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1.
Methods Mol Biol ; 1278: 391-405, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25859965

RESUMO

In the cell, homo- and hetero-associations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide for the selection of cell membrane permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.


Assuntos
Núcleosídeo-Difosfato Quinase/química , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Sequência de Aminoácidos , Permeabilidade da Membrana Celular , Humanos , Peptídeos/química , Proteínas/química
2.
Biochem Pharmacol ; 82(9): 1163-74, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21803029

RESUMO

Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Modelos Moleculares , NF-kappa B/antagonistas & inibidores , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
3.
Blood ; 118(4): 926-35, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21622647

RESUMO

Nuclear factor-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase complex, is a critical component of the NF-κB pathway. Hypomorphic mutations in the X-linked human NEMO gene cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). All known X-linked EDA-ID-causing mutations impair NEMO protein expression, folding, or both. We describe here 2 EDA-ID-causing missense mutations that affect the same residue in the CC2-LZ domain (D311N and D311G) that do not impair NEMO production or folding. Structural studies based on pull-down experiments showed a defect in noncovalent interaction with K63-linked and linear polyubiquitin chains for these mutant proteins. Functional studies on the patients' cells showed an impairment of the classic NF-κB signaling pathways after activation of 2 NEMO ubiquitin-binding-dependent receptors, the TNF and IL-1ß receptors, and in the CD40-dependent NF-κB pathway. We report the first human NEMO mutations responsible for X-linked EDA-ID found to affect the polyubiquitin binding of NEMO rather than its expression and folding. These experiments demonstrate that the binding of human NEMO to polyubiquitin is essential for NF-κB activation. They also demonstrate that the normal expression and folding of NEMO do not exclude a pathogenic role for NEMO mutations in patients with EDA-ID.


Assuntos
Displasia Ectodérmica Anidrótica Tipo 1/genética , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/genética , Ubiquitina/metabolismo , Western Blotting , Displasia Ectodérmica Anidrótica Tipo 1/metabolismo , Ativação Enzimática/genética , Feminino , Humanos , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/metabolismo , Masculino , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Linhagem , Ligação Proteica , Dobramento de Proteína , Transdução de Sinais/genética , Adulto Jovem
4.
J Mol Biol ; 395(1): 89-104, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-19854204

RESUMO

NEMO is an integral part of the IkappaB kinase complex and serves as a molecular switch by which the NF-kappaB signaling pathway can be regulated. Oligomerization and polyubiquitin (poly-Ub) binding, mediated through the regulatory CC2-LZ domain, were shown to be key features governing NEMO function, but the relationship between these two activities remains unclear. In this study, we solved the structure of this domain in complex with a designed ankyrin repeat protein, which helps its crystallization. We generated several NEMO mutants in this domain, including those associated with human diseases incontinentia pigmenti and immunodeficiency with or without anhidrotic ectodermal dysplasia. Analytical ultracentrifugation and thermal denaturation experiments were used to evaluate the dimerization properties of these mutants. A fluorescence-based assay was developed, as well, to quantify the interaction to monoubiquitin and poly-Ub chains. Moreover, the effect of these mutations was investigated for the full-length protein. We show that a proper folding of the ubiquitin-binding domain, termed NOA/UBAN/NUB, into a stable coiled-coil dimer is required but not sufficient for efficient interaction with poly-Ub. In addition, we show that binding to poly-Ub and, to a lesser extent, to monoubiquitin increases the stability of the NOA coiled-coil dimer. Collectively, these data provide structural insights into how several pathological mutations within and outside of the CC2-LZ's NOA ubiquitin binding site affect IkappaB kinase activation in the NF-kappaB signaling pathway.


Assuntos
Repetição de Anquirina , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Multimerização Proteica , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , NF-kappa B/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/farmacologia
5.
J Biol Chem ; 284(5): 2902-2907, 2009 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-19033441

RESUMO

NEMO (NF-kappaB essential modulator) is a regulatory protein essential to the canonical NF-kappaB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase eta ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-kappaB signaling in response to tumor necrosis factor-alpha. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type.


Assuntos
Ubiquitina/metabolismo , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , Western Blotting , Linhagem Celular , Teste de Complementação Genética , Humanos , Imunoprecipitação , Células Jurkat , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Homologia de Sequência de Aminoácidos , Ubiquitina/química
6.
J Mol Biol ; 377(5): 1419-32, 2008 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-18313693

RESUMO

The regulatory NEMO (NF-kappaB essential modulator) protein has a crucial role in the canonical NF-kappaB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global betabetaalpha fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1beta and TNF-alpha-induced NF-kappaB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-kappaB activation.


Assuntos
Sítios de Ligação , Quinase I-kappa B/química , Quinase I-kappa B/genética , Mutação Puntual , Dedos de Zinco , Dicroísmo Circular , Displasia Ectodérmica Hipo-Hidrótica Autossômica Recessiva/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/genética , Células Jurkat , Espectroscopia de Ressonância Magnética , Modelos Moleculares , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Zinco/metabolismo
7.
Protein Sci ; 16(9): 2013-22, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17766391

RESUMO

The link between the NF-kappaB signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the IkappaB kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) for NEMO, and we developed NEMO peptides inhibiting NF-kappaB activation at the level of the IKK complex. To improve the low-affinity inhibitors, we used ribosome display to select small and stable proteins with high affinity against the individual CC2-LZ because the entire NEMO protein is poorly soluble. Several binders with affinities in the low nanomolar range were obtained. When expressed in human cells, some of the selected molecules, despite their partial degradation, inhibited TNF-alpha-mediated NF-kappaB activation while having no effect on the basal activity. Controls with a naive library member or null plasmid had no effect. Furthermore, we could show that this NF-kappaB inhibition occurs through a specific interaction between the binders and the endogenous NEMO, resulting in decreased IKK activation. These results indicate that in vitro selections with the NEMO subdomain alone as a target may be sufficient to lead to interesting compounds that are able to inhibit NF-kappaB activation.


Assuntos
Repetição de Anquirina , Quinase I-kappa B/química , NF-kappa B/antagonistas & inibidores , NF-kappa B/química , Ubiquitina/metabolismo , Linhagem Celular , DNA Complementar , Escherichia coli/genética , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Luciferases/análise , Luciferases/metabolismo , Modelos Moleculares , NF-kappa B/genética , NF-kappa B/metabolismo , Plasmídeos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , beta-Galactosidase/análise , beta-Galactosidase/genética
8.
FEBS J ; 274(10): 2540-51, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17419723

RESUMO

NF-kappaB essential modulator (NEMO) plays an essential role in the nuclear factor kappaB (NF-kappaB) pathway as a modulator of the two other subunits of the IkappaB kinase (IKK) complex, i.e. the protein kinases, IKKalpha and IKKbeta. Previous reports all envision the IKK complex to be a static entity. Using glycerol-gradient ultracentrifugation, we observed stimulus-dependent dynamic IKK complex assembly. In wild-type fibroblasts, the kinases and a portion of cellular NEMO associate in a 350-kDa high-molecular-mass complex. In response to constitutive NF-kappaB stimulation by Tax, we observed NEMO recruitment and oligomerization to a shifted high-molecular-mass complex of 440 kDa which displayed increased IKK activity. This stimulus-dependent oligomerization of NEMO was also observed using fluorescence resonance energy transfer after a transient pulse with interleukin-1beta. In addition, fully activated, dimeric kinases not bound to NEMO were detected in these Tax-activated fibroblasts. By glycerol gradient ultracentrifugation, we also showed that: (a) in fibroblasts deficient in IKKalpha and IKKbeta, NEMO predominantly exists as a monomer; (b) in NEMO-deficient fibroblasts, IKKbeta dimers are present that are less stable than IKKalpha dimers. Intriguingly, in resting Rat-1 fibroblasts, 160-kDa IKKalpha-NEMO and IKKbeta-NEMO heterocomplexes were observed as well as a significant proportion of NEMO monomer. These results suggest that most NEMO molecules do not form a tripartite IKK complex with an IKKalpha-IKKbeta heterodimer as previously reported in the literature but, instead, NEMO is able to form a complex with the monomeric forms of IKKalpha and IKKbeta.


Assuntos
Quinase I-kappa B/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Centrifugação com Gradiente de Concentração , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Interleucina-1beta/farmacologia , Camundongos , NF-kappa B/fisiologia , Estrutura Quaternária de Proteína , Ratos , Proteínas Recombinantes/metabolismo
9.
J Exp Med ; 203(7): 1745-59, 2006 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-16818673

RESUMO

Germline mutations in five autosomal genes involved in interleukin (IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)-MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-kappaB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell-dependent IL-12 production, resulting in defective IFN-gamma secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-kappaB/c-Rel-mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-kappaB activators, such as tumor necrosis factor-alpha, IL-1beta, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell- and CD40L-triggered, CD40-, and NEMO/NF-kappaB/c-Rel-mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans.


Assuntos
Antígenos CD40/fisiologia , Genes Ligados ao Cromossomo X , Predisposição Genética para Doença , Quinase I-kappa B/genética , Interleucina-12/biossíntese , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Cromossomo X , Adolescente , Adulto , Animais , Linhagem Celular Transformada , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Células L , Masculino , Camundongos , Linhagem
10.
J Biol Chem ; 281(10): 6334-48, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16379012

RESUMO

The NEMO (NF-kappaB essential modulator) protein plays a crucial role in the canonical NF-kappaB pathway as the regulatory component of the IKK (IkappaB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-alpha and LPS-induced NF-kappaB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.


Assuntos
Displasia Ectodérmica/genética , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/genética , Lipopolissacarídeos/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Mutação Puntual , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Dicroísmo Circular , Displasia Ectodérmica/patologia , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/deficiência , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/patologia , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Temperatura , Tirosina/genética , Dedos de Zinco/genética
11.
J Biol Chem ; 281(5): 2711-20, 2006 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-16291743

RESUMO

Recombination is a major source of genetic heterogeneity in the human immunodeficiency virus type 1 (HIV-1) population. The main mechanism responsible for the generation of recombinant viruses is a process of copy choice between the two copies of genomic RNA during reverse transcription. We previously identified, after a single cycle of infection of cells in culture, a recombination hot spot within the gp120 gene, corresponding to the top portion of a RNA hairpin. Here, we determine that the hot region is circumscribed to 18 nucleotides located in the descending strand of the stem, following the sense of reverse transcription. Three factors appeared to be important, albeit at different extents, for the high rate of recombination observed in this region. The position of the hot sequence in the context of the RNA structure appears crucial, because changing its location within this structure triggered differences in recombination up to 20-fold. Another pivotal factor is the presence of a perfectly identical sequence between donor and acceptor RNA in the region of transfer, because single or double nucleotide differences in the hot spot were sufficient to almost completely abolish recombination in the region. Last, the primary structure of the hot region also influenced recombination, although with effects only in the 2-3-fold range. Altogether, these results provide the first molecular dissection of a hot spot in infected cells and indicate that several factors contribute to the generation of a site of preferential copy choice.


Assuntos
HIV-1/genética , Recombinação Genética , Linhagem Celular Tumoral , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV , Humanos , Cinética , Conformação de Ácido Nucleico , RNA/química , Transcrição Reversa
12.
J Biol Chem ; 279(52): 54248-57, 2004 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-15466857

RESUMO

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.


Assuntos
NF-kappa B/antagonistas & inibidores , NF-kappa B/química , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Morte Celular/efeitos dos fármacos , Linhagem Celular , Fenômenos Químicos , Físico-Química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Desenho de Fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte , Citometria de Fluxo , Humanos , Quinase I-kappa B , Interleucina-2/genética , Células Jurkat , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Mutagênese , NF-kappa B/metabolismo , Peptídeos/química , Peptídeos/genética , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão , Retinoblastoma , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/farmacologia , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/genética
13.
Biochem Pharmacol ; 68(9): 1749-56, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15450940

RESUMO

Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine L-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both D- and L-configuration. Human PGK demonstrated catalytic efficiencies in the 10(4)-10(5)M(-1)s(-1) range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in D- or L-configuration. In contrast, it was poorly active with natural pyrimidine D-nucleotides (less than 10(3)M(-1)s(-1)). Pyrimidine L-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their D-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, L-2'-deoxycytosine and L-2'-deoxythymidine.


Assuntos
Antivirais/farmacologia , Fosfoglicerato Quinase/metabolismo , Aciclovir/farmacologia , Humanos , Núcleosídeo-Difosfato Quinase/metabolismo , Fosfoglicerato Quinase/efeitos dos fármacos , Nucleotídeos de Purina/química , Nucleotídeos de Purina/metabolismo , Nucleotídeos de Purina/farmacologia , Nucleotídeos de Pirimidina/química , Nucleotídeos de Pirimidina/metabolismo , Nucleotídeos de Pirimidina/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Estavudina/farmacologia , Especificidade por Substrato , Zidovudina/farmacologia
14.
Biochem Pharmacol ; 68(9): 1879-88, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15450953

RESUMO

Amdoxovir [(-)-beta-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5'-nucleotidase (5'-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5'-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 microM DXG for 24 h, 0.015 pmole/10(6) cells (approximately 7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5'-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.


Assuntos
Dioxolanos/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Fosfotransferases/metabolismo , Nucleosídeos de Purina/metabolismo , Antivirais , Humanos , Fosforilação , Células Tumorais Cultivadas
15.
FEBS Lett ; 572(1-3): 276-80, 2004 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-15304362

RESUMO

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/química , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Epitopos/farmacologia , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
J Biol Chem ; 279(35): 36625-32, 2004 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-15218022

RESUMO

By frequently rearranging large regions of the genome, genetic recombination is a major determinant in the plasticity of the human immunodeficiency virus type I (HIV-1) population. In retroviruses, recombination mostly occurs by template switching during reverse transcription. The generation of retroviral vectors provides a means to study this process after a single cycle of infection of cells in culture. Using HIV-1-derived vectors, we present here the first characterization and estimate of the strength of a recombination hot spot in HIV-1 in vivo. In the hot spot region, located within the C2 portion of the gp120 envelope gene, the rate of recombination is up to ten times higher than in the surrounding regions. The hot region corresponds to a previously identified RNA hairpin structure. Although recombination breakpoints in vivo cluster in the top portion of the hairpin, the bias for template switching in this same region appears less marked in a cell-free system. By modulating the stability of this hairpin we were able to affect the local recombination rate both in vitro and in infected cells, indicating that the local folding of the genomic RNA is a major parameter in the recombination process. This characterization of reverse transcription products generated after a single cycle of infection provides insights in the understanding of the mechanism of recombination in vivo and suggests that specific regions of the genome might be prompted to yield different rates of evolution due to the presence of circumscribed recombination hot spots.


Assuntos
Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , RNA Viral , Recombinação Genética , Sequência de Bases , Linhagem Celular , Sistema Livre de Células , Clonagem Molecular , Vetores Genéticos , Heterozigoto , Homozigoto , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/metabolismo , Retroviridae/genética , Termodinâmica , Transfecção
17.
J Biol Chem ; 279(27): 27861-9, 2004 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-15107419

RESUMO

NEMO (NF-kappaB essential modulator) plays a key role in the canonical NF-kappaB pathway as the scaffold/regulatory component of the IkappaB kinase (IKK) complex. The self-association of NEMO involves the C-terminal halves of the polypeptide chains containing two putative coiled-coil motifs (a CC2 and a LZ leucine zipper), a proline-rich region, and a ZF zinc finger motif. Using purified truncation mutants, we showed that the minimal oligomerization domain of NEMO is the CC2-LZ segment and that both CC2 and LZ subdomains are necessary to restore the LPS-dependent activation of the NF-kappaB pathway in a NEMO-deficient cell line. We confirmed the association of the oligomerization domain in a trimer and investigated the specific role of CC2 and LZ subdomains in the building of the oligomer. Whereas a recombinant CC2-LZ polypeptide self-associated into a trimer with an association constant close to that of the wild-type protein, the isolated CC2 and LZ peptides, respectively, formed trimers and dimers with weaker association constants. Upon mixing, isolated CC2 and LZ peptides associated to form a stable hetero-hexamer as shown by gel filtration and fluorescence anisotropy experiments. We propose a structural model for the organization of the oligomerization domain of activated NEMO in which three C-terminal domains associate into a pseudo-hexamer forming a six-helix bundle. This model is discussed in relation to the mechanism of activation of the IKK complex by upstream activators.


Assuntos
Proteínas Serina-Treonina Quinases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anisotropia , Linhagem Celular , Cromatografia , Cromatografia em Gel , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Teste de Complementação Genética , Vetores Genéticos , Quinase I-kappa B , Cinética , Lipopolissacarídeos/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Peptídeos/química , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Transfecção , Dedos de Zinco
18.
Methods Mol Biol ; 261: 427-42, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15064474

RESUMO

In the cell, homo- and heteroassociations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide to the selection of the cell-membrane-permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.


Assuntos
Reagentes de Ligações Cruzadas/química , Proteínas/química , Células HeLa , Humanos , Modelos Moleculares , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/genética
19.
J Mol Biol ; 332(4): 915-26, 2003 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-12972261

RESUMO

NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.


Assuntos
Difosfato de Adenosina/metabolismo , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleotídeos/metabolismo , Proteínas Quinases/química , Difosfato de Adenosina/química , Animais , Cristalografia por Raios X , Corantes Fluorescentes/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Ligação Proteica , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína
20.
Eur J Biochem ; 270(8): 1784-90, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12694191

RESUMO

UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.


Assuntos
Núcleosídeo-Fosfato Quinase/metabolismo , Sequência de Bases , Clonagem Molecular , Monofosfato de Citidina/metabolismo , Primers do DNA , Humanos , Cinética , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Uridina Monofosfato/metabolismo
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