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1.
PLoS Pathog ; 19(11): e1011795, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38011215

RESUMO

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 µM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.


Assuntos
Infecção por Zika virus , Zika virus , Humanos , Zika virus/genética , Proteínas não Estruturais Virais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Peptídeos , RNA , Inibidores de Proteases
2.
Exp Cell Res ; 409(2): 112930, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34800542

RESUMO

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P2 and PI(3,4,5)P3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Apoptose , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Adesão Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Junções Intercelulares , Transdução de Sinais , Junções Íntimas , Células Tumorais Cultivadas
3.
Structure ; 29(9): 1029-1039.e3, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33878292

RESUMO

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.


Assuntos
Proteínas de Transporte/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Ligação Proteica
5.
Cancer Res ; 79(12): 3100-3111, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31040156

RESUMO

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain-containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS-driven cancer. SIGNIFICANCE: These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Domínios de Homologia à Plecstrina , Células Tumorais Cultivadas
6.
Cancer Lett ; 449: 145-162, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30771432

RESUMO

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.


Assuntos
Melanoma/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Espectrometria de Massas , Melanoma/genética , Camundongos , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 8: 16066, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28714476

RESUMO

Retinoid X receptor-alpha (RXRα) binds to DNA either as homodimers or heterodimers, but it also forms homotetramers whose function is poorly defined. We previously discovered that an N-terminally-cleaved form of RXRα (tRXRα), produced in tumour cells, activates phosphoinositide 3-kinase (PI3K) signalling by binding to the p85α subunit of PI3K and that K-80003, an anti-cancer agent, inhibits this process. Here, we report through crystallographic and biochemical studies that K-80003 binds to and stabilizes tRXRα tetramers via a 'three-pronged' combination of canonical and non-canonical mechanisms. K-80003 binding has no effect on tetramerization of RXRα, owing to the head-tail interaction that is absent in tRXRα. We also identify an LxxLL motif in p85α, which binds to the coactivator-binding groove on tRXRα and dissociates from tRXRα upon tRXRα tetramerization. These results identify conformational selection as the mechanism for inhibiting the nongenomic action of tRXRα and provide molecular insights into the development of RXRα cancer therapeutics.


Assuntos
Antineoplásicos/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptor X Retinoide alfa/antagonistas & inibidores , Sulindaco/análogos & derivados , Células A549 , Animais , Classe Ia de Fosfatidilinositol 3-Quinase , Cristalografia por Raios X , Células HEK293 , Células Hep G2 , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Receptor X Retinoide alfa/metabolismo , Transdução de Sinais , Sulindaco/farmacologia
8.
Cancer Res ; 76(14): 4259-4269, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27261507

RESUMO

The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.


Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias/tratamento farmacológico , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/fisiologia , Humanos , Camundongos , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Neurosci ; 34(45): 15123-31, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25378175

RESUMO

Emerging evidence suggests that oxidative/nitrosative stress, as occurs during aging, contributes to the pathogenesis of Parkinson's disease (PD). In contrast, detoxification of reactive oxygen species and reactive nitrogen species can protect neurons. DJ-1 has been identified as one of several recessively inherited genes whose mutation can cause familial PD, and inactivation of DJ-1 renders neurons more susceptible to oxidative stress and cell death. DJ-1 is also known to regulate the activity of the phosphatase and tensin homolog (PTEN), which plays a critical role in neuronal cell death in response to various insults. However, mechanistic details delineating how DJ-1 regulates PTEN activity remain unknown. Here, we report that PTEN phosphatase activity is inhibited via a transnitrosylation reaction [i.e., transfer of a nitric oxide (NO) group from the cysteine residue of one protein to another]. Specifically, we show that DJ-1 is S-nitrosylated (forming SNO-DJ-1); subsequently, the NO group is transferred from DJ-1 to PTEN by transnitrosylation. Moreover, we detect SNO-PTEN in human brains with sporadic PD. Using x-ray crystallography and site-directed mutagenesis, we find that Cys106 is the site of S-nitrosylation on DJ-1 and that mutation of this site inhibits transnitrosylation to PTEN. Importantly, S-nitrosylation of PTEN decreases its phosphatase activity, thus promoting cell survival. These findings provide mechanistic insight into the neuroprotective role of SNO-DJ-1 by elucidating how DJ-1 detoxifies NO via transnitrosylation to PTEN. Dysfunctional DJ-1, which lacks this transnitrosylation activity due to mutation or prior oxidation (e.g., sulfonation) of the critical cysteine thiol, could thus contribute to neurodegenerative disorders like PD.


Assuntos
Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Proteínas Oncogênicas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Motivos de Aminoácidos , Sequência de Aminoácidos , Estudos de Casos e Controles , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Dados de Sequência Molecular , Mutação , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteína Desglicase DJ-1
10.
Chem Biol ; 21(5): 596-607, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24704507

RESUMO

Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Receptor X Retinoide alfa/antagonistas & inibidores , Receptor X Retinoide alfa/química , Sulindaco/análogos & derivados , Sulindaco/farmacologia , Animais , Antineoplásicos/síntese química , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Receptor X Retinoide alfa/metabolismo , Relação Estrutura-Atividade , Sulindaco/química , Células Tumorais Cultivadas
11.
Chem Biol ; 20(8): 973-82, 2013 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-23891150

RESUMO

The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry, and functional data demonstrate that these peptide mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in the cell. The proposed strategy may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas Nucleares/antagonistas & inibidores , Peptídeos/química , Peptidomiméticos/química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
12.
Proc Natl Acad Sci U S A ; 110(19): 7808-13, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23603272

RESUMO

Host innate immune responses to DNA viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (NLRP) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (IL)-1ß and IL-18. We hypothesized that DNA viruses would target inflammasomes to overcome host defense. A Vaccinia virus (VACV) B-cell CLL/lymphoma 2 (Bcl-2) homolog, F1L, was demonstrated to bind and inhibit the NLR family member NLRP1 in vitro. Moreover, infection of macrophages in culture with virus lacking F1L (ΔF1L) caused increased caspase-1 activation and IL-1ß secretion compared with wild-type virus. Virulence of ΔF1L virus was attenuated in vivo, causing altered febrile responses, increased proteolytic processing of caspase-1, and more rapid inflammation in lungs of infected mice without affecting cell death or virus replication. Furthermore, we found that a hexapeptide from F1L is necessary and sufficient for inhibiting the NLRP1 inflammasome in vitro, thus identifying a peptidyl motif required for binding and inhibiting NLRP1. The functional importance of this NLRP1-binding motif was further confirmed by studies of recombinant ΔF1L viruses reconstituted either with the wild-type F1L or a F1L mutant that fails to bind NLRP1. Cellular infection with wild-type F1L reconstituted virus-suppressed IL-1ß production, whereas mutant F1L did not. In contrast, both wild-type and mutant versions of F1L equally suppressed apoptosis. In vivo, the NLR nonbinding F1L mutant virus exhibited an attenuated phenotype similar to ΔF1L virus, thus confirming the importance of F1L interactions with NLRP1 for viral pathogenicity in mice. Altogether, these findings reveal a unique viral mechanism for evading host innate immune responses.


Assuntos
Regulação Viral da Expressão Gênica , Imunidade Inata , Inflamassomos/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Caspases/metabolismo , Chlorocebus aethiops , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fenótipo , Proteínas Recombinantes/metabolismo , Células Vero , Virulência
13.
J Biol Chem ; 287(47): 39470-9, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23012361

RESUMO

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.


Assuntos
Antivirais/química , Sondas Moleculares/química , Peptídeo Hidrolases/química , Biblioteca de Peptídeos , Inibidores de Proteases/química , Vírus da Varíola/enzimologia , Proteínas Virais/antagonistas & inibidores , Motivos de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Desenho de Fármacos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Varíola/tratamento farmacológico , Varíola/enzimologia , Varíola/genética , Vírus da Varíola/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
J Med Chem ; 53(10): 3899-906, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20441222

RESUMO

The 14 kDa homodimeric N1L protein is a potent vaccinia and variola (smallpox) virulence factor. It is not essential for viral replication, but it causes a strong attenuation of viral production in culture when deleted. The N1L protein is predicted to contain the BH3-like binding domain characteristic of Bcl-2 family proteins, and it is able to bind the BH3 peptides. Its overexpression has been reported to prevent infected cells from committing apoptosis. Therefore, interfering with the N1L apoptotic blockade may be a legitimate therapeutic strategy affecting the viral growth. By using in silico ligand docking and an array of in vitro assays, we have identified submicromolar (600 nM) N1L antagonists belonging to the family of polyphenols. Their affinity is comparable to that of the BH3 peptides (70-1000 nM). We have also identified the natural polyphenol resveratrol as a moderate N1L inhibitor. Finally, we show that our ligands efficiently inhibit growth of vaccinia virus.


Assuntos
Antivirais/química , Fenóis/química , Proteínas Virais/antagonistas & inibidores , Fatores de Virulência/antagonistas & inibidores , Antivirais/síntese química , Antivirais/farmacologia , Proteínas Reguladoras de Apoptose/química , Proteína 11 Semelhante a Bcl-2 , Sítios de Ligação , Varredura Diferencial de Calorimetria , Linhagem Celular , Bases de Dados Factuais , Humanos , Ligantes , Proteínas de Membrana/química , Modelos Moleculares , Mutação , Fragmentos de Peptídeos/química , Fenóis/síntese química , Fenóis/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Resveratrol , Estilbenos/farmacologia , Relação Estrutura-Atividade , Termodinâmica , Ultracentrifugação , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/crescimento & desenvolvimento , Proteínas Virais/genética , Fatores de Virulência/genética
15.
Int J Biochem Cell Biol ; 42(6): 987-95, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20197107

RESUMO

Furin and related proprotein convertases cleave the multibasic motifs R-X-R/K/X-R in the precursor proteins and, as a result, transform the latent proproteins into biologically active proteins and peptides. Furin is present both in the intracellular secretory pathway and at the cell surface. Intracellular furin processes its multiple normal cellular targets in the Golgi and secretory vesicle compartments while cell-surface furin appears to be essential only for the processing of certain pathogenic proteins and, importantly, anthrax. To design potent, safe and selective inhibitors of furin, we evaluated the potency and selectivity of the derivatized peptidic inhibitors modeled from the extended furin cleavage sequence of avian influenza A H5N1. We determined that the N- and C-terminal modifications of the original RARRRKKRT inhibitory scaffold produced selective and potent, nanomolar range, inhibitors of furin. These inhibitors did not interfere with the normal cellular function of furin because of the likely functional redundancy existing between furin and other proprotein convertases. These furin inhibitors, however, were highly potent in blocking the furin-dependent cell-surface processing of anthrax protective antigen-83 both in vitro and cell-based assays and in vivo. We conclude that the inhibitors we have designed have a promising potential as selective anthrax inhibitors, without affecting major cell functions.


Assuntos
Antraz/metabolismo , Antígenos de Bactérias/metabolismo , Bacillus anthracis/fisiologia , Toxinas Bacterianas/metabolismo , Furina/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Antraz/prevenção & controle , Vacinas contra Antraz , Linhagem Celular Tumoral , Clonagem Molecular , Biologia Computacional , Drosophila , Humanos , Fragmentos de Peptídeos/síntese química , Especificidade por Substrato
16.
J Biol Chem ; 285(17): 13211-22, 2010 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-20167601

RESUMO

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.


Assuntos
Modelos Moleculares , Proteínas rac de Ligação ao GTP/química , Animais , Sítios de Ligação , Proteínas do Citoesqueleto , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Família Multigênica/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
17.
J Biol Chem ; 283(30): 20897-906, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18505722

RESUMO

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.


Assuntos
Furina/química , Pró-Proteína Convertases/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Biologia Computacional/métodos , Humanos , Espectrometria de Massas/métodos , Modelos Biológicos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Peptídeos/química , Pró-Proteína Convertases/fisiologia , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Especificidade por Substrato
18.
J Biol Chem ; 283(25): 17270-8, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18442976

RESUMO

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.


Assuntos
DNA/química , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/enzimologia , Sequência de Aminoácidos , Membrana Celular/virologia , Clonagem Molecular , Citoplasma/metabolismo , Cinética , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , RNA/química , RNA Helicases/química , Proteínas Recombinantes/química , Serina Endopeptidases/química
19.
J Biol Chem ; 282(29): 20847-53, 2007 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-17537721

RESUMO

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.


Assuntos
Furina/química , Pseudomonas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antraz/metabolismo , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Químicos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Espectrometria de Fluorescência/métodos
20.
Protein Sci ; 16(5): 795-806, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17400917

RESUMO

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an "induced fit" mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.


Assuntos
Evolução Molecular , Flaviviridae/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Cristalografia , Vírus da Dengue/enzimologia , Vírus da Dengue/genética , Flaviviridae/genética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Vírus do Nilo Ocidental/enzimologia , Vírus do Nilo Ocidental/genética
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