RESUMEN
N-myristoylation is one of the most widespread and important lipidation in eukaryotes and some prokaryotes, which is formed by covalently attaching various fatty acids (predominantly myristic acid C14:0) to the N-terminal glycine of proteins. Disorder of N-myristoylation is critically implicated in numerous physiological and pathological processes. Here, we presented a method for purification and comprehensive characterization of endogenous, intact N-glycine lipid-acylated peptides, which combined the negative selection method for N-terminome and the nanographite fluoride-based solid-phase extraction method (NeS-nGF SPE). After optimizing experimental conditions, we conducted the first global profiling of the endogenous and heterogeneous modification states for N-terminal glycine, pinpointing the precise sites and their associated lipid moieties. Totally, we obtained 76 N-glycine lipid-acylated peptides, including 51 peptides with myristate (C14:0), 10 with myristoleate (C14:1), 6 with tetradecadienoicate (C14:2), 5 with laurate (C12:0) and 4 with lauroleate (C12:1). Therefore, our proteomic methodology could significantly facilitate precise and in-depth analysis of the endogenous N-myristoylome and its heterogeneity.
Asunto(s)
Ácido Mirístico , Extracción en Fase Sólida , Extracción en Fase Sólida/métodos , Ácido Mirístico/química , Ácido Mirístico/análisis , Proteómica/métodos , Fluoruros/química , Fluoruros/análisis , Glicina/química , Glicina/análisis , Péptidos/química , Péptidos/análisisRESUMEN
Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery.
Asunto(s)
Lisina , Proteómica , Humanos , Lisina/metabolismo , Células HeLa , Ligandos , EndoglinaRESUMEN
Protein S-acylation is an important lipid modification characteristic for heterogeneity in the acyl chain and dynamicity in the acylation/deacylation cycle. Most S-acylproteomic research has been limited by indirect identification of modified proteins/peptides without attached fatty acids, resulting in the failure to precisely characterize S-acylated sites with attached fatty acids. The study of S-acylation turnover is still limited at the protein level. Herein, aiming to site-specifically profile both the heterogeneity and the turnover of S-acylation, we first developed a site-specific strategy for intact S-acylated peptide analysis by introducing an acid cleavable bioorthogonal tag into a metabolic labelling method (ssMLCC). The cleavable bioorthogonal tag allowed for the selective enrichment and efficient MS analysis of intact S-acylated peptides so that S-acylated sites and their attached fatty acids could be directly analysed, enabling the precise mapping of S-acylated sites, as well as circumventing false positives from previous studies. Moreover, 606 S-palmitoylated (C16:0) sites of 441 proteins in HeLa cells were identified. All types of S-acylated peptides were further characterized by an open search, providing site-specific profiling of acyl chain heterogeneity, including S-myristoylation, S-palmitoylation, S-palmitoleylation, and S-oleylation. Furthermore, site-specific monitoring of S-palmitoylation turnover was achieved by coupling with pulse-chase methods, facilitating the detailed observation of the dynamic event at each site in multi-palmitoylated proteins, and 85 rapidly cycling palmitoylated sites in 79 proteins were identified. This study provided a strategy for the precise and comprehensive analysis of protein S-acylation based on intact S-acylated peptide analysis, contributing to the further understanding of its complexity and biological functions.
Asunto(s)
Ácidos Grasos , Proteínas , Humanos , Células HeLa , Proteínas/metabolismo , Ácidos Grasos/metabolismo , Péptidos/metabolismo , AcilaciónRESUMEN
S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation.
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Fluoruros , Espectrometría de Masas en Tándem , Animales , Humanos , Cromatografía Liquida , Células HeLa , Acilación , Diferenciación Celular , MamíferosRESUMEN
Here, we have developed an approach termed methylamine stable isotope labeling (MeSIL) to relatively quantify N-glycopeptides through one step labeling. It is the first time that this approach is applied to measure N-glycopeptide changes in huh7 cells after Zika virus infection and it has been revealed that differentially expressed N-glycopeptides played important roles in virus infection at the glycosylation site-specific level.
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Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias de la Mama/patología , Clorhidrato de Erlotinib/uso terapéutico , Neoplasias Meníngeas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor ErbB-2/genética , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/genética , Femenino , Humanos , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/secundario , Persona de Mediana Edad , Terapia Molecular Dirigida , Mutación , Resultado del TratamientoRESUMEN
Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision. The mass tags lay on the cysteine residues, which were the potential palmitoylated sites, indicating that all the putative modified sites/peptides could be quantified, including the C-terminal peptide of one protein. Due to the isotopically labeled cysteine, the nonspecifically adsorbed peptide without cysteine was singlet in MS spectra, whereas pair peaks should be the signals of putative palmitoylated peptides, which could reduce spectral complexity and achieve double verification for the putative palmitoylated peptides. Finally, the palmitoylome in hepatocellular carcinoma (HCC) cells with different metastasis potentials (MHCC-97L and HCC-LM3 cells) were analyzed for the first time. Totally, 151 proteins were found to be differentially palmitoylated with high confidence, including many important proteins involved in a variety of biological processes, such as protein palmitoylation, cell proliferation, signal transduction, regulation of cell migration, and so on.
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Cisteína/metabolismo , Marcaje Isotópico , Lipoilación , Proteínas/metabolismo , Western Blotting , Línea Celular Tumoral , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de MasasAsunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Células Transicionales/tratamiento farmacológico , Indoles/administración & dosificación , Sulfonamidas/administración & dosificación , Neoplasias Urológicas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Carcinoma de Células Transicionales/genética , Femenino , Humanos , Indoles/uso terapéutico , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Sulfonamidas/uso terapéutico , Resultado del Tratamiento , Neoplasias Urológicas/genética , VemurafenibRESUMEN
Angiosarcoma is a rare and aggressive vascular malignancy with a poor prognosis. There is no standard chemotherapy regime for advanced angiosarcoma. Here, we report a case of advanced angiosarcoma that was successfully treated with apatinib, an oral tyrosine kinase inhibitor targeting the intracellular domain of vascular endothelial growth factor receptor-2 (VEGFR-2). To our knowledge, this is the first case to report the successful use of apatinib for angiosarcoma. Furthermore, the administration of apatinib results in fewer toxic effects than traditional cytotoxic chemotherapy. Quantitative polymerase chain reaction analysis revealed high expression of VEGFR-2 mRNA, suggesting that apatinib leads to clinical response by inhibiting VEGFR-2 tyrosine kinase activity and that VEGFR-2 plays a crucial role for angiosarcoma. Apatinib may be an additional option for angiosarcoma especially for the aged and patients with poor performance status. Further prospective studies are required to optimize the use of apatinib in patients with angiosarcoma and to identify which patients will benefit from the agent.
RESUMEN
Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus del Dengue/inmunología , Serogrupo , Acoplamiento Viral/efectos de los fármacos , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/farmacología , Células CHO , Cricetinae , Cricetulus , Dengue/inmunología , Humanos , RatonesRESUMEN
Malignant fibrous histiocytoma (MFH) is the most common soft-tissue sarcoma in late adult life. Unfortunately, advanced MFH has a poor prognosis due to a lack of effective drugs. We present here a case of advanced MFH with partial response to apatinib, a new potent oral small-molecule tyrosine kinase inhibitor targeting the intracellular domain of vascular endothelial growth factor receptor 2 (VEGFR-2). To the best of our knowledge, this is the first case report using apatinib for MFH. Quantitative polymerase chain reaction analysis revealed high expression of VEGFR-2 mRNA, suggesting that apatinib leads to clinical response by inhibiting VEGFR-2 tyrosine kinase activity and the crucial role of VEGFR-2 for MFH. Apatinib could be a new option for the treatment of MFH. Further studies are needed to optimize the treatment.
RESUMEN
The dengue virus (DENV) envelope protein domain III (ED3) has been suggested to contain receptor recognition sites and the critical neutralizing epitopes. Up to date, relatively little work has been done on fine mapping of neutralizing epitopes on ED3 for DENV4. In this study, a novel mouse type-specific neutralizing antibody 1G6 against DENV4 was obtained with both prophylactic and therapeutic effects. The epitope was mapped to residues 387-390 of DENV4 envelope protein. Furthermore, site-directed mutagenesis assay identified two critical residues (T388 and H390). The epitope is variable among different DENV serotypes but is highly conserved among four DENV4 genotypes. Affinity measurement showed that naturally occurring variations in ED3 outside the epitope region did not alter the binding of mAb 1G6. These findings expand our understanding of the interactions between neutralizing antibodies and the DENV4 and may be valuable for rational design of DENV vaccines and antiviral drugs.
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Virus del Dengue/inmunología , Epítopos/inmunología , Pruebas de Neutralización , Animales , Línea Celular , Cricetinae , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
BACKGROUND: Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed. METHODS AND FINDINGS: We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo. CONCLUSIONS: The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Toxinas Botulínicas/inmunología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Sitios de Unión , Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/toxicidad , Toxinas Botulínicas Tipo A , Secuencia Conservada , Mapeo Epitopo , Femenino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Infecciones por Flavivirus/prevención & control , Flavivirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Infecciones por Flavivirus/tratamiento farmacológico , Ratones , Internalización del VirusRESUMEN
Osteopontin (OPN) plays an important role in regulating lymphocyte adhesion and cytokine production associated with inflammatory processes and autoimmune diseases. Here we developed and characterized a monoclonal antibody F8E11 specific for human OPN (hOPN). F8E11 could inhibit OPN-induced lymphocyte activation and migration. Epitope mapping showed that F8E11 could specifically recognize the peptide QLYxxYP. In addition, a synthesized mimetic peptide F8P (EEKQLYNKYPDA) could block the binding of F8E11 to hOPN and significantly inhibit the hOPN-induced lymphocyte migration. Moreover, mutations on the QLYxxYP motif of hOPN also markedly diminished its activity for lymphocyte activation and migration. The functioning assay indicated that this novel epitope is critically involved in the lymphocyte migration through activating MAPK/ERK/AP-1 pathway, which can be inhibited by the motif QLYxxYP blocking antibody, F8E11. These results suggest that this novel epitope of OPN may provide a potential therapeutic target for the treatment of T cell mediated-immune diseases.
Asunto(s)
Movimiento Celular , Activación de Linfocitos , Osteopontina/fisiología , Linfocitos T/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Humanos , Imitación Molecular , Mutación , Oligopéptidos/química , Oligopéptidos/inmunología , Osteopontina/antagonistas & inhibidores , Osteopontina/genética , Estructura Terciaria de ProteínaRESUMEN
Osteopontin (OPN) is an extracellular matrix protein of pleiotropic properties and plays an important role in regulating lymphocyte adhesion and cytokine production associated with inflammatory processes and autoimmune diseases. Here we developed and characterized a monoclonal antibody (mAbs) (23C3D3) specific for human OPN. This antibody could inhibit OPN-induced lymphocyte adhesion, migration and survival. Epitope mapping showed that 23C3D3 could specifically recognize the phage displayed peptide (43)WLNPDP(48). In addition, a synthesized mimetic peptide (mimotope) 23P ((40)VATWLNPDPSQK(51)) could block the binding of 23C3D3 to hOPN and significantly inhibit the hOPN-induced lymphocyte adhesion, migration and survival. Moreover, mutations on the WLNPDP motif of hOPN also markedly diminished its activity for lymphocyte activation. Interestingly, this novel epitope is located in the extremely retained domain in all species. The functioning assay indicates that this novel epitope is critically involved in the lymphocyte migration and survival through activating ERK and the transcription factor NF-kappaB pathway, which can be inhibited by the motif (43)WLNPDP(48) blocking antibody, 23C3D3. These results suggest that this novel epitope of OPN may provide a potential therapeutic target for the treatment of T cell-mediated immune diseases.