RESUMO
Hepatitis A is an acute infection caused by Hepatitis A virus (HAV), which is widely distributed throughout the world. The HAV 3C cysteine protease (3Cpro), an important nonstructural protein, is responsible for most cleavage within the viral polyprotein and is critical for the processes of viral replication. Our group has previously demonstrated that HAV 3Cpro cleaves human NF-κB essential modulator (NEMO), a kinase required in interferon signaling. Based on this finding, we generated four luciferase-based biosensors containing the NEMO sequence (PVLKAQ↓ADIYKA) that is cleaved by HAV 3Cpro and/or the Nostoc punctiforme DnaE intein, to monitor the activity of HAV 3Cpro in human embryonic kidney cells (HEK-293T). Western blotting showed that HAV 3Cpro recognized and cleaved the NEMO cleavage sequence incorporated in the four biosensors, whereas only one cyclized luciferase-based biosensor (233-DnaE-HAV, 233DH) showed a measurable and reliable increase in firefly luciferase activity, with very low background, in the presence of HAV 3Cpro. With this biosensor (233DH), we monitored HAV 3Cpro activity in HEK-293T cells, and tested it against a catalytically deficient mutant HAV 3Cpro and other virus-encoded proteases. The results showed that the activity of this luciferase biosensor is specifically dependent on HAV 3Cpro. Collectively, our data demonstrate that the luciferase biosensor developed here might provide a rapid, sensitive, and efficient evaluation of HAV 3Cpro activity, and should extend our better understanding of the biological relevance of HAV 3Cpro.
Assuntos
Técnicas Biossensoriais , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , Luciferases/metabolismo , Proteínas Virais/análise , Proteínas Virais/metabolismo , Proteases Virais 3C , Linhagem Celular Tumoral , Células HEK293 , HumanosRESUMO
BACKGROUND: 3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus. RESULTS: Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis. CONCLUSIONS: 3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.
Assuntos
Caspases/metabolismo , Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , Lisossomos/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/química , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Macrolídeos/farmacologia , Microscopia Eletrônica , Mitocôndrias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas Virais/genéticaRESUMO
NEMO (NF-κB essential modulator) is a bridging adaptor indispensable for viral activation of interferon (IFN) antiviral response. Herein, we show that hepatitis A virus (HAV) 3C protease (3Cpro) cleaves NEMO at the Q304 residue, negating its signaling adaptor function and abrogating viral induction of IFN-ß synthesis via the retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5) and Toll-like receptor 3 (TLR3) pathways. NEMO cleavage and IFN antagonism, however, were lost upon ablation of the catalytic activity of 3Cpro. These data describe a novel immune evasion mechanism of HAV.
Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , Interações Hospedeiro-Patógeno , Quinase I-kappa B/metabolismo , Evasão da Resposta Imune , Interferon beta/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteases Virais 3C , Humanos , Hidrólise , Transdução de SinaisRESUMO
Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3C(pro)). Inhibition assays in vitro identified the hexanucleotide 5'-GGGGGT-3' (G(5)T) as a 3C(pro) protease inhibitor. Using (1)H NMR spectroscopy, G(5)T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of (1)H, (15)N-HSQC experiments the binding site for G(5)T was located to the C-terminal ß-barrel of HAV 3C(pro). Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G(5)T-binding site, nor does G(5)T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid-protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance.
Assuntos
Cisteína Endopeptidases/química , Desoxirribonucleotídeos/química , Vírus da Hepatite A/enzimologia , Proteínas Virais/química , Proteases Virais 3C , Antivirais/química , Sítios de Ligação , Cisteína Endopeptidases/metabolismo , Desoxirribonucleotídeos/metabolismo , Dimerização , Quadruplex G , Ressonância Magnética Nuclear Biomolecular , RNA Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-ß (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro), that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-ß promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro) protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro) and downstream 3D(pol) sequence, but not 3D(pol) polymerase activity. Cleavage occurs at two non-canonical 3C(pro) recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol) sequence modulates the substrate specificity of the upstream 3C(pro) protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro). HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , RNA Viral/genética , Receptor 3 Toll-Like/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Proteínas Adaptadoras de Transporte Vesicular/genética , Linhagem Celular , Cisteína Endopeptidases/genética , Vírus da Hepatite A/isolamento & purificação , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Luciferases/metabolismo , Plasmídeos/genética , Transdução de Sinais , Especificidade por Substrato , Receptor 3 Toll-Like/genética , Transfecção/métodos , Proteínas Virais/genéticaRESUMO
The Picornaviridae family contains a large number of human pathogens such as rhinovirus, poliovirus and hepatitis A virus (HAV). Hepatitis A is an infectious disease that causes liver inflammation. It is highly endemic in developing countries with poor sanitation, where infections often occur in children. As in other picornaviruses, the genome of HAV contains one open reading frame encoding a single polyprotein that is subsequently processed by viral proteinases to originate mature viral proteins during and after the translation process. In the polyprotein, the N-terminal P1 region generates the four capsid proteins, while the C-terminal P2 and P3 regions contain the enzymes, precursors and accessory proteins essential for polyprotein processing and virus replication. Here, the first crystals of protein 2AB of HAV are reported. The crystals belonged to space group P4(1) or P4(3), with unit-cell parameters a = b = 90.42, c = 73.43 Å, and contained two molecules in the asymmetric unit. Native and selenomethionine-derivative crystals diffracted to 2.7 and 3.2 Å resolution, respectively.
Assuntos
Vírus da Hepatite A/enzimologia , Peptídeo Hidrolases/química , Clonagem Molecular , Cristalografia por Raios X , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/isolamento & purificaçãoRESUMO
It is widely accepted that Hepatitis A virus (HAV) is responsible for liver failure and even death in older people and in people with other serious health issues; so, proposing new compounds with inhibitory activity can help to treated of these disease's. In current study, a new class of quinolines is proposed with inhibitor activity of the HAV proteinase. So, in the first step, fused quinoline derivatives has been synthesized in short reaction time (12.0 min) and high efficiency yields (94%) in presence of 1-carboxymethyl-2,3-dimethylimidazolium iodide ([cmdmim]I) ionic liquid catalyst using a new method. In the following, chemical reactivity and inhibitory activity of synthesized quinolines were evaluated in density functional theory (DFT) framework and molecular docking methodologies. High global softness (0.67 eV), low HOMOSWBNNT-LUMO4a gap (4.78 eV), and more negative adsorption energy (- 87.9 kJ mol-1) in these quinolines reveal that the 4a and 4b compounds have better delivery than other quinolines using SWBNNT as suitable carrier to target cells. Molecular docking shows that the best cavity of the HAV has - 134.2 kJ mol-1 interaction energy involving bonding and non-bonding interactions. In fact, these interactions are between fused quinolines with especial geometries and sidechain flexibility amino acids residues inside the best binding site of the HAV, as hydrogen bonding, steric, and electrostatic interactions. So, these interactions imply that proposed fused quinolines have good inhibitor activity for the HAV.
Assuntos
Proteases Virais 3C/antagonistas & inibidores , Antivirais/farmacologia , Hepatite A/tratamento farmacológico , Quinolinas/farmacologia , Antivirais/química , Antivirais/uso terapêutico , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/enzimologia , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Quinolinas/química , Quinolinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3C(pro)). However, PABP is cleaved by HAV 3C(pro) in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5' nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Regulação Viral da Expressão Gênica , Vírus da Hepatite A/genética , Iniciação Traducional da Cadeia Peptídica , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Regiões 5' não Traduzidas/química , Regiões 5' não Traduzidas/metabolismo , Linhagem Celular Tumoral , Fator de Iniciação Eucariótico 4G/metabolismo , Vírus da Hepatite A/enzimologia , Humanos , Modelos Genéticos , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/fisiologia , RNA Viral/química , RNA Viral/metabolismo , Deleção de SequênciaRESUMO
Development of novel antivirals, which requires knowledge of the viral life cycle in molecular detail, is a daunting task, involving extensive investments, and frequently resulting in failure. As there exist significant commonalities among virus families in the manner of host interaction, identifying and targeting common rather than specific features may lead to the development of broadly useful antivirals. Here, we have targeted the 3C protease of Hepatitis A Virus (HAV), a feco-orally transmitted virus of the family Picornaviridae, for identification of potential antivirals. The 3C protease is a viable drug target as it is required by HAV, as well as by other picornaviruses, for post-translational proteolysis of viral polyproteins and for inhibiting host innate immune pathways. Computational screening, followed by chemical synthesis and experimental validation resulted in identification of a few compounds which, at low micromolar concentrations, could inhibit HAV 3C activity. These compounds were further tested experimentally against the 3C protease of Human Rhinovirus, another member of the Picornaviridae family, with comparable results. Computational studies on 3C proteases from other members of the picornavirus family have indicated that the compounds identified could potentially be generic inhibitors for picornavirus 3C proteases.
Assuntos
Antivirais/química , Antivirais/farmacologia , Desenvolvimento de Medicamentos , Vírus da Hepatite A/enzimologia , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Cisteína Endopeptidases , Humanos , Conformação Molecular , Simulação de Dinâmica MolecularRESUMO
The poly(rC)-binding protein PCBP2 has multiple functions in post-transcriptional control of host and viral gene expression. Since it interacts with picornaviral RNA structures, it was proposed that PCBP2 regulates viral genome translation and replication. The hepatitis A virus (HAV), an atypical picornavirus, contains an unusual pyrimidine-rich tract (pY1) with unknown functions. Using in vivo and in vitro assays, we provide direct evidence that PCBP2 interacts with pY1 and that binding is mediated by KH domains 1 and 3. Proteolytic cleavage by the viral protease 3C generates a C-terminally truncated polypeptide with highly reduced RNA affinity. The results suggest that during HAV infection PCBP2 cleavage might specifically down-regulate viral protein synthesis, thereby giving way to viral RNA synthesis.
Assuntos
Vírus da Hepatite A/química , Vírus da Hepatite A/enzimologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Sítios de Ligação , Ligação ProteicaRESUMO
We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , Cetonas/metabolismo , Inibidores de Proteases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteases Virais 3C , Sítios de Ligação , Cristalografia por Raios X , Vírus da Hepatite A/química , Hidrólise , Cetonas/química , Modelos Moleculares , Inibidores de Proteases/química , Conformação ProteicaRESUMO
During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.
Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Patos , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.
Assuntos
Cisteína Endopeptidases/química , Inibidores Enzimáticos/química , Vírus da Hepatite A/enzimologia , Lactonas/química , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Proteases Virais 3C , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Lactonas/farmacologia , Espectroscopia de Ressonância Magnética , Engenharia de Proteínas , Serina , Proteínas Virais/genéticaRESUMO
Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.
Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Hepatite A/enzimologia , RNA Viral/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Dimerização , Enzimas Imobilizadas , Vírus da Hepatite A/genética , Humanos , Hidrólise , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Modelos Moleculares , Mutação/genética , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/genéticaRESUMO
BACKGROUND: Polyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches. METHODOLOGY/PRINCIPAL FINDINGS: Hepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2', or P3', but not substrates containing glutamine at the P2 or P1' positions. To accommodate glutamine at P2 and P1', key residues comprising the active sites of the S2 or S1' pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3'. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent. CONCLUSIONS/SIGNIFICANCE: These data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.
Assuntos
Morte Celular/efeitos dos fármacos , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Neurônios/citologia , Peptídeos/metabolismo , Peptídeos/toxicidade , Engenharia de Proteínas , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteases Virais 3C , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Vírus da Hepatite A/enzimologia , Humanos , Neurônios/efeitos dos fármacos , Proteólise , Especificidade por Substrato , Proteínas Virais/químicaRESUMO
A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be purified and used for further characterization. To establish the general utility of the screen, it was used to demonstrate that the cysteine proteinase of the hepatitis A virus, 3C(pro), prefers Ile, Val, or Leu at the P(4) position of the cleavage sequence and Gly, Ser, or Ala at the P'(1) position. The assay can also be used to screen small-molecule libraries for inhibitors.
Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína Proteases/metabolismo , Ensaios de Triagem em Larga Escala , Proteínas Virais/metabolismo , Proteases Virais 3C , Sequência de Aminoácidos , Sequência de Bases , Cisteína Endopeptidases/genética , Cisteína Proteases/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Vírus da Hepatite A/enzimologia , Cinética , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas , Espectrometria de Fluorescência , Especificidade por Substrato , Transformação Bacteriana , Proteínas Virais/genéticaRESUMO
We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.
Assuntos
Cisteína Endopeptidases/metabolismo , Vírus da Encefalomiocardite/enzimologia , Vírus da Hepatite A/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Linhagem Celular , Núcleo Celular/química , Sobrevivência Celular , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Citoplasma/química , Fibroblastos/virologia , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Mitochondrial antiviral signaling protein (MAVS) is an essential component of virus-activated signaling pathways that induce protective IFN responses. Its localization to the outer mitochondrial membrane suggests an important yet unexplained role for mitochondria in innate immunity. Here, we show that hepatitis A virus (HAV), a hepatotropic picornavirus, ablates type 1 IFN responses by targeting the 3ABC precursor of its 3C(pro) cysteine protease to mitochondria where it colocalizes with and cleaves MAVS, thereby disrupting activation of IRF3 through the MDA5 pathway. The 3ABC cleavage of MAVS requires both the protease activity of 3C(pro) and a transmembrane domain in 3A that directs 3ABC to mitochondria. Lacking this domain, mature 3C(pro) protease is incapable of MAVS proteolysis. HAV thus disrupts host signaling by a mechanism that parallels that of the serine NS3/4A protease of hepatitis C virus, but differs in its use of a stable, catalytically active polyprotein processing intermediate. The unique requirement for mitochondrial localization of 3ABC underscores the importance of mitochondria to host control of virus infections within the liver.
Assuntos
Cisteína Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Vírus da Hepatite A/enzimologia , Imunidade Inata/imunologia , Mitocôndrias/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cisteína Endopeptidases/química , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo/genética , Genoma Viral , Vírus da Hepatite A/fisiologia , Humanos , Fatores Reguladores de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon , Macaca mulatta , Dados de Sequência Molecular , RNA Viral/genética , Transdução de Sinais , Proteínas não Estruturais Virais/química , Proteínas Virais/metabolismo , Ativação Viral , Replicação ViralRESUMO
Fungi and viruses encode a variety of peptidases having a plethora of functions. Many fungal peptidases are extracellular and are likely used to degrade proteins in their environment. Viral peptidases are processing enzymes, intimately involved in the virus infectious cycle. The viral RNA genome is translated by the host-cell machinery into a large polyprotein that is cleaved by the viral peptidases into mature capsid proteins, non-structural proteins and enzymes. I review the structure and catalytic mechanism of scytalidoglutamic peptidase isolated from the wood-destroying fungus Scytalidium lignicolum. This enzyme has a unique beta-sandwich fold and a novel catalytic mechanism based on a glutamate, a glutamine and a nucleophilic water molecule. Hepatitis A virus (HAV) 3C peptidase was the first structure identified for a viral 3C enzyme that exhibited the three-dimensional fold of the chymotrypsin family of serine peptidases but had a cysteine sulfur atom instead of the serine oxygen as the nucleophile. The structure of HAV 3C was unusual in that the Asp residue expected as the third member of the catalytic triad did not interact with the general base His. The present structure is of a beta-lactone-inhibited version of HAV 3C that has a restored catalytic triad.
Assuntos
Ascomicetos/enzimologia , Vírus da Hepatite A/enzimologia , Peptídeo Hidrolases/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Ativação Enzimática , Peptídeo Hidrolases/isolamento & purificação , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.