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OBJECTIVES: Trauma predisposes to systemic sterile inflammation (systemic inflammatory response syndrome) as well as infection, but the mechanisms linking injury to infection are poorly understood. Mitochondrial debris contains formyl peptides. These bind formyl peptide receptor-1, trafficking neutrophils to wounds, initiating systemic inflammatory response syndrome, and wound healing. Bacterial formyl peptides, however, also attract neutrophils via formyl peptide receptor-1. Thus, mitochondrial formyl peptides might suppress neutrophils antimicrobial function. Also, formyl peptide receptor-1 blockade used to mitigate systemic inflammatory response syndrome might predispose to sepsis. We examined how mitochondrial formyl peptides impact neutrophils functions contributing to antimicrobial responses and how formyl peptide receptor-1 antagonists affect those functions. DESIGN: Prospective study of human and murine neutrophils and clinical cohort analysis. SETTING: University research laboratory and level 1 trauma center. PATIENTS: Trauma patients, volunteer controls. ANIMAL SUBJECTS: C57Bl/6, formyl peptide receptor-1, and formyl peptide receptor-2 knockout mice. INTERVENTIONS: Human and murine neutrophils functions were activated with autologous mitochondrial debris, mitochondrial formyl peptides, or bacterial formyl peptides followed by chemokines or leukotrienes. The experiments were repeated using formyl peptide receptor-1 antagonist cyclosporin H, "designer" human formyl peptide receptor-1 antagonists (POL7178 and POL7200), or anti-formyl peptide receptor-1 antibodies. Mouse injury/lung infection model was used to evaluate effect of formyl peptide receptor-1 inhibition. MEASUREMENTS AND MAIN RESULTS: Human neutrophils cytosolic calcium, chemotaxis, reactive oxygen species production, and phagocytosis were studied before and after exposure to mitochondrial debris, mitochondrial formyl peptides, and bacterial formyl peptides. Mitochondrial formyl peptide and bacterial formyl peptides had similar effects on neutrophils. Responses to chemokines and leukotrienes were suppressed by prior exposure to formyl peptides. POL7200 and POL7178 were specific antagonists of human formyl peptide receptor-1 and more effective than cyclosporin H or anti-formyl peptide receptor-1 antibodies. Formyl peptides inhibited mouse neutrophils responses to chemokines only if formyl peptide receptor-1 was present. Formyl peptide receptor-1 blockade did not inhibit neutrophils bacterial phagocytosis or reactive oxygen species production. Cyclosporin H increased bacterial clearance in lungs after injury. CONCLUSIONS: Formyl peptides both activate and desensitize neutrophils. Formyl peptide receptor-1 blockade prevents desensitization, potentially both diminishing systemic inflammatory response syndrome and protecting the host against secondary infection after tissue trauma or primary infection.
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Proteínas Mitocondriais/imunologia , Ativação de Neutrófilo/imunologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Animais , Ciclosporina/farmacologia , Humanos , Lesão Pulmonar/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções Respiratórias/fisiopatologiaRESUMO
Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.
Assuntos
DNA Mitocondrial , Doenças Mitocondriais , Fosforilação Oxidativa , Proibitinas , Humanos , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Masculino , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Doenças Mitocondriais/genética , Feminino , Adulto , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/genética , Transdução de Sinais , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
Mitochondrial dysfunction has been suggested to contribute to Parkinson's disease pathogenesis, though an understanding of the extent or exact mechanism of this contribution remains elusive. This has been complicated by challenging nature of pathway-based analysis and an inability simultaneously study multiple related proteins within human brain tissue. We used imaging mass cytometry (IMC) to overcome these challenges, measuring multiple protein targets, whilst retaining the spatial relationship between targets in post-mortem midbrain sections. We used IMC to simultaneously interrogate subunits of the mitochondrial oxidative phosphorylation complexes, and several key signalling pathways important for mitochondrial homoeostasis, in a large cohort of PD patient and control cases. We revealed a generalised and synergistic reduction in mitochondrial quality control proteins in dopaminergic neurons from Parkinson's patients. Further, protein-protein abundance relationships appeared significantly different between PD and disease control tissue. Our data showed a significant reduction in the abundance of PINK1, Parkin and phosphorylated ubiquitinSer65, integral to the mitophagy machinery; two mitochondrial chaperones, HSP60 and PHB1; and regulators of mitochondrial protein synthesis and the unfolded protein response, SIRT3 and TFAM. Further, SIRT3 and PINK1 did not show an adaptive response to an ATP synthase defect in the Parkinson's neurons. We also observed intraneuronal aggregates of phosphorylated ubiquitinSer65, alongside increased abundance of mitochondrial proteases, LONP1 and HTRA2, within the Parkinson's neurons with Lewy body pathology, compared to those without. Taken together, these findings suggest an inability to turnover mitochondria and maintain mitochondrial proteostasis in Parkinson's neurons. This may exacerbate the impact of oxidative phosphorylation defects and ageing related oxidative stress, leading to neuronal degeneration. Our data also suggest that that Lewy pathology may affect mitochondrial quality control regulation through the disturbance of mitophagy and intramitochondrial proteostasis.
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Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by reduced amounts of the mitochondrial protein frataxin. Frataxin levels in research studies are typically measured via Western blot analysis from patient fibroblasts, lymphocytes, or muscle biopsies; none of these is ideal for rapid detection in large scale clinical studies. Recently, a rapid, noninvasive lateral flow immunoassay was developed to accurately measure picogram levels of frataxin protein and shown to distinguish lymphoblastoid cells from FRDA carriers, patients and controls. We expanded the immunoassay to measure frataxin directly in buccal cells and whole blood from a large cohort of controls, known carriers and patients typical of a clinical trial population. The assay in buccal cells shared a similar degree of variability with previous studies conducted in lymphoblastoid cells (~10% coefficient of variation in controls). Significant differences in frataxin protein quantity were seen between the mean group values of controls, carriers, and patient buccal cells (100, 50.2, and 20.9% of control, respectively) and in protein extracted from whole blood (100, 75.3, and 32.2%, respectively), although there was some overlap between the groups. In addition, frataxin levels were inversely related to GAA repeat length and correlated directly with age of onset. Subjects with one expanded GAA repeat and an identified frataxin point mutation also carried frataxin levels in the disease range. Some patients displaying an FRDA phenotype but carrying only a single identifiable mutation had frataxin levels in the FRDA patient range. One patient from this group has a novel deletion that included exons 2 and 3 of the FXN gene based on multiplex ligation-dependent probe amplification (MLPA) analysis of the FXN gene. The lateral flow immunoassay may be a useful means to noninvasively assess frataxin levels repetitively with minimal discomfort in FRDA patients in specific situations such as clinical trials, and as a complementary diagnostic tool to aid in identification and characterization of atypical patients.
Assuntos
Ataxia de Friedreich/diagnóstico , Proteínas de Ligação ao Ferro/análise , Mucosa Bucal/citologia , Adolescente , Adulto , Criança , Feminino , Humanos , Imunoensaio/métodos , Masculino , Mucosa Bucal/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA+ corals had significantly higher mortality rates and faster disease progression. For VcpA- fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA+ fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.
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The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the beta-subunit. MAb3D5AB1 also binds to purified Escherichia coli F(1) with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F(1) ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy.
Assuntos
Angiostatinas/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , ATPases Mitocondriais Próton-Translocadoras/imunologia , Trifosfato de Adenosina/biossíntese , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Materiais Biomiméticos , Domínio Catalítico/imunologia , Bovinos , Membrana Corioalantoide/irrigação sanguínea , Neovascularização da Córnea/tratamento farmacológico , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Mapeamento de Epitopos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344RESUMO
Friedreich's Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials. Using recombinant frataxin we evaluated the accuracy and reproducibility of the assay. The assay measured recombinant human frataxin concentrations between 40 and 4000 pg/test or approximately 0.1-10 nM of sample. The intra and inter-assay error was <10% throughout the working range. To evaluate clinical utility of the assay we used genetically defined lymphoblastoid cells derived from FA patients, FA carriers and controls. Mean frataxin concentrations in FA patients and carriers were significantly different from controls and from one another (p=0.0001, p=0.003, p=0.005, respectively) with levels, on average, 29% (patients) and 64% (carriers) of the control group. As predicted, we observed an inverse relationship between GAA repeat number and frataxin protein concentrations within the FA patient cohort. The lateral flow immunoassay provides a simple, accurate and reproducible method to quantify frataxin protein in whole cell and tissue extracts, including primary samples obtained by non-invasive means, such as cheek swabs and whole blood. The assay is a novel tool for FA research that may facilitate improved diagnostic and prognostic evaluation of FA patients and could also be used to evaluate efficacy of therapies designed to cure FA by increasing frataxin protein levels.
Assuntos
Ataxia de Friedreich/diagnóstico , Heterozigoto , Imunoensaio/métodos , Proteínas de Ligação ao Ferro/metabolismo , Adolescente , Adulto , Idade de Início , Células Cultivadas , Criança , Estudos de Coortes , Feminino , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/análise , Proteínas de Ligação ao Ferro/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , FrataxinaRESUMO
BACKGROUND: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function. METHODS: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes. RESULTS: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs. CONCLUSION: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma. LEVEL OF EVIDENCE: Therapeutic, Level IV.
Assuntos
Quimiotaxia/efeitos dos fármacos , Neutrófilos/fisiologia , Peptídeos/sangue , Peptídeos/farmacologia , Ferimentos e Lesões/sangue , Cálcio/metabolismo , Células Cultivadas , Quimiocina CXCL1/farmacologia , Biologia Computacional , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Citosol/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Evolução Molecular , Humanos , Leucotrieno B4/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , N-Formilmetionina Leucil-Fenilalanina/química , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Peptídeos/química , Peptídeos/genética , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/metabolismo , Transdução de SinaisRESUMO
A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Proteínas/metabolismo , Animais , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Miocárdio/metabolismo , Ligação Proteica , Espectrometria de Massas em TandemRESUMO
COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membrane-embedded 13-subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments. Here, we introduce a simple, rapid, high-throughput 96-well plate protocol that uses a multiplex approach to determine the amount and activity of COX, which should find widespread use in evaluating the above diseases and in drug safety studies. Importantly, the method uses very small amounts of cell material or tissue and does not require the isolation of mitochondria. We show the utility of this approach by example of the analysis of fibroblasts from patients with COX activity deficiency and the effect of the antiretroviral drug ddC (2',3'-dideoxycytidine) on the biogenesis of the enzyme.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Extratos Celulares , Bases de Dados de Proteínas , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/química , Fibroblastos/química , Fibroblastos/citologia , Humanos , Imunoprecipitação , Espectrometria de Massas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Miocárdio/metabolismo , Especificidade de Órgãos , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Subunidades Proteicas/isolamento & purificação , Zalcitabina/toxicidadeRESUMO
The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281-1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.
Assuntos
Anticorpos Monoclonais , Doenças Mitocondriais/diagnóstico , Proteínas/análise , Proteômica/métodos , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Imuno-Histoquímica/métodos , Translocases Mitocondriais de ADP e ATP/análise , Translocases Mitocondriais de ADP e ATP/imunologia , Doenças Mitocondriais/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/imunologia , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/imunologiaRESUMO
The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.8-microm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.
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Citoesqueleto/fisiologia , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Técnicas Biossensoriais , Citoesqueleto/química , DNA Mitocondrial/química , Proteínas de Fluorescência Verde , Humanos , Membranas Intracelulares/química , Proteínas Luminescentes , Proteínas de Membrana/química , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Mitose , Complexo Piruvato Desidrogenase/química , Fase S , Células Tumorais CultivadasRESUMO
Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency. We also show that anti-E1 alpha and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2-5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1 alpha-subunit deficiency as a precursor to mutation analysis.
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Piruvato Desidrogenase (Lipoamida)/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Feminino , Fibroblastos/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Mosaicismo , Subunidades Proteicas , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/imunologia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/diagnóstico , Reprodutibilidade dos TestesRESUMO
Mitochondrial disorders can lead to a confusing array of symptoms, which frequently makes a diagnosis difficult. Traditional approaches to such diagnoses are based on enzyme activity assays, with further characterization provided by genetic analysis. However, these methods require relatively large sample sizes, are time-consuming, labor-intensive, and show variability between laboratories. Here, we report an immunocytochemical test that makes use of monoclonal antibodies to subunits from each of the oxidative phosphorylation complexes and pyruvate dehydrogenase to aid in the detection of mitochondrial disorders. It can be completed and data analyzed in less than 4 hr. We have used this test to study fibroblast cultures from patients with mitochondrial disorders arising from both mitochondrial DNA and nuclear DNA defects. We have also examined cases of Leigh syndrome arising from different genetic causes. We show that patients can be categorized on the basis of which complexes are affected and whether or not the defect being studied shows a mosaic distribution, an indicator of whether the causal mutation(s) is/are in the mitochondrial or nuclear genome. Immunocytochemical analysis as described here should be considered as an initial screen for mitochondrial disorders by which to direct (and limit) the subsequent enzymatic and genetic tests required to make an unambiguous diagnosis.
Assuntos
Doenças Mitocondriais/diagnóstico , Linhagem Celular , DNA Mitocondrial/genética , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Doença de Leigh/diagnóstico , Doença de Leigh/metabolismo , Doenças Mitocondriais/metabolismo , Mosaicismo , Mutação , Fosforilação , Complexo Piruvato Desidrogenase/metabolismoRESUMO
Monoclonal antibodies (mAbs) are important tools in the diagnosis and characterization of mitochondrial diseases. They can be used in immunohistochemical and/or Western blotting approaches to identify misassembled OXPHOS complexes or pyruvate dehydrogenase deficiencies where the intact complex is not formed which is the great majority of cases. The advantage of antibody based approaches is that they can be quantitative, require very small amounts of tissue sample and are fast, simple and relatively cheap to perform. Here we provide details of the mAbs currently available and describe optimized protocols for both immunohistochemistry using patient fibroblasts as well as Western blotting using either cell culture or biopsy material.
RESUMO
We are developing rapid immunoassays to measure the protein levels, enzymatic activities and post-translational modifications of mitochondrial proteins. These assays can be arrayed in multi-analyte panels for biomarker discovery and they can also be used individually at point of care where the level or activity of a small number proteins or even a single protein is highly informative. For example, we have characterized OXPHOS deficits associated with lipoatrophy, an adverse metabolic side-effect of anti-retroviral therapy, and have shown that OXPHOS deficits observed in vitro are also exhibited not only in clinically affected tissue (peripheral fat) but also in more easily accessible tissue (peripheral blood mononucleated cells). Similarly, we have shown that a small set of assays can be used to identify almost all patients with genetic deficits in OXPHOS complexes I or IV, the most common cause of inherited mitochondrial disease. Finally, we recently reported that Friedreich's Ataxia (FA) patients and carriers can be identified on the basis of a simple dipstick test to measure levels of a single protein, frataxin, an iron regulatory protein whose disrupted expression is the proximal cause of neurodegeneration in FA. Because each of these tests can be performed in an extremely simple, rapid dipstick format using non-invasive samples such as cheek swabs and fingerprick blood, they have potential for use as point of care diagnostics for mitochondrial disease and as front-line screening tools to help guide drug therapies and minimize adverse off-target drug effects.
Assuntos
Anticorpos , Doenças Mitocondriais/diagnóstico , Ataxia de Friedreich/diagnóstico , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/análise , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Modelos Biológicos , FrataxinaRESUMO
Drug-induced mitochondrial toxicity can occur as a result of inhibition of mitochondrial DNA (mtDNA) replication as with certain nucleoside reverse transcriptase inhibitors or inhibition of mtDNA-encoded protein synthesis as with certain antibacterials. Both types of dysfunction have the overall effect of reducing the level of proteins encoded by mtDNA. A lateral-flow immunoassay which measures the levels of both a mtDNA-encoded protein and a nuclear DNA-encoded protein allows simple and rapid determination of the ratio of these 2 proteins and, hence, identifies changes in mtDNA-encoded protein levels. Here, we describe an assay that compares the level of Complex IV (cytochrome c oxidase), a mitochondrial protein which has 3 subunits encoded by mtDNA and made by mitochondrial ribosomes, with that of frataxin, a protein encoded by nuclear DNA and made by cytosolic ribosomes. We tested a selection of antibacterials and antiretrovirals in cells and show that the ratio of Complex IV: frataxin decreases when a drug inhibits either mtDNA replication or mtDNA-encoded protein synthesis. The results obtained with the assay were confirmed by Western blotting and immunocytochemical analysis. The assay has high reproducibility, requires small amounts of sample, is quantitative, and is able to identify drugs which ultimately lead to a decrease in mtDNA-encoded proteins.
Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Antibacterianos/farmacologia , Antirretrovirais/farmacologia , Linhagem Celular , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Humanos , Imunoensaio , Proteínas de Ligação ao Ferro/biossíntese , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Reprodutibilidade dos Testes , FrataxinaRESUMO
High levels of free radicals produced by the mitochondrial respiratory chain, with subsequent damage to mitochondria have been implicated in a large and growing number of diseases. The underlying pathology of these diseases is oxidative damage to mitochondrial DNA, lipids and proteins which accumulate over time to produce a metabolic deficiency. We are developing an antibody based immunocapture array for many important mitochondrial proteins involved in free radical production, detoxification and mitochondrial energy production. Our array is capable of a multi-parameter measurement including enzyme activity, quantity, and oxidative protein modifications. Here we demonstrate the use of this array by analyzing the proteomic differences in OXPHOS (oxidative phosphorylation) enzymes between human heart and liver tissues, cells grown in media promoting aerobic versus anaerobic metabolism, and the catalytic/proteomic effects of mitochondria exposed to oxidative stress. Protein oxidation is identified as carbonyl formation arising from reactive oxygen species and 3-nitrotyrosine as a marker of reactive nitrogen species. Several identified modifications are confirmed by electrophoresis and mass spectrometry of immunocaptured material. We continue to expand this array as antibodies for enzyme isolation and detection become available.
Assuntos
Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Estresse Oxidativo/fisiologia , Proteoma/metabolismo , Tirosina/análogos & derivados , Anticorpos Monoclonais , Antioxidantes/uso terapêutico , Biomarcadores/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Metabolismo dos Lipídeos , Fígado/metabolismo , Doenças Mitocondriais/tratamento farmacológico , Miocárdio/metabolismo , Fosforilação Oxidativa , Análise Serial de Proteínas , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Tirosina/metabolismoRESUMO
Depletion of mitochondrial DNA (mtDNA) and mtDNA-encoded respiratory chain proteins in subcutaneous (SC) fat from patients with HIV lipoatrophy have clearly demonstrated the role of mitochondrial dysfunction in this syndrome. Research in HIV lipoatrophy, however, has been severely hampered by the lack of a suitable surrogate marker in blood or other easily obtained clinical specimens as fat biopsies are invasive and mtDNA levels in peripheral blood mononuclear cells (PBMC) do not consistently correlate with the disease process. We used a simple, rapid, quantitative 2-site dipstick immunoassay to measure OXPHOS enzymes Complex I (CI) and Complex IV (CIV), and rtPCR to measure mtDNA in 26 matched SC fat and PBMC specimens previously banked from individuals on potent antiretroviral (ARV) therapy with HIV lipoatrophy, on similar ARV therapy without lipoatrophy, and in HIV seronegative controls. Significant correlations were found between the respective PBMC and fat levels for both CI (r = 0.442, p = 0.024) and for CIV (r = 0.507, p = 0.008). Both CI and CIV protein levels were also significantly reduced in both PBMCs and fat in lipoatrophic subjects compared to HIV seronegative controls (p < or = 0.05), while a comparative reduction in mtDNA levels in lipoatrophic subjects was observed only in fat. We conclude that CI and CIV levels in PBMCs correlate to their respective levels in fat and may have utility as surrogate markers of mitochondrial dysfunction in lipoatrophy.
Assuntos
Infecções por HIV/complicações , Síndrome de Lipodistrofia Associada ao HIV/diagnóstico , Leucócitos Mononucleares/química , Proteínas Mitocondriais/análise , Proteínas/metabolismo , Gordura Subcutânea/química , Adulto , DNA Mitocondrial/análise , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Reação em Cadeia da Polimerase/métodos , Estatística como AssuntoRESUMO
Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.