RESUMEN
The plasma membrane Ca2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It has 10 transmembrane helices and 2 cytosolic loops, one of which contains the catalytic center. Its most distinctive feature is a C-terminal tail that contains most of the regulatory sites including that for calmodulin. The pump is also regulated by acidic phospholipids, kinases, a dimerization process, and numerous protein interactors. In mammals, four genes code for the four basic isoforms. Isoform complexity is increased by alternative splicing of primary transcripts. Pumps 2 and 3 are expressed preferentially in the nervous system. The pumps coexist with more powerful systems that clear Ca2+ from the bulk cytosol: their role is thus the regulation of Ca2+ in selected subplasma membrane microdomains, where a number of important Ca2+-dependent enzymes interact with them. Malfunctions of the pump lead to disease phenotypes that affect the nervous system preferentially.
Asunto(s)
Calcio/metabolismo , Células/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Señalización del Calcio , Humanos , Modelos Biológicos , Isoformas de Proteínas/metabolismoRESUMEN
Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca(2+) homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca(2+) uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca(2+) homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.
Asunto(s)
Autofagia , Señalización del Calcio , Complejo I de Transporte de Electrón/genética , Fibroblastos/fisiología , Proteínas Mitocondriales/genética , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Complejo I de Transporte de Electrón/metabolismo , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mutación Puntual , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
OBJECTIVE: Anaemia is a common complication of chronic kidney disease (CKD), particularly in dialysis patients. The recent European guidelines for anaemia treatment in CKD indicate the percentage of hypochromic red cells (%HYPO) and reticulocyte haemoglobin content (CHr) calculated by Siemens ADVIA haematology analysers as a useful tool indicating iron deficiency. The aim of this study was to evaluate the agreement between CHr and %HYPO parameters and the reticulocyte haemoglobin equivalent (RET-He) and red blood cell haemoglobin equivalent (RBC-He) calculated by the Sysmex XE-2100 haematology analyser in a cohort of 200 dialysis patients referred to the Nephrology Unit of our hospital. Furthermore, we evaluated a new index, the DF-Hypo XE, obtained from haemoglobin (Hb), haematocrit (Hct) and RET-He, provided by the Sysmex XE-2100, as a new potential marker of %HYPO in dialysed patients. MATERIAL AND METHODS: Blood samples collected in EDTA anticoagulant from 200 CKD patients receiving erythropoietin and iron to maintain haemoglobin level between 10 and 12 mg/dL were analysed on both the Siemens ADVIA 2120 and the Sysmex XE-2100 within 2 h of collection. RESULTS: There was good correlation between CHr and RET-He (r = 0.88; p<0.0001), %HYPO and DF-Hypo XE (r = 0.89; p<0.0001) and between RBC-He and CH (r = 0.96; p<0.0001), but there was a lower correlation, even though statistically significant, between RBC-He and %HYPO (r = -0.59; p<0.0001). The Altman-Bland analysis showed a very good level of agreement between CHr and RET-He (mean bias = 1.04 pg), %HYPO and DF-Hypo XE (mean bias = 1.73). Using a cut-off value of 29.4 pg for the RET-He and of 10.2 for the DF-Hypo XE, 15 out 17 patients with a CHr <29.0 pg and 9 out 11 patients with a %Hypo <10.0% were respectively correctly identified. CONCLUSIONS: Our study shows good correlation and agreement between CHr and RET-He and between %HYPO and DF-Hypo XE in evaluating CKD patients needing iron support.
Asunto(s)
Anemia Ferropénica/sangre , Índices de Eritrocitos , Enfermedades Renales/sangre , Recuento de Reticulocitos , Adulto , Anciano , Anemia Ferropénica/complicaciones , Anemia Ferropénica/diagnóstico , Biomarcadores/sangre , Estudios de Cohortes , Humanos , Enfermedades Renales/complicaciones , Persona de Mediana EdadRESUMEN
In mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Sordera/metabolismo , Enfermedades Genéticas Congénitas/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/genética , Membrana Celular/metabolismo , Sordera/genética , Enfermedades Genéticas Congénitas/genética , Humanos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.
Asunto(s)
Membrana Celular/metabolismo , Sordera/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , Animales , Células CHO , Calcio/metabolismo , Cóclea/metabolismo , Cricetinae , Cricetulus , Salud de la Familia , Femenino , Células Ciliadas Auditivas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación Missense , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Estructura Terciaria de ProteínaRESUMEN
B-cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disease caused by impaired apoptosis regulation that leads to an abnormal survival and an accumulation of B-lymphocytes. Anti-apoptotic Bcl-2 and proapoptotic Bax proteins are involved in the highly regulated mechanism of cell death. Bax and Bcl-2 intracellular levels were analyzed both in CD19+ and CD3+ cells from 28 B-CLL de novo patients and compared with cells from healthy donors. Our results were expressed as a ratio (Bax/Bcl-2) obtained by dividing Bax mean fluorescence intensity (MFI) and Bcl-2 MFI; obviously, a lower ratio is associated with an anti-apoptotic status, while a higher index correlates to apoptosis activation. In CD19+ B-CLL cells, the Bax/Bcl-2 ratio was lower than in the CD19+ normal counterpart (1.3 versus 3.51; P<.05), mainly due to a Bcl-2 over expression (17.65 versus 9.02; P<.001). In CD3+ cells from B-CLL patients, the Bax/Bcl-2 ratio was lower than in normal CD3+ cells (7.89 versus 8.96; P<.005), most importantly as a result of Bax suppression (77.22 versus 96.63; P<.001). These study data show an apoptosis inhibition not only in CD19+ cells, but also in CD3+ cells, suggesting a pivotal role of T-cells in B-CLL pathogenesis.
Asunto(s)
Apoptosis/fisiología , Linfocitos B/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Linfocitos T/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Adulto , Anciano , Antígenos CD19 , Linfocitos B/patología , Complejo CD3 , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana EdadRESUMEN
Many of the published reports of reference values in neonates are found in older medical literature. Recognition of abnormalities in blood cell morphology and hematologic parameters depend on well-established normative data; it is essential that each neonatal medical unit have its own reference ranges. We give the umbilical cord blood complete blood count reference values from 142 healthy, appropriate for gestational age (AGA) newborns and 58 small for gestational age (SGA) newborns (term and preterm). Our data, obtained by automated blood cell counter analysis of umbilical cord blood samples taken at birth, are comparable to other previously published data. The correlation between previous data and our reference data confirms that in term AGA newborns, values for red blood cells, hemoglobin, and hematocrit are higher and mean corpuscular volume values lower than in preterm AGA newborns. Also, we found that platelet levels are reduced in SGA newborns, in accordance with the literature. These findings further support the fact that preterm SGA infants are truly growth restricted, whereas term SGA infants are most likely small but otherwise healthy babies.
Asunto(s)
Sangre Fetal/citología , Pruebas Hematológicas/normas , Recién Nacido de Bajo Peso/sangre , Nacimiento Prematuro/sangre , Recuento de Células Sanguíneas/normas , Edad Gestacional , Humanos , Recién Nacido , Estándares de Referencia , Valores de ReferenciaRESUMEN
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is commonly overlooked or misdiagnosed owing to its recent identification. It is characterized clinically by recurrent cerebral infarcts, usually appearing between the ages of 30 and 50 years, subcortical dementia, and pseudobulbar palsy. It begins with migraine with aura in approximately one-third of patients. The pathological hallmark of angiopathy is the presence of characteristic granular osmiophilic material (GOM) within the basal lamina of smooth muscle cells. The defective gene in CADASIL is Notch3, which encodes a large transmembrane receptor, and 70% of missense mutations are in exons 3 and 4. Each gene defect leads to either a gain or loss of a cysteine residue in the extracellular N-terminal domain of the molecule. We report the case of a 53-year-old woman admitted to the hospital for transient ischemic attack and stroke-like episodes recurrent since age 43 years. The patient had pseudobulbar palsy, pyramidal signs, and cognitive impairment but not frank dementia. Cerebral MRI showed periventricular diffuse and confluent ischemic lesions. Ultrastructural study revealed an abnormal deposition of granular osmiophilic material (GOM) within the basal lamina in skin capillaries. Direct sequence analysis of the Notch3 gene was performed. Since no mutation was detected in exons 3 and 4, the remaining exons were sequenced and a missense mutation, CGC-TGC in codon 1006 of exon 19 was found. The mutation led to a gain of a cysteine residue. This is the first missense mutation in codon 1006 of exon 19 of the Notch3 gene to be described in Italy and the second reported in the literature.
Asunto(s)
Codón , Demencia por Múltiples Infartos/genética , Demencia por Múltiples Infartos/patología , Mutación Missense , Proteínas Proto-Oncogénicas/genética , Receptores de Superficie Celular , Encéfalo/patología , Exones , Salud de la Familia , Femenino , Humanos , Italia/etnología , Imagen por Resonancia Magnética/métodos , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Receptor Notch3 , Receptores Notch , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.
Asunto(s)
Apoptosis , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Empalme del ARN , Adulto , Secuencia de Aminoácidos , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/genética , Clonación Molecular , Dimerización , Expresión Génica , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Distribución Tisular , Células Tumorales CultivadasRESUMEN
In the course of evolution, Ca2+ has emerged as the most versatile intracellular messenger. Its concentration within cells is controlled by reversible binding to specific classes of proteins that act as Ca2+ sensors to decode its information before passing it on to targets. The decoding operation is based on specific conformational changes in the sensor proteins. Other proteins intrinsic to membranes simply control Ca2+ concentration without processing its message, by transporting it across membrane boundaries. They are located in the plasma membrane and in the membranes of the organelles (the endo(sarco)plasmic reticulum, the mitochondria, the nuclear envelope), which play distinctive roles in the cellular homeostasis of Ca2+. Ca2+ is an ambivalent signaling agent. It carries information to virtually all processes important to cell life (e.g., it couples excitation to contraction, secretion, gene transcription, and controls enzyme activity through protein phosphorylation-dephosphorylation), but also transmits signals that promote the programmed demise of cells. When escaping control, Ca2+ also precipitates toxic cell death.
Asunto(s)
Señalización del Calcio/fisiología , Animales , Proteínas de Unión al Calcio/fisiología , Membrana Celular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Transporte IónicoRESUMEN
The dynamic interactions of the main pathways for active Ca(2+) transport have been analysed in living cells by altering the expression of their components. The plasma membrane (PMCA) and the endoplasmic reticulum (ER) (SERCA) Ca(2+) pumps were transiently overexpressed in CHO cells, and the Ca(2+) homeostasis in the subcellular compartments was investigated using specifically targeted chimaeras of the Ca(2+)- sensitive photoprotein aequorin. In resting cells, overexpression of the PMCA and SERCA pumps caused a reduction and an increase in ER [Ca(2+)] levels, respectively, while no significant differences were detected in cytosolic and mitochondrial [Ca(2+)]. Upon stimulation with an inositol 1,4, 5-trisphosphate (IP(3))-generating agonist, the amplitude of the mitochondrial and cytosolic Ca(2+) rises correlated with the ER [Ca(2+)] only up to a threshold value, above which the feedback inhibition of the IP(3) channel by Ca(2+) appeared to be limiting.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Adenosina Trifosfato/metabolismo , Aequorina/química , Aequorina/metabolismo , Animales , Western Blotting , Células CHO , Cloruro de Calcio/farmacología , Proteínas de Transporte de Catión , Membrana Celular/metabolismo , Cricetinae , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/farmacología , Fura-2/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Microscopía Fluorescente , Mitocondrias/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Plásmidos/metabolismo , Isoformas de Proteínas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transducción de Señal , TransfecciónRESUMEN
Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca(2+)-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria 'buy time' by precipitating inside Ca2+ and phosphate. If injury persists, Ca2(+)-death eventually ensues.
Asunto(s)
Calcio/fisiología , Transducción de Señal , Animales , Humanos , Transporte Iónico , Sistemas de Mensajero SecundarioRESUMEN
OBJECTIVE: Intercellular adhesion molecule 1 (ICAM-1) is widely distributed in shoulder synovial membrane of active polymyalgia rheumatica (PMR) and strongly expressed in granulomatous inflammatory infiltrate of the temporal artery in giant cell arteritis (GCA). ICAM-1 genes may contribute to the inflammatory PMR/GCA processes. We examined potential associations of ICAM-1 gene polymorphisms with PMR/GCA susceptibility and severity. METHODS: We enrolled 121 consecutive patients with "pure" PMR and 56 patients with biopsy positive GCA residing in Reggio Emilia, Italy. Among patients with PMR, 91 had a followup duration of at least one year. Selected as control subjects were 228 healthy blood donors, 75 patients with nonarteritic central retinal artery occlusion, and 116 cataract surgery patients from the same geographic area. All PMR/GCA patients and controls were genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for ICAM-1 polymorphism at codon 241 (exon 4) and codon 469 (exon 6). RESULTS: The frequency of R241 was significantly higher in PMR/GCA patients [p = 0.00001, odds ratio (OR) 5.0 (95% confidence intervals, CI 2.6-9.6) ], in pure PMR patients [p = 0.00001, OR 5.0 (95% CI 2.5-10.0)], and in GCA patients [p = 0.00005; OR 5.0 (95% CI 2.2-11.5)] compared to the healthy controls. The frequency of R241 was significantly higher in total PMR/GCA patients compared to patients with nonarteritic central retinal artery occlusion [p = 0.0007; OR 5.3 (95% CI 1.8-15.5)] and cataract surgery patients [p = 0.0003; OR 4.1 (95% CI 1.8-9.0)]. The distribution of E/K 469 genotype was similar in PMR/GCA patients and in the 3 control groups. Cox proportional hazards modeling identified 2 variables that independently increased the risk of PMR relapse/recurrence: erythrocyte sedimentation rate at diagnosis > 72 mm/h [relative risk 1.6 (95% CI 1.1-2.3)] and the presence of R241 allele [relative risk 1.6 (95% CI 1.1-2.4)]. CONCLUSION: Our findings show that G/R 241 polymorphism of ICAM-1 is associated with PMR/GCA susceptibility and confers an increased risk of relapse/recurrence in PMR.
Asunto(s)
Arteritis de Células Gigantes/genética , Molécula 1 de Adhesión Intercelular/genética , Polimorfismo Genético , Polimialgia Reumática/genética , Alelos , Catarata/etiología , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Arteritis de Células Gigantes/metabolismo , Arteritis de Células Gigantes/fisiopatología , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Masculino , Persona de Mediana Edad , Polimialgia Reumática/metabolismo , Polimialgia Reumática/fisiopatología , Arteria Renal , Factores de Riesgo , Índice de Severidad de la Enfermedad , Trombosis/etiologíaRESUMEN
Eukaryotic cells remove calcium from the cytosol using P-type pumps in the plasma membrane and in the sarco(endo)plasmic reticulum. These pumps share membrane topography and general mechanism of action, but differ in regulatory properties. Recent advances in the field include the three-dimensional structure of the sarco(endo)plasmic reticulum and further understanding of the transcriptional regulation of the plasma membrane P-type pump by calcium.
Asunto(s)
Canales de Calcio/química , ATPasas Transportadoras de Calcio/química , Secuencia de Aminoácidos , Animales , Canales de Calcio Tipo P/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Isoformas de Proteínas/química , Estructura Secundaria de ProteínaRESUMEN
The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.
Asunto(s)
Calpaína/química , Músculo Esquelético/enzimología , Animales , Baculoviridae , Calcio/metabolismo , Calpaína/genética , Células Cultivadas , Clonación Molecular , Estabilidad de Enzimas , Humanos , Isoenzimas/metabolismo , Microscopía Fluorescente , Microscopía de Contraste de Fase , Proteínas Musculares/química , Músculo Esquelético/ultraestructura , Mutagénesis Sitio-Dirigida , Unión Proteica , Ratas , Proteínas Recombinantes/química , SpodopteraRESUMEN
In the last decade, the study of Ca2+ homeostasis within organelles in living cells has been greatly enhanced by the utilisation of a recombinant Ca(2+)-sensitive photoprotein, aequorin. Aequorin is a Ca2+ sensitive photoprotein of a coelenterate that, in the past, was widely employed to measure Ca2+ concentration in living cells. In fact, the purified protein was widely used to monitor cytoplasmic [Ca2+] changes in invertebrate muscle cells after microinjection. However, due to the time-consuming and traumatic procedure of microinjection, the role of aequorin in the study of Ca2+ homeostasis remained confined to a limited number of cells (giant cells) susceptible to microinjection. Thus, in most instances, it was replaced by the fluorescent indicators developed by Roger Tsien and coworkers. The cloning of aequorin cDNA [Inouye et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3154-3158] and the explosive development of molecular biology offered new possibilities in the use of aequorin, as microinjection has been replaced by the simpler technique of cDNA transfection. As a polypeptide, aequorin allows the endogenous production of the photoprotein in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localise it within the cell by including defined targeting signals in the amino acid sequence. Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles. In this review, we will not discuss the procedure of aequorin microinjection and its use as purified protein but we will present the new advances provided by recombinant aequorin in the study of intracellular Ca2+ homeostasis, discussing in greater detail the advantages and disadvantages in the use of this probe.
Asunto(s)
Aequorina/metabolismo , Calcio/análisis , Animales , Calcio/metabolismo , Retículo Endoplásmico/química , Homeostasis , Humanos , Mitocondrias/química , Proteínas Recombinantes/metabolismoRESUMEN
In recent years, genetic defects of the mitochondrial genome (mtDNA) were shown to be associated with a heterogeneous group of disorders, known as mitochondrial diseases, but the cellular events deriving from the molecular lesions and the mechanistic basis of the specificity of the syndromes are still incompletely understood. Mitochondrial calcium (Ca2+) homeostasis depends on close contacts with the endoplasmic reticulum and is essential in modulating organelle function. Given the strong dependence of mitochondrial Ca2+ uptake on the membrane potential and the intracellular distribution of the organelle, both of which may be altered in mitochondrial diseases, we investigated the occurrence of defects in mitochondrial Ca2+ handling in living cells with either the tRNALys mutation of MERRF (myoclonic epilepsy with ragged-red fibers) or the ATPase mutation of NARP (neurogenic muscle weakness, ataxia and retinitis pigmentosa). There was a derangement of mitochondrial Ca2+ homeostasis in MERRF, but not in NARP cells, whereas cytosolic Ca2+ responses were normal in both cell types. Treatment of MERRF cells with drugs affecting organellar Ca2+ transport mostly restored both the agonist-dependent mitochondrial Ca2+ uptake and the ensuing stimulation of ATP production. These results emphasize the differences in the cellular pathogenesis of the various mtDNA defects and indicate specific pharmacological approaches to the treatment of some mitochondrial diseases.
Asunto(s)
Señalización del Calcio/genética , ADN Mitocondrial , Encefalomiopatías Mitocondriales/metabolismo , Fosforilación Oxidativa , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Línea Celular , Clonazepam/análogos & derivados , Clonazepam/farmacología , Histamina/farmacología , Humanos , Síndrome MERRF/genética , Síndrome MERRF/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Encefalomiopatías Mitocondriales/genética , Oligomicinas/farmacología , ARN de Transferencia de Lisina/genética , Tiazepinas/farmacología , TransfecciónRESUMEN
The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.