RESUMEN
Despite therapeutic advancements, cervical cancer caused by high-risk subtypes of the human papillomavirus (HPV) remains a leading cause of cancer-related deaths among women worldwide. This study aimed to discover potential drug candidates from the Asian medicinal plant Andrographis paniculata, demonstrating efficacy against the E6 protein of high-risk HPV-16 subtype through an in-silico computational approach. The 3D structures of 32 compounds (selected from 42) derived from A. paniculata, exhibiting higher binding affinity, were obtained from the PubChem database. These structures underwent subsequent analysis and screening based on criteria including binding energy, molecular docking, drug likeness and toxicity prediction using computational techniques. Considering the spectrometry, pharmacokinetic properties, docking results, drug likeliness, and toxicological effects, five compounds-stigmasterol, 1H-Indole-3-carboxylic acid, 5-methoxy-, methyl ester (AP7), andrographolide, apigenin and wogonin-were selected as the potential inhibitors against the E6 protein of HPV-16. We also performed 200 ns molecular dynamics simulations of the compounds to analyze their stability and interactions as protein-ligand complexes using imiquimod (CID-57469) as a control. Screened compounds showed favorable characteristics, including stable root mean square deviation values, minimal root mean square fluctuations and consistent radius of gyration values. Intermolecular interactions, such as hydrogen bonds and hydrophobic contacts, were sustained throughout the simulations. The compounds displayed potential affinity, as indicated by negative binding free energy values. Overall, findings of this study suggest that the selected compounds have the potential to act as inhibitors against the E6 protein of HPV-16, offering promising prospects for the treatment and management of CC.
Asunto(s)
Andrographis , Papillomavirus Humano 16 , Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales , Neoplasias del Cuello Uterino , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/virología , Humanos , Femenino , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/química , Andrographis/química , Papillomavirus Humano 16/efectos de los fármacos , Proteínas Represoras/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Fitoquímicos/farmacología , Fitoquímicos/química , Simulación por Computador , Extractos Vegetales/química , Extractos Vegetales/farmacología , Simulación de Dinámica Molecular , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/virología , Diterpenos/farmacología , Diterpenos/química , Unión ProteicaRESUMEN
Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.
Asunto(s)
Péptidos , Péptidos/química , Proteínas Oncogénicas Virales/química , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Espectrometría de Masas en Tándem/métodos , Unión ProteicaRESUMEN
E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.
Asunto(s)
Multimerización de Proteína , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Humanos , Ubiquitina/metabolismo , Ubiquitina/química , Ubiquitinación , Modelos Moleculares , Cristalografía por Rayos X , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Unión Proteica , Conformación Proteica en Hélice alfaRESUMEN
The activity of proteins is thought to be invariably determined by their amino acid sequence or composition, but we show that a long segment of a viral protein can support infection independent of its sequence or composition. During virus entry, the papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm to bind cellular factors such as retromer required for intracellular virus trafficking. Here, we show that an ~110 amino acid segment of L2 is predicted to be disordered and that large deletions in this segment abolish infectivity of HPV16 pseudoviruses by inhibiting cytoplasmic protrusion of L2, association with retromer, and proper virus trafficking. The activity of these mutants can be restored by insertion of protein segments with diverse sequences, compositions, and chemical properties, including scrambled amino acid sequences, a tandem array of a short sequence, and the intrinsically disordered region of an unrelated cellular protein. The infectivity of mutants with small in-frame deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or composition, determines its activity during HPV16 pseudovirus infection. We propose that a minimal length of L2 is required for it to protrude far enough into the cytoplasm to bind cytoplasmic trafficking factors, but the sequence of this segment is largely irrelevant. Thus, protein segments can carry out complex biological functions such as Human papillomavirus pseudovirus infection in a sequence-independent manner. This finding has important implications for protein function and evolution.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Proteínas de la Cápside/química , Virus del Papiloma Humano , Internalización del Virus , Células HeLa , Cápside/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/químicaRESUMEN
The Human papillomaviruses type 16 E7 oncoprotein is a 98-amino-acid, 11-kilodalton acidic oncoprotein with three conserved portions. Due to its interaction with the pRb-E2F complex, CKII, CKI (mostly p21), and even HDAC1, it possesses strong transformative and carcinogenic qualities that inhibit normal differentiation and cell cycle regulation. Here, we target the E7 oncoprotein using two prior research active compounds: asarinin and thiazolo[3,2-a]benzimidazole-3(2H)-one,2-(2-fluorobenzylideno)-7,8-dimethyl (thiazolo), and valproic acid as a control. We are performing molecular docking followed by molecular dynamic analysis. By acting as competitive inhibitors in the binding site, it was hypothesized that both drugs would inhibit E7-mediated pRb degradation and E7-mediated p21 degradation, resulting in decreased cell cycle progression, immortalization, and proliferation. In addition, we expect that the direct inhibitory action of valproic acid in E7 will target the CKII-mediated phosphorylation pathway necessary for destabilizing p130 and pRb. According to the results of the dynamic simulation, stable interactions exist between every compound. Despite the instability of E7 protein, stability results indicate that both natural chemicals are preferable, with thiazolo outperforming valproic acid.
Asunto(s)
Proteínas Oncogénicas Virales , Neoplasias del Cuello Uterino , Femenino , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Simulación del Acoplamiento Molecular , Virus del Papiloma Humano , Proteína de Retinoblastoma , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ligandos , Ácido Valproico/farmacologíaRESUMEN
p53 exerts its tumour suppressor activity by modulating hundreds of genes and it can also repress viral replication. Such is the case of human papillomavirus (HPV) through targeting the E2 master regulator, but the biochemical mechanism is not known. We show that the C-terminal DNA binding domain of HPV16 E2 protein (E2C) triggers heterotypic condensation with p53 at a precise 2/1 E2C/p53 stoichiometry at the onset for demixing, yielding large regular spherical droplets that increase in size with E2C concentration. Interestingly, transfection experiments show that E2 co-localizes with p53 in the nucleus with a grainy pattern, and recruits p53 to chromatin-associated foci, a function independent of the DNA binding capacity of p53 as judged by a DNA binding impaired mutant. Depending on the length, DNA can either completely dissolve or reshape heterotypic droplets into irregular condensates containing p53, E2C, and DNA, and reminiscent of that observed linked to chromatin. We propose that p53 is a scaffold for condensation in line with its structural and functional features, in particular as a promiscuous hub that binds multiple cellular proteins. E2 appears as both client and modulator, likely based on its homodimeric DNA binding nature. Our results, in line with the known role of condensation in eukaryotic gene enhancement and silencing, point at biomolecular condensation of E2 with p53 as a means to modulate HPV gene function, strictly dependent on host cell replication and transcription machinery.
Asunto(s)
Condensados Biomoleculares , Replicación del ADN , Proteínas de Unión al ADN , Papillomavirus Humano 16 , Proteínas Oncogénicas Virales , Proteína p53 Supresora de Tumor , Replicación Viral , Humanos , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 16/fisiología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/virología , Dominios Proteicos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Replicación Viral/fisiología , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/virologíaRESUMEN
OBJECTIVE: To characterize HPV16 E6/E7 mutation and its association with p53 expression among Indonesian women with cervical cancer. METHODS: This is a cross-sectional study involving 31 Indonesian women with pathologically proven cervical cancer and HPV16 infection. Data about the clinical characteristics of the study population were obtained from the medical records. Biopsy specimen of the cervical cancer mass from each study participant was obtained for DNA isolation. The ORFs of E6 and E7 genes were amplified using specific primer designed according to K02718/HPV16R gene sequence obtained from GenBank. Sequencing was performed using software program MEGA10. HPV16 E6 and E7 prototype sequences for nucleotide alignment (HPv16. P, GenBank Access code: NC_001526) was selected from European variant. The sequence of nucleotide and amino acid was aligned using software program BioEdit. p53 expression was evaluated through immunohistochemistry and quantified using immunoreactivity score (IRS). RESULTS: Twelve subjects (38.7%) present with E6 and E7 mutation. Median age, parity, stage and histologic type of the tumour did not associate with E6/E7 mutation. E6 and E7 mutation rate was 25.8% (8/31) and 12.9% (4/31), respectively. Seven single nucleotide changes were identified within the E6 and E7 oncogenes, including four non-synonymous and three synonymous mutations. E6 T27C was the most prevalent mutation (16.1%). Nonsynonymous mutations were more prevalent within E7 gene (9.6%) (N29T, N29S, and R77C). Median IRS did not differ between HPV16-E6/E7 variants and wildtype (p value = 0.990). There was no association between E6/E7 mutations and p53 expression in Indonesian women with cervical cancer (PR 1.4, 95% CI: 0.29-6.77, p value = 0.704). CONCLUSIONS: HPV16 E6 mutation was more prevalent than E7 mutation among Indonesian women. There was no association between E6/E7 mutation and p53 expression level.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Estudios Transversales , Femenino , Humanos , Indonesia/epidemiología , Mutación , Nucleótidos/metabolismo , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Oncogenes , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias del Cuello Uterino/complicaciones , Neoplasias del Cuello Uterino/genéticaRESUMEN
BACKGROUND: Human papillomavirus type 16 (HPV16) is the most prevalent etiology of cervical cancer in Indonesian women. The L2 minor capsid protein has considerable potential as a broad-protective antigen target of the cervical cancer vaccine strategies, yet the data on L2 gene variation is still minimal. In this research, we determined the variations of the HPV16 L2 gene sequences in Indonesian cervical cancer specimens. METHODS: We cross-sectionally observed 23 DNA isolates of HPV16 positive cervical cancer specimens stored in the laboratory of the Center for Diagnostic and Research on Infectious Diseases (PDRPI Lab), Faculty of Medicine, Universitas Andalas, Padang, Indonesia. We detected and amplified the HPV16 L2 gene sequences in the samples, followed by sequencing, DNA alignment, single nucleotide polymorphisms (SNPs) analysis, and phylogenetic tree reconstruction. RESULTS: As many as 35 SNPs were found, consist of 18 synonymous SNPs (sSNPs) and 17 non-synonymous SNPs (nsSNPs). Amino acid variations were mostly detected at S269P (100%) and L330F (43.48%) with no variation in the immuno-protective region near L2 N-terminus. A total of 5 HPV16 phylogenetic sub-lineages were found closely related to A1 (n=5), A2 (n=12), A3 (n=2), A4 (n=3), and C (n=1). CONCLUSION: The variations of HPV16 L2 gene sequences are mainly located in the central region of the L2 sequences, and the cross-protective region near the L2 N-terminus is remarkably conserved. This study should enhance the information about HPV16 L2 gene variation in Indonesia.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Femenino , Papillomavirus Humano 16 , Humanos , Indonesia/epidemiología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Filogenia , Neoplasias del Cuello Uterino/genéticaRESUMEN
In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally â¼13 µs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47RE6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47E6 and R290p53, which is unfavorable for E6-p53 binding. R102AE6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47E6 and E286p53/L298p53/R290p53. L50EE6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Mutación , Proteínas Oncogénicas Virales/química , Unión Proteica , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The tumor suppressor p53 is regulated by various posttranslational modifications including different types of ubiquitylation, which exert distinct effects on p53. While modification by ubiquitin chains targets p53 for degradation, attachment of single ubiquitin moieties (mono-ubiquitylation) affects the intracellular location of p53 and/or its interaction with chromatin. However, how this is achieved at the molecular level remains largely unknown. Similarly, since p53 can be ubiquitylated at different lysine residues, it remains unclear if the eventual effect depends on the position of the lysine modified. Here, we combined genetic code expansion with oxime ligation to generate p53 site-specifically mono-ubiquitylated at position 120. We found that mono-ubiquitylation at this position neither interferes with p53 ubiquitylation by the E3 ligases HDM2 and E6AP in complex with the viral E6 oncoprotein nor affects p53 binding to a cognate DNA sequence. Thus, ubiquitylation per se does not affect physiologically relevant properties of p53.
Asunto(s)
Proteínas Oncogénicas Virales , Proteína p53 Supresora de Tumor , Lisina/metabolismo , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteína p53 Supresora de Tumor/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: The Alphapapillomavirus 9 (α-9 HPV) is a member of the Alphapapillomavirus genus and Papillomaviridae family. These viruses are almost all carcinogenic HPV, which is closely related to 75% of invasive cervical cancer worldwide, and has a high prevalence in Sichuan. The carcinogenic function is mainly realized by its E6 oncoprotein. METHODS: Cell samples were collected by cervical scraped for HPV detecting and typing. HPV-16, HPV-31, HPV-33, HPV-52, HPV-58 5 α-9 genus HPV subtype positive samples were selected, their E6 gene was sequenced and analyzed. The positive selection sites of HPV E6 genes were estimated by PAML 4.8 server. The secondary and tertiary structure of E6 protein were predicted by PSIPred and Swiss-model. The T-cell antigen epitopes of E6 protein were predicted by IEDB. RESULTS: α-9 HPV has a high prevalence in Sichuan, China. From 2012 to 2017, 18,067 cell cervical samples were collected, and 3135 were detected with α-9 HPV infection. Among which, 250 cases HPV-16 E6, 96 cases HPV-31 E6, 216 cases HPV-33 E6, 288 cases HPV-52 E6 and 405 cases HPV-58 E6 were successfully amplified, 17, 6, 6, 13, and 4 non-synonymous nucleotide mutations were respectively detected in HPV-16, 31, 33, 52, and 58 E6, 7 positive selection sites of α-9 HPV E6 were selected out (D32E of HPV-16 E6, K35N, K93N and R145I of HPV-33 E6, K93R of HPV-52 E6, K93N and R145K of HPV-58 E6). The structure and antigen epitopes of E6 protein with amino acid substitution differ from those of wild-type E6 protein, especially for the mutation located in the E6 positive selection site. CONCLUSIONS: HPV E6 nucleotide non-synonymous mutation in the positive selection site influence the protein structure and decrease the antigen epitopes affinity of the E6 protein overall, making it more difficult for the HPV-infected cells to be detected by the immune system, and enhancing the HPV adaptability to the environment. Mutations influence the validity of HPV clinical diagnostic probes, the polymorphism analysis of α-9 HPV E6 enrich the data of HR-risk HPV in Sichuan China, and the detection probes designed with the polymorphism data in mind can improve the efficiency of clinical detection; Mutations influence epitopes affinity, the association of E6 polymorphism and epitope affinity can improve the design of therapeutic vaccine with good immunity and high generality antigen epitope; The above study all provide a good theoretical basis for the prevention and treatment of HPV-related diseases.
Asunto(s)
Alphapapillomavirus , Proteínas Oncogénicas Virales , Proteínas Represoras , Alphapapillomavirus/genética , China/epidemiología , Epítopos de Linfocito T/genética , Femenino , Papillomavirus Humano 16 , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Infecciones por Papillomavirus , Filogenia , Polimorfismo de Nucleótido Simple , Proteínas Represoras/química , Proteínas Represoras/genética , Neoplasias del Cuello UterinoRESUMEN
High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes.
Asunto(s)
Alphapapillomavirus , Proteínas Oncogénicas Virales , Papillomaviridae , Proteína Tirosina Fosfatasa no Receptora Tipo 4 , Proteínas Represoras , Alphapapillomavirus/química , Alphapapillomavirus/metabolismo , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ , Papillomaviridae/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 4/química , Proteína Tirosina Fosfatasa no Receptora Tipo 4/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismoRESUMEN
HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.
Asunto(s)
Alphapapillomavirus/genética , Mutación , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/epidemiología , Alphapapillomavirus/química , Alphapapillomavirus/clasificación , Alphapapillomavirus/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Linfocitos B/inmunología , Linfocitos B/virología , Sitios de Unión , Cuello del Útero/inmunología , Cuello del Útero/virología , China/epidemiología , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Femenino , Genotipo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Modelos Moleculares , Tipificación Molecular , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/virología , Filogenia , Prevalencia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Linfocitos T/inmunología , Linfocitos T/virologíaRESUMEN
Human papillomavirus type 16 (HPV-16) is one of the most important cause of developing cervical cancer. Therefore, effective epitope-based vaccine design for HPV-16 would be of major medical benefit. The aim of our study was to identify B- and T-cell epitopes of HPV-16 L1 protein. In this study, the HPV-16 L1 gene was isolated from HPV recovered from five vaginal swab samples using specific primers and finally sequenced. The ExPASy translate tool (http://web.expasy.org/translate/) was used to convert nucleotide sequence into amino acid sequence. Bioinformatic analysis was employed to predict suitable B- and T-cell epitopes and immunogenicity, allergenicity, and toxicity of predicted epitopes were then evaluated. Afterward, the selected T-cell epitopes were docked using Molegro Virtual Docker software. The two epitopes 207 AMDFTTLQA215 and 200 MVDTGFGAM208 have showed a very strong binding affinity to HLA-A0201 and HLA-B3501 molecules, respectively. Outcome of B-cell epitope prediction showed that epitope 475 KAKPKFTLGKRK ATPTTSSTSTTAKRKK502 contained overlapped epitope, which might be the epitope associated with the production of neutralizing antibody response. Based on this finding, the predicted B- and T-cell epitopes are promising targets for epitope-based vaccine development against HPV-16. Further in vivo and in vitro experiments are needed to confirm our findings.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Proteínas de la Cápside , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Femenino , Papillomavirus Humano 16/química , Papillomavirus Humano 16/genética , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Linfocitos TRESUMEN
HPV16 is the most prominent cause of cervical cancer. HPV16 E1, a helicase required for HPV replication exhibits increased expression in association with cervical cancer progression, suggesting that E1 has a similar effect on the host as the HPV16 E6 and E7 oncoproteins. This study aimed to determine whether expression of HPV16 E1 correlated with carcinogenesis by modulating cellular pathways involved in cervical cancer. HEK293T cells were transfected with pEGFP, pEGFPE1 or truncated forms of HPV16 E1. Cell proliferation, cell death, and the impact of HPV16 E1 on host gene expression was then evaluated. HPV16 E1 overexpression resulted in a significant reduction of cell viability and cellular proliferation (p-value<0.0001). Moreover, prolonged expression of HPV16 E1 significantly induced both apoptotic and necrotic cell death, which was partially inhibited by QVD-OPH, a broad-spectrum caspase inhibitor. Microarray, real time RT-PCR and kinetic host gene expression analyses revealed that HPV16 E1 overexpression resulted in the downregulation of genes involved in protein synthesis (RPL36A), metabolism (ALDOC), cellular proliferation (CREB5, HIF1A, JMJDIC, FOXO3, NFKB1, PIK3CA, TSC22D3), DNA damage (ATR, BRCA1 and CHEK1) and immune response (ISG20) pathways. How these genetic changes contribute to HPV16 E1-mediated cervical carcinogenesis warrants further studies.
Asunto(s)
Carcinogénesis/genética , Daño del ADN/genética , Regulación Neoplásica de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Proteínas Oncogénicas Virales/metabolismo , Neoplasias del Cuello Uterino/patología , Apoptosis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Células HEK293 , Humanos , Necrosis , Proteínas Oncogénicas Virales/química , Dominios Proteicos , Transducción de Señal/genéticaRESUMEN
The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.
Asunto(s)
Inmunomodulación , Canales Iónicos/metabolismo , Proteínas Viroporinas/metabolismo , Virosis/tratamiento farmacológico , Virus/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Autofagia , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Evasión Inmune , Inflamasomas/inmunología , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Proteínas Viroporinas/química , Virosis/inmunología , Virosis/virología , Virus/efectos de los fármacos , Virus/inmunología , Virus/patogenicidadRESUMEN
The high-risk α-type papillomaviruses have a C-terminal PDZ-binding motif (PBM) on one of the two major oncoproteins E6 or E7; the vast majority on E6. The PBM is essential for the high-risk HPV life cycle, for episomal maintenance of the virus genome, and for maintaining the mitotic stability of the infected cell. The question is why only these viruses have PBMs - are there specific constraints imposed by the mucosal epithelium in which these viruses replicate? However the low-risk α-HPVs, such as HPV-6 and HPV-11 replicate extremely efficiently without a PBM, while viruses of the alpha8 group, such as HPV-40, replicate well with a very primitive PBM. So what does PDZ-binding capacity contribute to the fitness of the virus?
Asunto(s)
Alphapapillomavirus/metabolismo , Aptitud Genética , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ , Secuencias de Aminoácidos , Animales , Sitios de Unión , Humanos , Proteínas Oncogénicas Virales/química , Infecciones por Papillomavirus/virología , Unión ProteicaRESUMEN
Human papillomaviruses (HPV)-16 and 18 are the most prevalent types associated with cervical cancer. HPV L1 and L2 capsid proteins and E7 oncoprotein play crucial roles in HPV-related diseases. Hence, these proteins were proposed as target antigens for preventive and therapeutic vaccines. In this study, two multiepitope DNA-based HPV vaccine candidates were designed using in silico analysis including the immunogenic and conserved epitopes of HPV16/18 L1, L2 and E7 proteins (the L1-L2-E7 fusion DNA), and of heat shock protein 70 (HSP70) linked to the L1-L2-E7 DNA construct (the HSP70-L1-L2-E7 fusion DNA). Next, the expression of the L1-L2-E7 and HSP70-L1-L2-E7 multiepitope DNA constructs was evaluated in a mammalian cell line. Finally, immunological responses and antitumor effects of the DNA constructs were investigated in C57BL/6 mice. Our data indicated high expression rates of the designed multiepitope L1-L2-E7 DNA (~ 56.16%) and HSP70-L1-L2-E7 DNA (~ 80.45%) constructs in vitro. The linkage of HSP70 epitopes to the L1-L2-E7 DNA construct significantly increased the gene expression. Moreover, the HSP70-L1-L2-E7 DNA construct could significantly increase immune responses toward Th1 response and CTL activity, and induce stronger antitumor effects in mouse model. Thus, the designed HSP70-L1-L2-E7 DNA construct represents promising results for development of HPV DNA vaccine candidates.
Asunto(s)
Proteínas de la Cápside/química , Epítopos/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas E7 de Papillomavirus/química , Infecciones por Papillomavirus/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Neoplasias del Cuello Uterino/prevención & control , Vacunas de ADN/administración & dosificación , Animales , Diseño Asistido por Computadora , Femenino , Células HEK293 , Papillomavirus Humano 16/genética , Humanos , Ratones , Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales/química , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/administración & dosificación , Vacunas contra Papillomavirus/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Neoplasias del Cuello Uterino/inmunología , Vacunas de ADN/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Epítopos/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/prevención & control , Vacunas contra el Cáncer/química , Biología Computacional , Epítopos/química , Femenino , Humanos , Inmunogenicidad Vacunal , Simulación del Acoplamiento Molecular , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/inmunología , Vacunas contra Papillomavirus/química , Proteínas Represoras/química , Proteínas Represoras/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/virologíaRESUMEN
BACKGROUND: Cancers of cervix, head and neck regions have been found to be associated with Human Papilloma Virus (HPV) infection. E1 protein makes an important papillomavirus replication factor. Among the ORFs of papillomaviruses, the most conserved sequence is that of the E1 ORF. It is the viral helicase with being a member of class of ATPases associated with diverse cellular activities (AAA+) helicases. The interactions of E1 with human DNA and proteins occurs in the presence of short linear peptide motifs on E1 identical to those on human proteins. METHODS: Different Motifs were identified on HPV16 E1 by using ELMs. Elastic network models were generated by using 3D structures of E1. Their dynamic fluctuations were analyzed on the basis of B factors, correlation analysis and deformation energies. RESULTS: 3 motifs were identified on E1 which can interact with Cdk and Cyclin domains of human proteins. 11 motifs identified on E1 have their CDs of Pkinase on human proteins. LIG_MYND_2 has been identified as involved in stabilizing interaction of E1 with Hsp40 and Hsp70. These motifs and amino acids comprising these motifs play a major role in maintaining interactions with human proteins, ultimately causing infections leading to cancers. CONCLUSION: Our study identified various motifs on E1 which interact with specific counter domains found in human proteins, already reported having the interactions with E1. We also validated the involvement of these specific motifs containing regions of E1 by modeling elastic networks of E1. These motif involving interactions could be used as drug targets.