RESUMO
Parkinson's disease (PD) is a common neurodegenerative disorder which affects dopaminergic neurons leading to alteration of numerous cellular pathways. Several reports highlight that PD disturbs also other cells than CNS neurons including PBMCs, which could lead, among other things, to dysfunctions of immune functions. Because autophagy could be altered in PD, a monocentric pilot study was performed to quantify the transcripts levels of several autophagy genes in blood cells. MAP1LC3B, GABARAP, GABARAPL1, GABARAPL2 and P62/SQSTM1 were found to be overexpressed in patients. On the contrary, transcripts for HSPA8 and GAPDH were both decreased. Expression of MAP1LC3B and GABARAP was able to successfully segregate PD patients from healthy controls. The accuracy of this segregation was substantially increased when combined expressions of MAP1LC3B and GAPDH or GABARAP and GAPDH were used as categorical variables. This pilot study suggests that autophagy genes expression is dysregulated in PD patients and may open new perspectives for the characterisation of prediction markers.
Assuntos
Autofagia/genética , Doença de Parkinson/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/genética , Biomarcadores/sangue , Neurônios Dopaminérgicos/metabolismo , Feminino , França , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , Leucócitos Mononucleares , Aprendizado de Máquina , Masculino , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Projetos Piloto , Proteína Sequestossoma-1/genéticaRESUMO
OBJECTIVE: We measure the transcript levels of the proapoptotic GALIG, antiapoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV-infected patients effectively treated with combined antiretroviral therapy (cART). DESIGN: Monocentric pilot study conducted on peripheral blood mononuclear cell (PBMC) of 40 uninfected donors and 27 HIV-positive patients effectively treated by cART for at least 8.4 years. METHODS: Transcripts of the various genes were quantified by reverse transcription (RT)-quantitative PCR (qPCR) and RT-droplet digital PCR and compared using the standard statistical Mann-Whitney U test and machine learning algorithms. RESULTS: A concomitant overexpression of GALIG and MCL1 is detected in PBMC of effectively cART-treated patients. Overexpression of MAP1LC3B and GABARAPL1 is also measured, whereas BECN1 is underexpressed. Finally, accurate classification (94.5%) of our PBMC samples as HIV-negative donors or HIV-positive cART-treated is obtained in three separate machine-learning algorithms with GABARAPL1 and ATG9a as input variables. CONCLUSION: cART-treated HIV patients display altered transcript levels for three genes of basal autophagy. Some of these alterations may appear contradictory: BECN1 and ATG9a, both key actors in the formation of mammalian autophagosome, exhibit decreased amount of transcripts, whereas mRNA from the ATG8 family increase. Given the known role of impaired basal autophagy in immune senescence and chronic inflammation, the functional significance of our findings should be explored in larger studies.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Apoptose , Autofagia , Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Leucócitos Mononucleares/patologia , Terapia Antirretroviral de Alta Atividade , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
GALIG, an internal gene to the human galectin-3 gene, encodes two distinct proteins, Mitogaligin and Cytogaligin through translation of a unique mRNA in two overlapping alternative reading frames. When overexpressed GALIG induces apoptosis. In cultured cells, Mitogaligin destabilizes mitochondria membranes through interaction with cardiolipin. Little is known regarding the role of Cytogaligin. This protein displays multiple subcellular localizations; cytosol, nucleus, and mitochondria. We illustrate here that Cytogaligin is also secreted in the extracellular medium. Cytogaligin is shown to interact with α-Synuclein, the major component of Lewy bodies in Parkinson's disease. Overexpression of Cytogaligin reduces α-Synuclein dimerization raising a possible role in the evolution of α-Synuclein aggregation, a key molecular event underlying the pathogenesis of Parkinson's disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Líquido Extracelular/metabolismo , Galectinas/metabolismo , Frações Subcelulares/metabolismo , alfa-Sinucleína/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose , Células HeLa , Humanos , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
Objective There appears to be differences in the clinical presentation of hereditary angioedema (HAE) and angiotensin-converting enzyme inhibitor-induced (ACE-I) angioedema (AE). The aim of this study was to compare the clinical characteristics of these two AE forms. Methods We conducted a retrospective study of consecutive patients with HAE or ACE-I AE. The attack characteristics experienced by the patients were compared by a logistic regression analysis using generalized estimating equations. Results A total of 56 patients were included in this study (ACE-I AE, n=25; HAE, n=31). A total of 534 attacks were documented. Severe attacks were more common in the patients who had an acute episode of ACE-I AE than HAE. Swelling of the tongue, lips and larynx were significantly associated with ACE-I AE [OR: 8.70 (95% CI, 1.04-73.70), OR: 20.4 (95% CI, 4.9-84.2) and OR: 7.50 (95% CI, 1.20-48.30), respectively]. Conclusion Swelling of the tongue, lips and larynx are significantly more frequent in drug-induced AE than HAE.
Assuntos
Angioedema/patologia , Angioedemas Hereditários/patologia , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Laringe/patologia , Lábio/patologia , Língua/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Angioedema/induzido quimicamente , Angioedema/diagnóstico , Angioedemas Hereditários/diagnóstico , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Feminino , Humanos , Imunossupressores/efeitos adversos , Laringe/efeitos dos fármacos , Lábio/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Língua/efeitos dos fármacos , Adulto JovemRESUMO
GALIG gene expression induces apoptosis in cultured cells through a pathway still under investigation. It is highly expressed in leukocytes but weakly detectable in bone marrow, suggesting a role in the myeloid lineage homeostasis. We show here that GALIG-induced cell death is counteracted by the overexpression of MCL-1, a pro-survival member of the Bcl2 family. Moreover, during spontaneous neutrophil apoptosis, a substantial increase in GALIG gene expression is observed: GALIG still opposes MCL-1. Finally, in bone marrow and peripheral blood cells from patients with Acute Myeloid Leukemia type 2, the level of GALIG transcripts is massively down-regulated when compared to their normal counterparts, while MCL-1 is expressed to the same extent. These data suggest that GALIG could be a key player in the cell death pathway involved in leukocytes homeostasis and myeloid malignancies.
Assuntos
Apoptose/genética , Proteínas Sanguíneas/genética , Galectinas/genética , Leucemia Mieloide Aguda/genética , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Sanguíneas/metabolismo , Células da Medula Óssea/metabolismo , Sobrevivência Celular/genética , Galectinas/metabolismo , Regulação Leucêmica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/metabolismo , Microscopia de Fluorescência , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Mitogaligin, a protein encoded by galig, an internal cytotoxic gene of the galectin-3 locus, is mostly a mitochondrial protein. Mitochondrial targeting is due to an already identified mitochondrial localization signal. Interaction of mitogaligin with mitochondria leads to cytochrome c cytosolic leakage and ultimately to cell death. We have previously pointed out that mitogaligin can also be directed to the nucleus when the mitochondrial addressing signal is inactivated, indicating a possible dual intracellular localization of the protein. When expressed in the nucleus, mitogaligin exhibits also apoptotic properties leading to cell death. In this report, we show that nuclear addressing of mitogaligin depends on a sequence differing from classical signals containing basic, lysine or proline-tyrosine rich residues. The signal consists of a long sequence of amino acids residues based on a series of a short repetitive degenerated sequence.
Assuntos
Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Galectinas/metabolismo , Sinais de Localização Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Galectinas/química , Galectinas/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Sequential detections of different proteins on Western blot save time and precious samples. The main problem concerning reprobing is that stripping buffers can unbind both the antibody and the tested antigen. An original reprobing method has been set up based on horseradish peroxidase (HRP) inhibition after enhanced chemiluminescence detection. Instead of removing previously fixed antibodies as common stripping buffers do, the HRP activity linked to the secondary antibody is irreversibly inhibited by excess of hydrogen peroxide. A 15-min incubation allows one to perform at least five different sequential detections without losing significant amounts of blotted proteins.
Assuntos
Western Blotting/métodos , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/metabolismo , Sondas Moleculares/análise , Armoracia/enzimologia , Ativação Enzimática , Especificidade por SubstratoRESUMO
Galig, an internal gene to the galectin-3 gene, encodes two proteins and induces cell death in human cells. Mitogaligin, one of these proteins, contains a mitochondrial targeting sequence and promotes the release of cytochrome c into the cytosol. Here, we show that mitogaligin can also localize to nucleus. The nuclear form of mitogaligin induced cell death through a pathway exhibiting typical properties of apoptosis. These observations indicate for the first time that mitogaligin expresses cytotoxic properties not only when addressed to mitochondria but also when targeted to the nucleus.
Assuntos
Apoptose , Proteínas Sanguíneas/metabolismo , Núcleo Celular/metabolismo , Galectinas/metabolismo , Proteínas Sanguíneas/genética , Dano ao DNA , Galectinas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , Proteína X Associada a bcl-2/metabolismoRESUMO
Galig, a gene embedded within the galectin-3 gene, induces cell death when transfected in human cells. This death is associated with cell shrinkage, nuclei condensation, and aggregation of mitochondria. Galig contains two different overlapping open reading frames encoding two unrelated proteins. Previous observations have shown that one of these proteins, named mitogaligin, binds to mitochondria and promotes the release of cytochrome c. However, the mechanism of action of this cytotoxic protein remains still obscure. The present study provides evidence that synthetic peptides enclosing the mitochondrial localization signal of mitogaligin bind to anionic biological membranes leading to membrane destabilization, aggregation, and content leakage of mitochondria or liposomes. This binding to anionic phospholipids is the most efficient when cardiolipin, a specific phospholipid of mitochondria, is inserted in the membranes. Thus, cardiolipin may constitute a target of choice for mitogaligin sorting and membrane destabilization activity.
Assuntos
Cardiolipinas/química , Membrana Celular/química , Membrana Celular/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Sanguíneas/genética , Morte Celular/genética , Citocromos c/metabolismo , Citosol/metabolismo , Galectinas/genética , Células HeLa , Humanos , Lipossomos/metabolismo , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Ligação Proteica , TransfecçãoRESUMO
Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.