in the transcription of a small array of genes, including IFN-ß. Inducible NO synthase (iNOS), which catalyzes the production of NO, is induced by a range of stimuli, including cytokines and microbes. NO is a potent source of reactive nitrogen species that play an important role in killing intracellular pathogens and forms a crucial component of host defense. We have recently identified iNOS as a target of the mammalian SPSB2 protein. The SOCS box is a peptide motif, which, in conjunction with elongins B and C, recruits cullin-5 and Rbx-2 to form an active E3 ubiquitin ligase complex. In this study, we show that SPSB1 is the only SPSB family member to be regulated by the same TLR pathways that induce iNOS expression and characterize the interaction between SPSB1 and iNOS. Through the use of SPSB1 transgenic mouse macrophages and short hairpin RNA knockdown of SPSB1, we show that SPSB1 controls both the induction of iNOS and the subsequent production of NO downstream of TLR3 and TLR4. Further, we demonstrate that regulation of iNOS by SPSB1 is dependent on the proteasome. These results suggest that SPSB1 acts through a negative-feedback loop that, together with SPSB2, controls the extent of iNOS induction and NO production.


Assuntos
Regulação da Expressão Gênica/imunologia , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismo , Animais , Western Blotting , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Imunoprecipitação , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
7.
J Biol Chem ; 286(25): 22546-57, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21504902

RESUMO

Predatory marine cone snails (genus Conus) utilize complex venoms mainly composed of small peptide toxins that target voltage- and ligand-gated ion channels in their prey. Although the venoms of a number of cone snail species have been intensively profiled and functionally characterized, nothing is known about the initiation of venom expression at an early developmental stage. Here, we report on the expression of venom mRNA in embryos of Conus victoriae and the identification of novel α- and O-conotoxin sequences. Embryonic toxin mRNA expression is initiated well before differentiation of the venom gland, the organ of venom biosynthesis. Structural and functional studies revealed that the embryonic α-conotoxins exhibit the same basic three-dimensional structure as the most abundant adult toxin but significantly differ in their neurological targets. Based on these findings, we postulate that the venom repertoire of cone snails undergoes ontogenetic changes most likely reflecting differences in the biotic interactions of these animals with their prey, predators, or competitors. To our knowledge, this is the first study to show toxin mRNA transcripts in embryos, a finding that extends our understanding of the early onset of venom expression in animals and may suggest alternative functions of peptide toxins during development.


Assuntos
Conotoxinas/genética , Conotoxinas/metabolismo , Caramujo Conus/embriologia , Caramujo Conus/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Conotoxinas/química , Caramujo Conus/anatomia & histologia , Caramujo Conus/genética , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência
8.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 412-418, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204687

RESUMO

The SPRY domain-containing SOCS box protein 2 (SPSB2) is one of four mammalian SPSB proteins that are characterized by a C-terminal SOCS box and a central SPRY/B30.2 domain. SPSB2 interacts with inducible nitric oxide synthase (iNOS) via the SPRY domain and polyubiquitinates iNOS, resulting in its proteasomal degradation. Inhibitors that can disrupt SPSB2-iNOS interaction and augment NO production may serve as novel anti-infective and anticancer agents. The previously determined murine SPSB2 structure may not reflect the true apo conformation of the iNOS-binding site. Here, the crystal structure of human SPSB2 SPRY domain in the apo state is reported at a resolution of 1.9 Å. Comparison of the apo and ligand-bound structures reveals that the iNOS-binding site is highly preformed and that major conformational changes do not occur upon ligand binding. Moreover, the C-terminal His6 tag of the recombinant protein binds to a shallow pocket adjacent to the iNOS-binding site on a crystallographically related SPSB2 molecule. These findings may help in structure-based and fragment-based SPSB2 inhibitor design in the future.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Modelos Moleculares , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Sequência de Aminoácidos , Domínio B30.2-SPRY , Cristalografia por Raios X , Humanos , Óxido Nítrico Sintase Tipo II/química , Conformação Proteica
9.
Biomol NMR Assign ; 13(2): 299-304, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31065957

RESUMO

RING finger protein 135 (RNF135, also named Riplet or REUL) exerts multiple biological functions and its C-terminal PRY-SPRY/B30.2 domain is indispensable for most of these functions. RNF135 interacts with RIG-I (retinoic acid-inducible gene-I) via the PRY-SPRY domain and ubiquitinates RIG-I to promote innate anti-viral signaling, while mutations in the RNF135 gene can cause the Macrocephaly, macrosomia, facial dysmorphism (MMFD) syndrome, and RNF135 reportedly regulates the proliferation of glioblastoma cells as well as tongue cancer cells. Nevertheless, structure of full-length RNF135 or its PRY-SPRY domain has not been determined, and structural basis for molecular interactions involving RNF135 is largely unknown. Here we report the backbone 1H, 13C, and 15N chemical shift assignments of the PRY-SPRY domain of RNF135 and the secondary structure elements predicted based on chemical shifts, as well as the perturbations caused by the R286H mutation that is associated with MMFD syndrome. We found that the mutation did not alter the gross structure of the PRY-SPRY domain, so it may have impaired RNF135 function by affecting protein-protein interactions mediated by the domain.


Assuntos
Domínio B30.2-SPRY , Ressonância Magnética Nuclear Biomolecular , Ubiquitina-Proteína Ligases/química , Mutação , Ubiquitina-Proteína Ligases/genética
10.
J Mol Biol ; 364(4): 690-704, 2006 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-17020769

RESUMO

Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on 15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an alpha-helix, a three-stranded anti-parallel beta-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.


Assuntos
Heparina/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Sítios de Ligação , Glicosaminoglicanos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Espectroscopia de Ressonância Magnética , Movimento (Física) , Oligopeptídeos , Estrutura Terciária de Proteína , Soluções
11.
Enzyme Microb Technol ; 97: 82-89, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28010776

RESUMO

ß-Mannanase has been widely used in industries such as food and feed processing and thus has been a target enzyme for biotechnological development. In this study, we sought to improve the stability and protease resistance of a recombinant ß-mannanase, MAN47 from Armillariella tabescens, through rationally designed N-glycosylation. Based on homology modeling, molecular docking, secondary structure analysis and glycosylation feasibility analysis, an enhanced aromatic sequon sequence was introduced into specific MAN47 loop regions to facilitate N-glycosylation. The mutant enzymes were expressed in Pichia pastoris SMD1168, and their thermal stability, pH stability, trypsin resistance and pepsin resistance were determined. Two mutant MAN47 enzymes, g-123 and g-347, were glycosylated as expected when expressed in yeast, and their thermal stability, pH stability, and protease resistance were significantly improved compared to the wild-type enzyme. An enzyme with multiple stability characterizations has broad prospects in practical applications, and the rational design N-glycosylation strategy may have applications in simultaneously improving several properties of other biotechnological targets.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , beta-Manosidase/química , beta-Manosidase/metabolismo , Proteínas de Bactérias/genética , Biotecnologia , Domínio Catalítico , Clostridiales/genética , Estabilidade Enzimática , Glicosilação , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Manosidase/genética
12.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 21(1): 43-6, 2004 Feb.
Artigo em Zh | MEDLINE | ID: mdl-14767908

RESUMO

OBJECTIVE: To explore the mutation and amplification of RIT1 gene and their correlation with carcinogenesis of hepatocellular carcinoma (HCC). METHODS: The polymerase chain reactioindirect sequencing method was used for detecting the mutations in the sequence of all 6 exons in the RIT1 gene of 50 HCC tissues and paratumor tissues. And the amplification of RIT1 gene was examined by fluorescence quantitative polymerase chain reaction method. RESULTS: A nucleotide 241 G --> C substitution in exon 5 of RIT1 gene was detected in one patient's HCC tissue, but not in paratumor tissue; this 241 G --> C substitution leads to Glu81Gln amino acid alteration in the conservative domain binding GTP. A nucleotide G --> C substitution in 5'-UTR (-21 bp from initial codon) was detected in all of the 50 HCC tissues and paratumor tissues, and 2- to 297-fold amplification of RIT1 gene was detected in 11 of 43 qualified cases, the amplification frequency being 25.6%. CONCLUSION: Gene amplification is one of the main activating ways of RIT1 gene in HCC, and its amplification might be correlated with HCC carcinogenesis, while point mutation might be not.


Assuntos
Carcinoma Hepatocelular/genética , Amplificação de Genes , Neoplasias Hepáticas/genética , Mutação , Proteínas ras/genética , Adulto , Idoso , Sequência de Bases , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Humanos , Pessoa de Meia-Idade , Mutação Puntual
13.
ACS Chem Biol ; 8(6): 1344-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23557677

RESUMO

Among the µ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, µ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23, and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in µ-BuIIIB, which is also present in µ-BuIIIA and µ-BuIIIC but absent in other µ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related µ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of µ-BuIIIB for NaV1.3. One analogue, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. On the basis of previous results for related µ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated and suggest that further gains in potency might be achieved by additional modifications of this region.


Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Sequência de Aminoácidos , Animais , Dissulfetos/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.3/metabolismo , Xenopus
14.
J Mol Biol ; 401(3): 389-402, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20561531

RESUMO

The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4((59-77)) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.


Assuntos
RNA Helicases DEAD-box/química , Receptores de Trombina/química , Proteínas Supressoras da Sinalização de Citocina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Consenso , Cristalografia por Raios X , RNA Helicases DEAD-box/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Ligação Proteica , Conformação Proteica , Receptores de Trombina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo
15.
J Cell Biol ; 190(1): 129-41, 2010 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-20603330

RESUMO

Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain-containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB-iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Leishmania major , Leishmaniose Cutânea/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação Enzimológica da Expressão Gênica/genética , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniose Cutânea/genética , Macrófagos/parasitologia , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis , Óxido Nítrico Sintase Tipo II/genética , Complexo de Endopeptidases do Proteassoma/genética , Estrutura Terciária de Proteína , Proteínas Supressoras da Sinalização de Citocina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética
16.
Growth Horm IGF Res ; 19(3): 226-31, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19056307

RESUMO

OBJECTIVE: Insulin-like growth factor-I (IGF-I) plays important roles in normal growth and development, as well as in disease states, and its structure and function have been studied extensively using nuclear magnetic resonance (NMR) spectroscopy. However, IGF-I typically gives poor quality NMR spectra containing many broad peaks, because of aggregation at the protein concentrations generally required for NMR experiments as well as the internal dynamics of the molecule. The present study was undertaken to determine a reliable set of assignments under more physiological conditions. DESIGN: Several reports of chemical shift assignments have been published previously for IGF-I either bound to a ligand or at relatively low pH (approximately 3-4), but there are many contradictions among them, reflecting the poor behaviour of IGF-I. Low pH conditions are also suboptimal for the analysis of interactions between IGF-I and IGF binding proteins (IGFBP) or IGFBP fragments. Spectra were recorded at low concentrations in order to identify conditions of temperature and pH where all peaks could be observed. RESULTS: We show that good quality 2D (1)H-(15)N HSQC spectra of (15)N-labelled IGF-I can be obtained at pH 6 and 37 degrees C, much closer to physiological conditions, by using lower IGF-I concentrations (0.05 mM). Surprisingly, at this concentration and temperature, spectra were of better quality at pH 6 than at pH 4, in contrast to previous observations made at millimolar concentrations of IGF-I. We were then also able to assign the chemical shifts of IGF-I at pH 6 and 37 degrees C using 3D heteronuclear spectra recorded on a 0.7 mM (15)N/(13)C-labelled IGF-I sample. CONCLUSION: These results provide a valuable resource for future studies of the structure, dynamics, folding, and binding interactions of IGF-I, as well as analogues thereof, by means of NMR spectroscopy.


Assuntos
Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Ressonância Magnética Nuclear Biomolecular , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Ligação Proteica , Soluções , Temperatura
17.
J Mol Biol ; 386(3): 662-74, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19154741

RESUMO

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent beta-sandwich, consisting of two seven-stranded beta-sheets wrapped around a long loop that extends from the centre strands of the inner or concave beta-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.


Assuntos
Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Análise Mutacional de DNA , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores Ativados por Proteinase/metabolismo , Alinhamento de Sequência
18.
Biochemistry ; 46(48): 13720-32, 2007 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-17985932

RESUMO

A family of six insulin-like growth factor (IGF) binding proteins (IGFBP-1-6) binds IGF-I and IGF-II with high affinity and thus regulates their bioavailability and biological functions. IGFBPs consist of N- and C-terminal domains, which are highly conserved and cysteine-rich, joined by a variable linker domain. The role of the C-domain in IGF binding is not completely understood in that C-domain fragments have very low or even undetectable IGF binding affinity, but loss of the C-domain dramatically disrupts IGF binding by IGFBPs. We recently reported the solution structure and backbone dynamics of the C-domain of IGFBP-2 (C-BP-2) and identified a pH-dependent heparin binding site [Kuang, Z., Yao, S., Keizer, D. W., Wang, C. C., Bach, L. A., Forbes, B. E., Wallace, J. C., and Norton, R. S. (2006) Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2), J. Mol. Biol. 364, 690-704]. Here, we have analyzed the molecular interactions among the N-domain of IGFBP-2 (N-BP-2), C-BP-2, and IGFs using cross-linking and nuclear magnetic resonance (NMR) spectroscopy. The binding of C-BP-2 to the IGF-I.N-BP-2 binary complex was significantly stronger than the binding of C-BP-2 to IGF-I alone, switching from intermediate exchange to slow exchange on the NMR time scale. A conformational change or stabilization of the IGF-I Phe49-Leu54 region and the Phe49 aromatic ring upon binding to the N-domains, as well as an interdomain interaction between N-BP-2 and C-BP-2 (which is also detectable in the absence of ligand), may contribute to this cooperativity in IGF binding. Glycosaminoglycan binding by IGFBPs can affect their IGF binding although the effects appear to differ among different IGFBPs; here, we found that heparin bound to the IGF-I.N-BP-2.C-BP-2 ternary complex, but did not cause it to dissociate.


Assuntos
Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Fator de Crescimento Insulin-Like I/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica
19.
Ai Zheng ; 22(7): 695-9, 2003 Jul.
Artigo em Zh | MEDLINE | ID: mdl-12866958

RESUMO

BACKGROUND & OBJECTIVE: Previous study has demonstrated that high frequent gain of 1q was detected in hepatocellular carcinoma (HCC), 1q21-22 was identified as the minimum overlapping amplified region and might contain the candidate oncogenes involved in HCC. RIT1 gene is located in 1q21.3 region and is a member of Ras subfamily. RIT1 protein is similar to Ras protein in molecular structure and functions. It was speculated that RIT1 gene might be a candidate oncogene in HCC. So, the amplification of RIT1 gene was examined in HCC and was linked with the clinical indicators in this study to explore the possible functions of RIT1 gene in HCC development and progression. METHODS: The fluorescence quantitative polymerase chain reaction(FQ-PCR) method was established successfully. The number of RIT1 gene DNA copies was examined in the tumor tissues and its paratumor tissues from 43 patients with HCC by PE ABI 7000 Sequence Detector. The ratio of the number of RIT1 gene DNA copies between the tumor tissue and its paratumor tissue represented the extent of amplification of RIT1 gene DNA. RESULTS: RIT1 gene DNA was amplified in 11 cases (25.6%)among 43 patients. The mean survival time (15 months) of the RIT1 gene-amplification group is significantly shorter than that (34 months) of the non-amplification group (P = 0.0009); furthermore, the pathological grade and the extent of liver cirrhosis were significantly different between the RIT1 gene-amplification group and the non-amplification group (P< 0.01). CONCLUSION: The amplification of RIT1 gene might be one of the activation ways in HCC and might play an important role in HCC development and progression.


Assuntos
Carcinoma Hepatocelular/genética , Amplificação de Genes , Neoplasias Hepáticas/genética , Proteínas ras/genética , Adulto , Idoso , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Cromossomos Humanos Par 1 , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA