Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576104

RESUMO

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.


Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fenômenos Biofísicos , Sinalização do Cálcio , Citosol/metabolismo , Células HeLa , Humanos , Cinética
2.
Methods Mol Biol ; 2275: 187-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118039

RESUMO

Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes .


Assuntos
Quelantes de Cálcio/química , Cálcio/análise , Fura-2/química , Mitocôndrias/química , Ciclosporina , Citosol/química , Células HeLa , Humanos , Microscopia de Fluorescência
3.
Aging Clin Exp Res ; 33(6): 1705-1708, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31606858

RESUMO

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.


Assuntos
Doença de Alzheimer , Idoso , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Homeostase , Humanos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo
4.
Front Neurosci ; 14: 547746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33177972

RESUMO

The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.

5.
Cells ; 9(10)2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32992716

RESUMO

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-ß (Aß) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).


Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Membrana Celular/genética , Flavina-Adenina Dinucleotídeo/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/genética , Presenilina-2/metabolismo
6.
Adv Exp Med Biol ; 1131: 881-900, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646538

RESUMO

Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca2+ signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca2+ signaling molecules and the disclosure of conserved Ca2+ signaling pathways. In this review, we will analyze the applications of Ca2+ imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.


Assuntos
Cálcio , Drosophila melanogaster , Animais , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Cálcio/metabolismo , Drosophila melanogaster/fisiologia , Humanos , Modelos Animais
7.
Angew Chem Int Ed Engl ; 58(29): 9917-9922, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132197

RESUMO

Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 µm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.


Assuntos
Cálcio/metabolismo , Corantes Fluorescentes/uso terapêutico , Mitocôndrias/metabolismo , Corantes Fluorescentes/metabolismo , Humanos
8.
Front Physiol ; 10: 1544, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31920731

RESUMO

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.

9.
Calcif Tissue Int ; 93(1): 39-47, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23494409

RESUMO

Few studies have provided information on the prevalence of vertebral fractures (VFs) and their risk factors in hemodialysis patients. A multicenter, cross-sectional, observational study was carried out to assess the prevalence of VFs and vascular calcifications (VCs) in 387 hemodialysis patients (mean age 64.2 ± 14.1 years, 63 % males) and in a control group of 51 osteoporotic subjects. Biochemical tests included 25(OH) vitamin D, bone Gla protein (total and undercarboxylated), and total matrix Gla protein. Vertebral quantitative morphometry was carried out centrally for the detection of VF, defined as reduction by ≥20 % of one of the vertebral body dimensions. In the same radiograph, aortic and iliac VC scores were calculated. Prevalence of VF was 55.3 % in hemodialysis patients and 51.0 % in the control group. Multivariate analysis disclosed that male gender (59.8 vs. 47.6 %, p = 0.02; OR = 1.78, 95 % CI 1.15-2.75) and age (mean ± SD 66.7 ± 13.1 vs. 61.0 ± 14.7 years, p < 0.001; OR = 1.03, 95 % CI 1.01-1.05) were significantly associated with VF. The prevalence of aortic VC was significantly higher in hemodialysis patients than in controls (80.6 vs. 68.4 %, p = 0.001). The factors with the strongest association with VC, apart from atrial fibrillation, were serum 25(OH)vitamin D levels below 29 ng/mL for aortic VC (OR = 1.85, 95 % CI 1.04-3.29) and VF both for aortic (OR = 1.77, 95 % CI 1.00-3.14) and iliac (OR = 1.96, 95 % CI 1.27-3.04) VC. In conclusion, the prevalence of VF, especially in males, and VC, in both genders, is high in hemodialysis patients. VF is associated with VC. Vitamin D deficiency is also associated with VC. Further longitudinal studies are warranted to investigate fractures in renal patients.


Assuntos
Fraturas da Coluna Vertebral/epidemiologia , Calcificação Vascular/complicações , Adulto , Idoso , Aorta Abdominal/patologia , Estudos Transversais , Feminino , Humanos , Artéria Ilíaca/patologia , Masculino , Pessoa de Meia-Idade , Prevalência , Diálise Renal , Fraturas da Coluna Vertebral/etiologia , Calcificação Vascular/patologia , Vitamina D/sangue
10.
J Bone Miner Res ; 27(11): 2271-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22692665

RESUMO

Vitamin K (vitamin K1 or phylloquinone and vitamin K2, a series of menaquinones [MKs]) is involved in the production of bone and matrix amino acid γ-carboxy-glutamic acid (Gla) proteins, regulating bone and vascular calcification. Low vitamin K concentrations are associated with increased risks of fractures and vascular calcification, and frequent complications in hemodialysis patients. We carried out an observational study to establish the prevalence of vitamin K deficiency and to assess the relationship between vitamin K status, vertebral fractures, vascular calcification, and survival in 387 patients on hemodialysis for ≥1 year. We determined plasma levels of vitamin K compound, bone-Gla-protein, matrix-Gla-protein, and routine biochemistry. Vertebral fractures (reduction in vertebral body height by ≥20%) and aortic and iliac calcifications were also investigated in a spine (D(5) -L(4)) radiograph. Three-year patient survival was analyzed. Important proportions of patients had deficiency of MK7 (35.4%), vitamin K1 (23.5%), and MK4 (14.5%). A total of 55.3% of patients had vertebral fractures, 80.6% had abdominal aorta calcification, and 56.1% had iliac calcification. Vitamin K1 deficiency was the strongest predictor of vertebral fractures (odds ratio [OR], 2.94; 95% confidence interval [CI], 1.38-6.26). MK4 deficiency was a predictor of aortic calcification (OR, 2.82; 95% CI, 1.14-7.01), whereas MK5 deficiency actually protected against it (OR, 0.38; 95% CI, 0.15-0.95). MK7 deficiency was a predictor of iliac calcification (OR, 1.64; 95% CI, 1.03-2.60). The presence of vertebral fractures was also a predictor of vascular calcifications (OR, 1.76; 95% CI, 1.00-3.08). Increased alkaline phosphatase and C reactive protein (CRP), age, and cerebrovascular events were predictors of mortality. Our study suggests that the vitamin K system may be important for preserving bone mass and avoiding vascular calcification in hemodialysis patients, pointing out a possible role of vitamin K in bone and vascular health. Based on our results, we suggest that the general population should also be studied for vitamin K deficiency as a possible cause of both vertebral fractures and vascular calcification.


Assuntos
Diálise Renal , Fraturas da Coluna Vertebral/complicações , Fraturas da Coluna Vertebral/mortalidade , Calcificação Vascular/complicações , Calcificação Vascular/mortalidade , Vitamina K/sangue , Adulto , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Itália/epidemiologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fraturas da Coluna Vertebral/sangue , Análise de Sobrevida , Calcificação Vascular/sangue
11.
Blood ; 116(1): 54-62, 2010 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-20395415

RESUMO

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1-infected cells. ROS measurements showed an increasing gradient from resting to mitogen-activated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of "normal" infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas dos Retroviridae/metabolismo , Linfócitos T/metabolismo , Linhagem Celular , Células Cultivadas , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Lentivirus/genética , Microscopia Confocal , Mitocôndrias/metabolismo , Oxirredução , Interferência de RNA , Proteínas dos Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/virologia , Transdução Genética
12.
Apoptosis ; 14(2): 153-63, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19089619

RESUMO

Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.


Assuntos
Apoptose , Citoproteção , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Apoptose/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citoproteção/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Fosfatos de Poli-Isoprenil/farmacologia , Prenilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Proteína X Associada a bcl-2/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo
13.
Exp Cell Res ; 312(8): 1390-400, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16483570

RESUMO

Defects in the regulation of programmed cell death play a fundamental role in the development of neoplasia and neurological disorders, both of which are linked to the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection. We previously showed that the HTLV-1 Tax protein protects from apoptosis induced by serum starvation by preventing cytochrome c release and Bax relocation to mitochondria, two early events in the mitochondrial apoptotic pathway. As a natural extension of these findings, and to better define the action of Tax, in the present study, we investigated the outcome of Tax and two mutants which are inactive in CREB/ATF (M47) or NF-kappaB (M22) pathways, in the control of apoptosis induced by the proapoptotic Bax protein. We found that activation of CREB, rather than NF-kappaB, is a key phenomenon in preventing apoptosis. Furthermore, the importance of CREB activation is strengthened by experiments with CREB mutants, treatment with forskolin, and in situ analysis of P-CREB status in cells transfected with Tax or its nonprotecting M47 mutant. Considered together, these results underscore a primary role of CREB in preventing apoptosis triggered by Bax, and suggest that Tax might act by affecting the phosphorylation state of CREB.


Assuntos
Apoptose/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Produtos do Gene tax/metabolismo , Infecções por HTLV-I/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Fatores de Transcrição/fisiologia , Apoptose/genética , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene tax/genética , Infecções por HTLV-I/genética , Infecções por HTLV-I/fisiopatologia , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Mutação/fisiologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...