RESUMO
GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in many key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution.
Assuntos
Anticorpos , Animais , Camundongos , Anticorpos/imunologia , Anticorpos/metabolismo , Fígado/metabolismo , Fígado/imunologia , Camundongos Knockout , Proteínas Mitocondriais/imunologia , Proteínas do Tecido Nervoso/imunologiaRESUMO
BACKGROUND AND AIMS: The increased frequency of urinary tract infections in patients with primary biliary cholangitis (PBC) and the cross-reactivity between the lipoyl domains (LD) of human pyruvate dehydrogenase complex (hPDC-E2) and Escherichia coli PDC-E2 (ePDC-E2) have long suggested a role of E. coli in causality of PBC. This issue, however, has remained speculative. We hypothesized that by generating specific constructs of human and E. coli PDC-E2, we would be able to assess the specificity of autoantibody responses and define whether exposure to E. coli in susceptible hosts is the basis for the antimitochondrial antibody (AMA) response. APPROACH AND RESULTS: Importantly, the reactivity of hPDC-E2 LD (hPDC-E2LD) affinity-purified antibodies against hPDC-E2LD could only be removed by prior absorption with hPDC-E2LD and not ePDC-E2, suggesting the presence of unique human PDC-E2 epitopes distinct from E. coli PDC-E2. To identify the autoepitope(s) present in hPDC-E2LD, a more detailed study using a variety of PDC-E2 constructs was tested, including the effect of lipoic acid (LA) on ePDC-E2 conformation and AMA recognition. Individual recombinant ePDCE2 LD domains LD1, LD2 and LD3 did not react with either AMA or antibodies to LA (anti-LA), but in contrast, anti-LA was readily reactive against purified recombinant LD1, LD2, and LD3 expressed in tandem (LP); such reactivity increased when LP was precultured with LA. Moreover, when the three LD (LD1, LD2, LD3) domains were expressed in tandem in pET28a or when LD1 was expressed in another plasmid pGEX, they were lipoylated and reactive to PBC sera. CONCLUSIONS: In conclusion, our data are consistent with an exposure to E. coli that elicits specific antibody to ePDC-E2 resulting in determinant spreading and the classic autoantibody to hPDC-E2LD. We argue this is the first step to development of human PBC.
Assuntos
Autoantígenos/imunologia , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/imunologia , Infecções por Escherichia coli/complicações , Escherichia coli/imunologia , Cirrose Hepática Biliar/microbiologia , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , Autoanticorpos/sangue , Estudos de Casos e Controles , Reações Cruzadas/imunologia , Epitopos/imunologia , Escherichia coli/enzimologia , Hepatite Autoimune/sangue , Humanos , Lipoilação , Conformação Molecular/efeitos dos fármacos , Ácido Tióctico/imunologia , Ácido Tióctico/farmacologiaRESUMO
Angioedema is characterized by swelling of the skin or mucous membranes. Overproduction of the vasodilator bradykinin (BK) is an important contributor to the disease pathology, which causes rapid increase in vascular permeability. BK formation on endothelial cells results from high molecular weight kininogen (HK) interacting with gC1qR, the receptor for the globular heads of C1q, the first component of the classical pathway of complement. Endothelial cells are sensitive to blood-flow-induced shear stress and it has been shown that shear stress can modulate gC1qR expression. This study aimed to determine the following: (1) how BK or angioedema patients' (HAE) plasma affected endothelial cell permeability and gC1qR expression under shear stress, and (2) if monoclonal antibody (mAb) 74.5.2, which recognizes the HK binding site on gC1qR, had an inhibitory effect in HK binding to endothelial cells. Human dermal microvascular endothelial cells (HDMECs) grown on Transwell inserts were exposed to shear stress in the presence of HAE patients' plasma. Endothelial cell permeability was measured using FITC-conjugated bovine serum albumin. gC1qR expression and HK binding to endothelial cell surface was measured using solid-phase ELISA. Cell morphology was quantified using immunofluorescence microscopy. The results demonstrated that BK at 1 µg/mL, but not HAE patients' plasma and/or shear stress, caused significant increases in HDMEC permeability. The mAb 74.5.2 could effectively inhibit HK binding to recombinant gC1qR, and reduce HAE patients' plasma-induced HDMEC permeability change. These results suggested that monoclonal antibody to gC1qR, i.e., 74.5.2, could be potentially used as an effective therapeutic reagent to prevent angioedema.
Assuntos
Angioedema/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Bradicinina/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Proteínas de Transporte/imunologia , Células Endoteliais/efeitos dos fármacos , Proteínas Mitocondriais/imunologia , Angioedema/imunologia , Angioedema/metabolismo , Angioedema/fisiopatologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores/metabolismo , Permeabilidade Capilar/imunologia , Fármacos Cardiovasculares/uso terapêutico , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Permeabilidade/efeitos dos fármacos , Resistência ao Cisalhamento/efeitos dos fármacosRESUMO
Infection with SARS-CoV-2 triggers the simultaneous activation of innate inflammatory pathways including the complement system and the kallikrein-kinin system (KKS) generating in the process potent vasoactive peptides that contribute to severe acute respiratory syndrome (SARS) and multi-organ failure. The genome of SARS-CoV-2 encodes four major structural proteins - the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. However, the role of these proteins in either binding to or activation of the complement system and/or the KKS is still incompletely understood. In these studies, we used: solid phase ELISA, hemolytic assay and surface plasmon resonance (SPR) techniques to examine if recombinant proteins corresponding to S1, N, M and E: (a) bind to C1q, gC1qR, FXII and high molecular weight kininogen (HK), and (b) activate complement and/or the KKS. Our data show that the viral proteins: (a) bind C1q and activate the classical pathway of complement, (b) bind FXII and HK, and activate the KKS in normal human plasma to generate bradykinin and (c) bind to gC1qR, the receptor for the globular heads of C1q (gC1q) which in turn could serve as a platform for the activation of both the complement system and KKS. Collectively, our data indicate that the SARS-CoV-2 viral particle can independently activate major innate inflammatory pathways for maximal damage and efficiency. Therefore, if efficient therapeutic modalities for the treatment of COVID-19 are to be designed, a strategy that includes blockade of the four major structural proteins may provide the best option.
Assuntos
Antígenos Virais/imunologia , COVID-19/imunologia , Proteínas do Sistema Complemento/imunologia , Sistema Calicreína-Cinina , SARS-CoV-2/imunologia , Proteínas Estruturais Virais/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Hemólise , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/imunologia , Proteínas Recombinantes/imunologia , Proteínas Estruturais Virais/genéticaRESUMO
OBJECTIVES: Heat shock proteins are overexpressed in many human malignancies. The role of heat shock proteins as a therapeutic target in cancer as well as their association with drug resistance were widely documented. The aim of this study was to evaluate the concentration of IgG class HSP27 and HSP60 antibodies in serum of patients with endometrial and cervical cancer, as well as to analyse the variability of concentrations of the examined antibodies depending on the cancer stage. MATERIAL AND METHODS: The study included 59 women with adenocarcinoma of the endometrium and 36 women with cervical cancer, the control group consisted of 54 healthy women. The concentrations of IgG class antibodies against the tested heat shock proteins were determined by an immunoenzymatic assay (ELISA) using commercial assays. RESULTS: In both endometrial and cervical cancer, the serum concentration of IgG anti-HSP27 antibody was significantly higher than in the healthy control group. The concentration of IgG anti-HSP60 antibody in endometrial cancer, cervical cancer and healthy control was similar. The median IgG anti-HSP27 antibody serum concentration of endometrial cancer patients was not correlated with FIGO-stage. In cervical cancer inverse correlation between concentration of this antibody and FIGO stage was observed. The median IgG anti-HSP60 antibody concentration in serum of endometrial cancer patients was lower in FIGO stage I and II compared to FIGO stage IV and in FIGO stage IA compared to FIGO stage IB. Concentrations of examined antibodies correlated positively with each other, both in the group of women with cancer and in the group of healthy women. The strongest correlations were found in the group of patients with endometrial cancer. CONCLUSIONS: Concentration of anti-HSP27 antibody could help in detection of cervical and endometrial cancer. We need to look for the cut-off point in large cohort studies. Anti-HSP27 and anti-HSP60 antibodies should be further evaluated for their potential usage as biomarkers in cervical and endometrial cancer as they shown some correlation with stage of disease.
Assuntos
Chaperonina 60 , Neoplasias do Endométrio , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico , Proteínas Mitocondriais , Chaperonas Moleculares , Neoplasias do Colo do Útero , Biomarcadores Tumorais/imunologia , Chaperonina 60/imunologia , Neoplasias do Endométrio/imunologia , Feminino , Proteínas de Choque Térmico HSP27/imunologia , Proteínas de Choque Térmico/imunologia , Humanos , Imunoglobulina G/imunologia , Proteínas Mitocondriais/imunologia , Chaperonas Moleculares/imunologia , Neoplasias do Colo do Útero/imunologiaRESUMO
High expression of p32 in certain tumors makes it a potential target for immunotherapy. In the present study, the first goal was to design multi-epitope peptides from the P32 protein and the second goal was to compare the prophylactic effects of DCs- and PBMCs- based vaccines by pulsing them with designed peptides. For these purposes, 160 BALB/c mice were vaccinated in 5 different subgroups of each 4 peptides using PBS (F1-4a), F peptides alone (F1-4b), F peptides with CpG-ODN (F1-4c), F peptides with CpGODN and DCs (F1-4d), and F peptides with CpG-ODN and PBMCs (F1-4e). We found a significantly higher interferon-γ (IFN-γ) and granzyme B levels in T cells of F4d and F4e subgroups compared to control (p ≤ 0.05). The result of challenging spleen PBMCs of vaccinated mice with 4T1 cells showed significant up- and down- regulation of Fas ligand (FasL) and forkhead box P3 (Foxp3) gene expression between F4d and F4e subgroups with control, respectively. In addition, a significant change was seen in Caspase3 gene expression of F4d subgroup compared to control (p ≤ 0.05). Supernatant levels of IFN-γ and perforin were significantly increased in F4d and F4e subgroups compared to control. Consequently, significantly lower tumor sizes and prolonged survival time were detected in F4d and F4e subgroups compared to control after challenging mice with 4T1 cells. Accordingly, these results demonstrated that PBMCs pulsed F4 peptide-based vaccine could induce a protective immune response while it is a simple and less expensive vaccine.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Leucócitos Mononucleares/imunologia , Neoplasias Mamárias Animais/imunologia , Proteínas Mitocondriais/imunologia , Peptídeos/imunologia , Animais , Apresentação de Antígeno , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades AntigênicasRESUMO
Dysfunction of CD4+ T cells by HIV infection can cause serious immune defects. Recently, Campbell and colleagues described an intriguing and simple therapeutic method for HIV-infected resting central memory CD4+ T cells (HIV-TCM), dependently on inhibitor of apoptosis (IAP) family proteins-targeted and second mitochondria-derived activator of caspases (SMAC) mimetics-mediated apoptosis, which is only triggered in HIV-TCM and not uninfected ones. Autophagy induction and subsequent formation of a ripoptosome-like death signaling complex were observed after such treatment, which may partially explain the potential mechanism. However, the direct intracellular inhibitory effects of IAPs on autophagy, as well as the critical roles of autophagy in activating extracellular anti-infection immune responses, warrant further investigation. Thus, this pointer aims to provide potential alternative mechanisms and to suggest important avenues for follow-up study.
Assuntos
Autofagia/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Mitocondriais/imunologia , Proteínas Mitocondriais/metabolismoRESUMO
BACKGROUND: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. METHODS: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. RESULTS: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. CONCLUSIONS: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.
Assuntos
Interações entre Hospedeiro e Microrganismos , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteômica , Células A549 , Animais , Galinhas/virologia , Humanos , Influenza Aviária/virologia , Mitocôndrias/química , Mitocôndrias/imunologia , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/imunologiaRESUMO
The NOD-like receptor X1 (NLRX1) is a member of highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family (known as NLR), that localizes to the mitochondrial outer membrane and regulate the innate immunity by interacting with mitochondrial antiviral-signaling protein (MAVS). As one of cytoplasmic PRRs, NLRX1 plays key roles for pathogen recognition, autophagy and regulating of subsequent immune signaling pathways. In this study, we identified the nlrx1 in turbot as well as its expression profiles in mucosal surfaces following bacterial infection. In our results, the full-length nlrx1 transcript consists of an open reading frame (ORF) of 4,886 bp encoding the putative peptide of 966 amino acids. The phylogenetic analysis revealed the SmNlrx1 showed the closest relationship to Cynoglossus semilaevis. In addition, the Nlrx1 mRNA expression could be detected in all the examined tissues, with the most abundant expression level in head kidney, and the lowest expression level in liver. Moreover, Nlrx1 showed similar expression patterns following Vibrio anguillarum and Streptococcus iniae infection, that were both significantly up-regulated following challenge, especially post S. iniae challenge. Finally, fluorescence microscopy unveiled that the SmNlrx1 localized to mitochondria in HEK293T by N-terminal mitochondrial targeting sequence. Characterization of Nlrx1 might have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity and will probably contribute to the development of novel intervention strategies for farming turbot.
Assuntos
Linguados/imunologia , Proteínas Mitocondriais/imunologia , Mucosa/imunologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Linguados/microbiologia , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mucosa/microbiologia , Filogenia , Mapas de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Streptococcus iniae/fisiologia , Vibrio/fisiologiaRESUMO
NLRX1 is a member of the NOD-like receptor family, a set of pattern recognition receptors associated with innate immunity. Interestingly, NLRX1 exists in somewhat of an exile from its NLR counterparts with unique features that mediate atypical functions compared with traditional NOD-like receptors (NLRs). Aside from a mitochondrial targeting sequence, the N-terminal region is yet to be characterized. Mitochondrially located, NLRX1 sits within a subgroup of regulatory NLRs responsible for negatively regulating cellular inflammatory signalling. As well as modulating pathogen response, emerging evidence is implicating NLRX1 as a central homeostatic gatekeeper between mitochondrial biology and immunological response. More recently, NLRX1 has been implicated in a wide range of disease, both pathogen-driven and otherwise. Emerging links of NLRX1 in cancer biology, autoimmunity and other inflammatory conditions are raising the potential of targeting NLRX1 therapeutically, with recent studies in inflammatory bowel disease showing great promise. Within this review, we address the unique features of NLRX1, its roles in innate immune signalling and its involvement in a range of inflammatory, metabolic and oncology disease indications with a focus on areas that could benefit from therapeutic targeting of NLRX1.
Assuntos
Imunidade Inata/imunologia , Proteínas Mitocondriais/imunologia , Animais , HumanosRESUMO
BACKGROUND AND OBJECTIVE: The relationship between antimitochondrial antibody (AMA) levels and the severity or prognosis of primary biliary cholangitis (PBC) is unclear. This study explored the clinical significance of serum IgG antimitochondrial M2 antibody (IgG-M2) levels. METHODS: From 2008 to 2017, a retrospective analysis was conducted with PBC patients who had available quantitative values of serum IgG-M2 levels obtained with ELISA based on triple expression hybrid clones. The patients were divided into two groups based on high and low concentrations of IgG-M2. Baseline parameters, the incidence of adverse events, and prognosis were compared. RESULTS: Among the 530 PBC patients, the levels of albumin, cholinesterase, hemoglobin, fibrinogen and triglycerides and the red blood cell count were significantly lower in the high-concentration group than in the low-concentration group (n = 263, 49.6%). The red cell distribution width (RDW) and levels of serum immunoglobulin (Ig) G, IgM and IgA were significantly higher in the high-concentration group than in the low-concentration group. Spearman's correlation analysis suggested that the correlation between the above baseline indicators and IgG-M2 levels was statistically significant but weak (r < 0.2, P < 0.05). In total, 203 patients were followed up, of whom 87 (42.9%) were in the high-concentration group. During the median follow-up period of 52 months (range: 28-75), 121 (59.6%) experienced hepatic decompensation, and 37 (18.2%) died or underwent liver transplantation. There was no significant difference in the incidence of complications or survival (log-rank test: P = 0.079) between the two groups. One year after ursodeoxycholic acid (UDCA) treatment, the two groups had similar responses. In addition, the levels of IgG-M2 did not fluctuate significantly during treatment. CONCLUSION: IgG-M2 levels were not related to the disease severity, prognosis or efficacy of UDCA. The levels of IgG-M2 did not change significantly during treatment.
Assuntos
Autoantígenos/imunologia , Imunoglobulina G/imunologia , Cirrose Hepática Biliar/imunologia , Proteínas Mitocondriais/imunologia , Idoso , Autoantígenos/sangue , Biomarcadores/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Cirrose Hepática Biliar/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.
Assuntos
Aspergillus fumigatus/imunologia , Células Dendríticas/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas Mitocondriais/imunologia , Aspergilose Pulmonar/imunologia , Células Th2/imunologia , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Neutrófilos/imunologia , Neutrófilos/patologia , Aspergilose Pulmonar/genética , Aspergilose Pulmonar/patologia , Células Th2/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Hsp60sp, a signal peptide derived from the leader sequence of heat shock protein 60 kDa (Hsp60), is a Qa-1/HLA-E-binding peptide. We previously showed that Hsp60sp-specific CD8+ T cells are involved in the immunoregulation of autoimmune diseases by controlling the response of self-reactive lymphocytes. Here, we report that Hsp60sp-specific CD8+ T cells killed malignant lymphocytes in vitro independently of transporter associated with antigen processing (TAP) and classical MHC-I expression. Induction of this cytotoxic T lymphocyte (CTL) response in vivo, either by adoptive transfer of in vitro-amplified CTLs or peptide-loaded dendritic cell immunization, resulted in effective control of lymphoid tumors, including TAP- or classical MHC-I-deficient cells. Hsp60sp-specific immune activation combined with programmed cell death protein 1 (PD-1) blocking synergistically restrained mouse lymphoma development. Importantly, Hsp60sp-specific CD8+ T cells did not negatively affect normal tissues and cells. Our data suggest that Hsp60sp-based immunotherapy is an inviting strategy to control lymphoid malignancies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Chaperonina 60/química , Células Dendríticas/imunologia , Inibidores de Checkpoint Imunológico/administração & dosagem , Linfoma/terapia , Proteínas Mitocondriais/química , Sinais Direcionadores de Proteínas/fisiologia , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação de Antígeno , Linfócitos T CD8-Positivos/transplante , Linhagem Celular , Chaperonina 60/imunologia , Terapia Combinada , Células Dendríticas/transplante , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Imunização , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/imunologia , Linfócitos T Citotóxicos/transplanteRESUMO
Severe acute respiratory syndrome-related coronavirus 2 infection has been associated with Guillain-Barré syndrome. We investigated here the potential mechanism underlying the virus-induced damage of the peripheral nervous systems by searching the viral amino acid sequence for peptides common to human autoantigens associated with immune-mediated polyneuropathies. Our results show molecular mimicry between the virus and human heat shock proteins 90 and 60, which are associated with Guillain-Barré syndrome and other autoimmune diseases. Crucially, the shared peptides are embedded in immunoreactive epitopes that have been experimentally validated in the human host.
Assuntos
Betacoronavirus/metabolismo , Chaperonina 60 , Infecções por Coronavirus/virologia , Síndrome de Guillain-Barré/metabolismo , Proteínas de Choque Térmico HSP90 , Proteínas Mitocondriais , Pneumonia Viral/virologia , Proteínas Virais , Sequência de Aminoácidos , Autoantígenos , COVID-19 , Chaperonina 60/química , Chaperonina 60/imunologia , Bases de Dados de Proteínas , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Epitopos Imunodominantes , Proteínas Mitocondriais/química , Proteínas Mitocondriais/imunologia , Mimetismo Molecular , Pandemias , SARS-CoV-2 , Proteínas Virais/química , Proteínas Virais/imunologiaRESUMO
Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress70 protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer prosurvival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complementdependent cytotoxicity (CDC) and may therefore protect cells from antibodybased immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a Cterminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, MotP2 and MotP7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, MotP2 and MotP7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, MotP7, but not MotP2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At subtoxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalinbinding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with MotP2 or MotP7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complementactivating antibody therapy to target mortalin for precision cancer therapy.
Assuntos
Proteínas do Sistema Complemento/imunologia , Proteínas de Choque Térmico HSP70/farmacologia , Proteínas Mitocondriais/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Peptídeos/farmacologia , Peptidomiméticos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/química , Ligação ProteicaRESUMO
INTRODUCTION: Antibodies to hexokinase 1 (HK1) and kelch-like 12 (KLHL12) have been identified as potential biomarkers in primary biliary cholangitis (PBC), and this study assesses changes of these antibodies over time and if they are associated with clinical outcomes. METHODS: Two hundred fifty-four PBC patients (93.3% female, 51 ± 12.3 years old) were tested for anti-HK1 and anti-KLHL12, antimitochondrial (AMA), anti-gp210, and anti-sp100 antibodies. One hundred sixty-nine patients were tested twice and 49 three times within 4.2 (0.8-10.0) years. Biochemistry and clinical features at diagnosis, response to therapy, events of decompensation, and liver-related death or transplantation were evaluated. RESULTS: Anti-HK1 and anti-KLHL2 were detected in 46.1% and 22.8% patients, respectively. AMA were positive in 93.7%, anti-sp100 in 26.4%, and anti-gp210 in 21.3% of patients. Anti-HK1 and anti-KLHL12 positivity changed over time in 13.3% and 5.5% of patients, respectively. Anti-HK1 or anti-KLHL12 were present in 37.5% of AMA-negative patients, and in 40% of AMA, anti-gp210, and anti-sp100 negative. No significant differences were observed between those with or without HK1 and KLHL12 antibodies, but transplant-free survival and time to liver decompensation were significantly lower in patients anti-HK1 positive (P = 0.039; P = 0.04) and in those anti-sp100 positive (P = 0.01; P = 0.007). No changes in survival and events of liver decompensation were observed according to the positivity of AMA, anti-KLHL12, or anti-gp210 antibodies. DISCUSSION: HK1 and KLHL12 antibodies are present in 40% of PBC patients who are seronegative by the conventional PBC-specific antibodies. The novel antibodies remain rather steady during the course of the disease, and HK1 antibodies are associated with unfavourable outcomes.
Assuntos
Autoanticorpos/imunologia , Hexoquinase/imunologia , Cirrose Hepática Biliar/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adulto , Antígenos Nucleares/imunologia , Autoantígenos/imunologia , Colagogos e Coleréticos/uso terapêutico , Progressão da Doença , Feminino , Humanos , Imunossupressores/uso terapêutico , Cirrose Hepática Biliar/terapia , Transplante de Fígado , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/imunologia , Complexo de Proteínas Formadoras de Poros Nucleares/imunologia , Prognóstico , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
Viral infections trigger host innate immune responses, characterized by the production of type-I interferons (IFN) including IFNß. IFNß induces cellular antiviral defense mechanisms and thereby contributes to pathogen clearance. Accumulating evidence suggests that mitochondria constitute a crucial platform for the induction of antiviral immunity. Here we demonstrate that the mitochondrial protein phosphoglycerate mutase family member 5 (PGAM5) is important for the antiviral cellular response. Following challenge of HeLa cells with the dsRNA-analog poly(I:C), PGAM5 oligomers and high levels of PGAM5 were found in mitochondrial aggregates. Using immunoprecipitation, a direct interaction of PGAM5 with the mitochondrial antiviral-signaling protein (MAVS) was demonstrated. In addition, PGAM5 deficient cells showed diminished expression of IFNß and IFNß target genes as compared to WT cells. Moreover, PGAM5 deficient mouse embryonic fibroblasts (MEFs) exhibited decreased phosphorylation levels of IRF3 and TBK1 when challenged with poly(I:C) intracellularly. Finally, PGAM5 deficient MEFs, upon infection with vesicular stomatitis virus (VSV), revealed diminished IFNß expression and increased VSV replication. Collectively, our study highlights PGAM5 as an important regulator for IFNß production mediated via the TBK1/IRF3 signaling pathway in response to viral infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Células Cultivadas , Fibroblastos/virologia , Células HeLa , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Camundongos , Proteínas Mitocondriais/imunologia , Fosfoproteínas Fosfatases/imunologia , Poli I-C/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Infecções por Rhabdoviridae/imunologia , Transdução de Sinais , Replicação Viral/imunologiaRESUMO
Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8+ T cells in vivo. We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Proteínas Mitocondriais/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Neoplasias/terapiaRESUMO
HSP60 is a mitochondrial chaperone protein that is associated with decreased overall survival of ovarian cancer patients. We determined whether targeting HSP60 with its monoclonal antibody would induce cytotoxicity in sensitive and chemoresistant ovarian cancer cells and whether it is synergistic when combined with chemotherapeutic drugs. Epithelial ovarian cancer (EOC) cells and their docetaxel- or cisplatin-resistant counterparts were utilized. HSP60 mRNA levels were determined by real-time RT-PCR. Cytotoxicity of HSP60 antibody (0.5 or 1.5 µg/ml) alone and in combination with chemotherapy were assessed by MTT Cell Proliferation Assay. Unpaired t tests were used to compare groups for real-time RT-PCR. One-way ANOVA followed by Tukey's post hoc tests with Bonferroni correction was performed for cytotoxicity comparisons. Significant synergistic effects of the antibody combined with chemotherapy were determined by the CompuSyn Software. Basal HSP60 mRNA levels were increased in chemoresistant EOC cells as compared with their sensitive counterparts (p < 0.05). There was no significant difference in cytotoxicity between EOC cell types; however, treatment with the HSP60 antibody for 24 h showed a dose response (0.5 and 1.5 µg/ml) cytotoxic effect to both sensitive and chemoresistant EOC cells as compared with the isotype control (p < 0.05). Importantly, treatment with both doses of HSP60 antibody was not cytotoxic to normal macrophages. Combination of the HSP60 antibody with docetaxel or cisplatin was significantly synergistic in both sensitive and chemoresistant EOC cells. Here, we identify a novel target that may serve not only for ovarian cancer treatment but also for sensitization of patients to chemotherapy. The cytotoxic effect of HSP60 monoclonal antibody and its synergism with chemotherapeutic agents highlight HSP60 as a promising target for therapy and chemosensitization in ovarian cancer treatment.
Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Carcinoma Epitelial do Ovário/tratamento farmacológico , Chaperonina 60/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Mitocondriais/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 60/imunologia , Chaperonina 60/metabolismo , Cisplatino/administração & dosagem , Docetaxel/administração & dosagem , Quimioterapia Combinada/métodos , Feminino , Humanos , Proteínas Mitocondriais/imunologia , Proteínas Mitocondriais/metabolismo , RNA Mensageiro/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by immune complexes. Because these complexes contain mitochondrial components, we assessed the presence of antibodies to whole mitochondria (wMITO) using an ELISA in which mitochondria from mouse liver are bound to microtiter plates pre-coated with poly-l-lysine. Studies with this ELISA demonstrated that SLE plasmas contain abundant anti-wMITO activity. While digestion with DNase 1 did not affect anti-wMITO activity, adsorption of plasma on DNA affinity columns could reduce binding activity. Assay for anti-mitochondrial antibodies (AMA) by immunofluorescence and an ELISA with the M2 antigen (2-oxo-acid dehydrogenase protein complex) showed a low frequency of positivity, indicating that AMA and anti-wMITO are distinct specificities. In the study of 204 patients with SLE, the levels of anti-wMITO were higher in active SLE and correlated with levels of anti-DNA. These findings suggest that anti-wMITO can form immune complexes with mitochondria which may drive pathogenesis.