RESUMO
Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.
Assuntos
Suscetibilidade a Doenças , Imunidade Inata , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Sepse/etiologia , Animais , Biomarcadores , Morte Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Edição de Genes , Técnicas de Silenciamento de Genes , Marcação de Genes , Genômica/métodos , Imunofenotipagem , Inflamação/etiologia , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fenótipo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Chronic activation of beta-adrenergic receptors by the sympathetic nervous system results in the apoptosis of cardiomyocytes. Due to the inability of cardiomyocytes to regenerate, this can result in heart failure. Upregulation of the pro-apoptotic protein Bim has been implicated as the cause of cardiomyocyte apoptosis. Beta blockers are the frontline drug used to negate this apoptotic pathway, as no direct inhibitors of Bim expression currently exist. Unfortunately, treatment of heart failure using beta blockers is not optimal. Therefore, direct inhibition of Bim expression is an attractive strategy to provide protection against stress-induced apoptosis of cardiomyocytes. Herein we explore a class of N-benzylsulfonyl-2-phenylazepanes to obtain anti-apoptotic compounds capable of reducing Bim expression levels to 7% of the control at 10 µM in cardiomyocytes under conditions of chronic beta-adrenergic receptor activation with little inhibitory effect upon protein kinase A activity and minimal toxicity.
Assuntos
Insuficiência Cardíaca , Proteínas Proto-Oncogênicas , Animais , Apoptose , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 11 Semelhante a Bcl-2/farmacologia , Fibroblastos/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
BCL-2 family proteins regulate the mitochondrial apoptotic pathway. BOK, a multidomain BCL-2 family protein, is generally believed to be an adaptor protein similar to BAK and BAX, regulating the mitochondrial permeability transition during apoptosis. Here we report that BOK is a positive regulator of a key enzyme involved in uridine biosynthesis; namely, uridine monophosphate synthetase (UMPS). Our data suggest that BOK expression enhances UMPS activity, cell proliferation, and chemosensitivity. Genetic deletion of Bok results in chemoresistance to 5-fluorouracil (5-FU) in different cell lines and in mice. Conversely, cancer cells and primary tissues that acquire resistance to 5-FU down-regulate BOK expression. Furthermore, we also provide evidence for a role for BOK in nucleotide metabolism and cell cycle regulation. Our results have implications in developing BOK as a biomarker for 5-FU resistance and have the potential for the development of BOK-mimetics for sensitizing 5-FU-resistant cancers.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Uridina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Mamíferos , Camundongos , Complexos Multienzimáticos/metabolismo , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Neutrophils are rapidly deployed innate immune cells, and excessive recruitment is causally associated with influenza-induced pathologic conditions. Despite this, the complete set of influenza lethality-associated neutrophil effector proteins is currently unknown. Whether the expression of these proteins is predetermined during bone marrow (BM) neutrophil maturation or further modulated by tissue compartment transitions has also not been comprehensively characterized at a proteome-wide scale. In this study, we used high-resolution mass spectrometry to map how the proteomes of murine neutrophils change comparatively across BM, blood, and the alveolar airspaces to deploy an influenza lethality-associated response. Following lethal influenza infection, mature neutrophils undergo two infection-dependent and one context-independent compartmental transitions. Translation of type I IFN-stimulated genes is first elevated in the BM, preceding the context-independent downregulation of ribosomal proteins observed in blood neutrophils. Following alveolar airspace infiltration, the bronchoalveolar lavage (BAL) neutrophil proteome is further characterized by a limited increase in type I IFN-stimulated and metal-sequestering proteins as well as a decrease in degranulation-associated proteins. An influenza-selective and dose-dependent increase in antiviral and lipid metabolism-associated proteins was also observed in BAL neutrophils, indicative of a modest capacity for pathogen response tuning. Altogether, our study provides new and comprehensive evidence that the BAL neutrophil proteome is shaped by BM neutrophil maturation as well as subsequent compartmental transitions following lethal influenza infection.
Assuntos
Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteômica/métodos , Animais , Células da Medula Óssea/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Rapidly identifying cachexia-inducing factors that directly induce muscle wasting is an existing challenge. We developed two reporter cell lines that allow swift detection of such factors in blood from patients. C2C12 myoblasts were used for the establishment of reporter cells. A luciferase reporter gene, driven by promoters of wasting genes, Muscle RING-finger protein-1 (MuRF1) and Muscle Atrophy F-Box Protein (MAFbx/Atrogin-1) were used for the construction of reporter constructs. Increased expression of these genes in muscle tissue under wasting conditions was shown in vivo and in vitro. We found these reporter cell lines could detect factors associated with cancer cachexia, such as myostatin (Mstn), activin A, and TNF-α. We further investigated the capacity to directly detect a cachectic state using plasma samples from cachectic mice and cancer patients. Activation of the reporter cell lines was observed by the addition of plasma from mice with cancer cachexia and serum samples from patients with pancreatic or colorectal cancer. These results indicate that the reporter cell lines are competent as a tool for screening cachexia-inducing factors and potentially distinguishing a cachectic state induced by cancer.
Assuntos
Caquexia/sangue , Caquexia/genética , Atrofia Muscular/sangue , Atrofia Muscular/genética , Neoplasias/complicações , Ativinas/metabolismo , Animais , Caquexia/diagnóstico , Caquexia/etiologia , Linhagem Celular Transformada , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Muscular/diagnóstico , Atrofia Muscular/etiologia , Mioblastos/metabolismo , Miostatina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The role of the immunoproteasome is perceived as confined to adaptive immune responses given its ability to produce peptides ideal for MHC Class-I binding. Here, we demonstrate that the immunoproteasome subunit, LMP2, has functions beyond its immunomodulatory role. Using LMP2-deficient mice, we demonstrate that LMP2 is crucial for lymphocyte development and survival in the periphery. Moreover, LMP2-deficient lymphocytes show impaired degradation of key BH3-only proteins, resulting in elevated levels of pro-apoptotic BIM and increased cell death. Interestingly, LMP2 is the sole immunoproteasome subunit required for BIM degradation. Together, our results suggest LMP2 has important housekeeping functions and represents a viable therapeutic target for cancer.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/imunologia , Cisteína Endopeptidases/imunologia , Linfócitos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Animais , Western Blotting , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Cisteína Endopeptidases/deficiência , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/deficiênciaRESUMO
Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/patologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteína 11 Semelhante a Bcl-2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Transposable elements (TEs) have been active in the mammalian genome for millions of years and the silencing of these elements in the germline is important for the survival of the host. Mice carrying reporter transgenes can be used to model transcriptional silencing. A mutagenesis screen for modifiers of epigenetic gene silencing produced a line with a mutation in Trim33; the mutants displayed increased expression of the reporter transgene. ChIP-seq of Trim33 in testis revealed 9,109 peaks, mostly at promoters. This is the first report of ChIP-seq for Trim33 in any tissue. Comparison with ENCODE datasets showed that regions of high read density for Trim33 had high read density for histone marks associated with transcriptional activity and mapping to TE consensus sequences revealed Trim33 enrichment at RLTR10B, the LTR of one of the youngest retrotransposons in the mouse genome, MMERVK10C. We identified consensus sequences from the 266 regions at which Trim33 ChIP-seq peaks overlapped RLTR10B elements and found a match to the A-Myb DNA-binding site. We found that TRIM33 has E3 ubiquitin ligase activity for A-MYB and regulates its abundance. RNA-seq revealed that mice haploinsufficient for Trim33 had altered expression of a small group of genes in the testis and the gene with the most significant increase was found to be transcribed from an upstream RLTR10B. These studies provide the first evidence that A-Myb has a role in the actions of Trim33 and suggest a role for both A-Myb and Trim33 in the arms race between the transposon and the host. This the first report of any factor specifically regulating RLTR10B and adds to the current literature on the silencing of MMERVK10C retrotransposons. This is also the first report that A-Myb has a role in the transcription of any retrotransposon.
Assuntos
Inativação Gênica , Retroelementos/genética , Testículo/metabolismo , Fatores de Transcrição/metabolismo , Animais , Genoma , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas c-myb/metabolismo , Retroviridae/genética , Sequências Repetidas Terminais , Transativadores/metabolismo , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.
Assuntos
Apoptose/fisiologia , Conformação Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células Cultivadas , Dimerização , Dissulfetos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium.
Assuntos
Antígenos de Protozoários , Eritrócitos , Fígado , Proteínas de Membrana , Camundongos Endogâmicos BALB C , Proteínas de Protozoários , Animais , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Feminino , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Humanos , Eritrócitos/parasitologia , Eritrócitos/imunologia , Fígado/parasitologia , Fígado/imunologia , Fígado/metabolismo , Vacinas Antimaláricas/imunologia , Malária/imunologia , Malária/parasitologia , Malária/prevenção & controle , Reações Cruzadas/imunologia , Plasmodium falciparum/imunologia , Plasmodium berghei/imunologia , Epitopos/imunologia , Hepatócitos/parasitologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Plasmodium/imunologia , Merozoítos/imunologia , Merozoítos/metabolismoRESUMO
Deregulated ß-adrenoceptor/cAMP-PKA pathway is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. We recently had demonstrated that the ß-adrenoceptor/cAMP-PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member BIM in tissues, such as the thymus and the heart. Induction of BIM is driven by the transcriptional co-activator CBP (CREB Binding Protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the BIM promoter site [Lee et al., Cell Death Difference 20(7):941-952 (2013)]. However since CBP is a co-factor for multiple transcription factors, BH-3 only proteins other than Bim could also contribute to this apoptosis pathway. Here we provide evidence for the involvement of p53-CBP axis in apoptosis through Puma/Noxa induction, in response to ß-adrenoceptor activation. Our findings highlight the molecular complexity of pathophysiology associated with a deregulated neuro-endocrine system and for developing novel therapeutic strategies for these diseases.
Assuntos
Apoptose , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Camundongos , Proto-Oncogene Mas , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Animais , Proteína 11 Semelhante a Bcl-2 , Humanos , Camundongos , Fosforilação , Ligação Proteica , Estabilidade Proteica , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Monepantel is an anti-helminthic drug that also has anti-cancer properties. Despite several studies over the years, the molecular target of monepantel in mammalian cells is still unknown, and its mechanism-of-action is not fully understood, though effects on cell cycle, mTOR signalling and autophagy have been implicated. METHODS: Viability assays were performed on >20 solid cancer cell cells, and apoptosis assays were performed on a subset of these, including 3D cultures. Genetic deletion of BAX/BAK and ATG were used to establish roles of apoptosis and autophagy in killing activity. RNA-sequencing was performed on four cell lines after monepantel treatment, and differentially regulated genes were confirmed by Western blotting. RESULTS: We showed that monepantel has anti-proliferative activity on a broad range of cancer cell lines. In some, this was associated with induction of apoptosis which was confirmed using a BAX/BAK-deficient cell line. However, proliferation is still inhibited in these cells following monepantel treatment, indicating cell-cycle disruption as the major anti-cancer effect. Previous studies have also indicated autophagic cell death occurs following monepantel treatment. We showed autophagy induction in multiple cell lines; however, deletion of a key autophagy regulator ATG7 had minimal impact on monepantel's anti-proliferative activity, suggesting autophagy is associated with, but not required for its anti-tumour effects. Transcriptomic analysis of four cell lines treated with monepantel revealed downregulation of many genes involved in the cell cycle, and upregulation of genes linked to ATF4-mediated ER stress responses, especially those involved in amino-acid metabolism and protein synthesis. CONCLUSIONS: As these outcomes are all associated with mTOR signalling, cell cycle and autophagy, we now provide a likely triggering mechanism for the anti-cancer activity of monepantel.
Assuntos
Estresse do Retículo Endoplasmático , Neoplasias , Animais , Humanos , Proteína X Associada a bcl-2 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Apoptose , Serina-Treonina Quinases TOR/metabolismo , Autofagia , Linhagem Celular Tumoral , Mamíferos/metabolismoRESUMO
Cellular apoptosis is a key pathological mechanism contributing to neuronal death following ischemic stroke. The pro-apoptotic Bcl-2 family protein, Bim, is an important regulator of apoptosis. In this study we investigated the effect of Bim expression on post-stroke functional outcomes, brain injury and inflammatory mechanisms. Wild type (WT) and Bim-deficient mice underwent 1-h middle cerebral artery occlusion (MCAO) followed by 23 h of reperfusion. At 24-h post-stroke, we assessed functional deficit, infarct volume, immune cell death, as well as the number of infiltrating immune cells in the brain and circulating immune cells. Bim deficiency did not affect infarct volume (P > 0.05), but resulted in less motor impairment (~ threefold greater latency to fall in hanging grip strength test, P < 0.05) and a lower median clinical score than WT mice (P < 0.05). Additionally following MCAO, Bim-deficient mice exhibited fewer myeloid cells (particularly neutrophils) in the ischemic brain hemisphere and less apoptosis of CD3+ T cells in the spleen and thymus compared with WT (all P < 0.05). After MCAO, Bim-deficient mice also tended to have more M2-polarised macrophages in the brain than WT mice. In sham-operated mice, we found that Bim deficiency resulted in greater numbers of circulating total CD45+ leukocytes, Ly6Clo+ monocytes and CD3+ T cells, although MCAO did not affect the number of circulating cells at 24 h in either genotype. Our findings suggest that Bim deficiency modulates post-stroke outcomes, including reductions in motor impairment, brain inflammation and systemic post-stroke leukocyte apoptosis. Bim could therefore serve as a potential therapeutic target for stroke.
Assuntos
Proteína 11 Semelhante a Bcl-2 , Isquemia Encefálica , AVC Isquêmico , Animais , Camundongos , Apoptose/genética , Encéfalo , Isquemia Encefálica/complicações , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Inflamação/genética , Inflamação/complicações , AVC Isquêmico/patologia , Camundongos Endogâmicos C57BL , Deleção de Genes , Proteína 11 Semelhante a Bcl-2/genéticaRESUMO
Inflammation is a natural defence mechanism of the body to protect against pathogens. It is induced by immune cells, such as macrophages and neutrophils, which are rapidly recruited to the site of infection, mediating host defence. The processes for eliminating inflammatory cells after pathogen clearance are critical in preventing sustained inflammation, which can instigate diverse pathologies. During chronic inflammation, the excessive and uncontrollable activity of the immune system can cause extensive tissue damage. New therapies aimed at preventing this over-activity of the immune system could have major clinical benefits. Here, we investigated the role of the pro-survival Bcl-2 family member A1 in the survival of inflammatory cells under normal and inflammatory conditions using murine models of lung and peritoneal inflammation. Despite the robust upregulation of A1 protein levels in wild-type cells upon induction of inflammation, the survival of inflammatory cells was not impacted in A1-deficient mice compared to wild-type controls. These findings indicate that A1 does not play a major role in immune cell homoeostasis during inflammation and therefore does not constitute an attractive therapeutic target for such morbidities.
Assuntos
Peritonite , Pneumonia , Animais , Apoptose/fisiologia , Sobrevivência Celular , Inflamação/patologia , CamundongosRESUMO
BACKGROUND: Developing therapies for cancer cachexia has not been successful to date, in part due to the challenges of achieving robust quantitative measures as a readout of patient treatment. Hence, identifying biomarkers to assess the outcomes of treatments for cancer cachexia is of great interest and important for accelerating future clinical trials. METHODS: We established a novel xenograft model for cancer cachexia with a cachectic human PC3* cell line, which was responsive to anti-Fn14 mAb treatment. Using RNA-seq and secretomic analysis, genes differentially expressed in cachectic and non-cachectic tumors were identified and validated by digital droplet PCR (ddPCR). Correlation analysis was performed to investigate their impact on survival in cancer patients. RESULTS: A total of 46 genes were highly expressed in cachectic PC3* tumors, which were downregulated by anti-Fn14 mAb treatment. High expression of the top 10 candidates was correlated with low survival and high cachexia risk in different cancer types. Elevated levels of LCN2 were observed in serum samples from cachectic patients compared with non-cachectic cancer patients. CONCLUSION: The top 10 candidates identified in this study are candidates as potential biomarkers for cancer cachexia. The diagnostic value of LCN2 in detecting cancer cachexia is confirmed in patient samples.
RESUMO
In many cell types polarized transport directs the movement of mRNAs and proteins from their site of synthesis to their site of action, thus conferring cell polarity. The cytoplasmic dynein microtubule motor complex is involved in this process. In Drosophila melanogaster, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for the transport of macromolecules to the oocyte and to the apical surface of the blastoderm embryo. Hence, Egl and BicD, which have been shown to associate, may be part of a conserved core localization machinery in Drosophila, although a direct association between these molecules and the dynein motor complex has not been shown. Here we report that Egl interacts directly with Drosophila dynein light chain (Dlc), a microtubule motor component, through an Egl domain distinct from that which binds BicD. We propose that the Egl-BicD complex is loaded through Dlc onto the dynein motor complex thereby facilitating transport of cargo. Consistent with this model, point mutations that specifically disrupt Egl-Dlc association also disrupt microtubule-dependant trafficking both to and within the oocyte, resulting in a loss of oocyte fate maintenance and polarity. Our data provide a direct link between a molecule necessary for oocyte specification and the microtubule motor complex, and supports the hypothesis that microtubule-mediated transport is important for preserving oocyte fate.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Dineínas/metabolismo , Oócitos/fisiologia , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Dineínas/química , Dineínas/genética , Feminino , Humanos , Microtúbulos/metabolismo , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Oócitos/citologia , Oogênese , Ovário/citologia , Ovário/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Subpopulações de Linfócitos B/citologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mitógenos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Subpopulações de Linfócitos T/citologia , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/imunologia , Células Cultivadas , Fragmentos Fab das Imunoglobulinas/farmacologia , Ionomicina/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Wistar , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.
Assuntos
Apresentação de Antígeno/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Macroautofagia/genética , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Brefeldina A/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macroautofagia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , TransfecçãoRESUMO
Sepsis remains to be a major contributor to mortality in ICUs, and immune suppression caused by immune cell apoptosis determines the overall patient survival. However, diagnosis of sepsis-induced lymphopenia remains problematic with no accurate prognostic techniques or biomarkers for cell death available. Developing reliable prognostic tools for sepsis-mediated cell death is not only important for identifying patients at increased risk of immune suppression but also to monitor treatment progress of currently trialed immunotherapy strategies. We have previously shown an important role for endoplasmic reticulum stress (ER stress) in inducing sepsis-mediated cell death and here report on the identification of a secreted form of the ER chaperone BiP (immunoglobulin binding protein) as a novel circulating prognostic biomarker for immune cell death and ER stress during sepsis. Using biochemical purification and mass spectrometry coupled with an established in vitro sepsis cell death assay, we identified BiP/Grp78 as a factor secreted by lipopolysaccharide-activated macrophages that is capable of inducing cell death in target cells. Quantitative ELISA analysis showed significantly elevated levels of circulating BiP in mice undergoing polymicrobial sepsis, which was absent in Bim-/- mice that are protected from sepsis-induced lymphopenia. Using blood serum from human sepsis patients, we could detect a significant difference in levels of secreted BiP in sepsis patients compared to nonseptic controls, suggesting that secreted circulating BiP could indeed be used as a prognostic marker that is directly correlative to immune cell death during sepsis.