RESUMO
We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La(3+)- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca(2+) concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca(2+) concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Calsequestrina/metabolismo , Proteínas de Membrana/metabolismo , Fibras Musculares de Contração Lenta/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Calsequestrina/genética , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Camundongos , Contração Muscular , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismoRESUMO
The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Canais de Cálcio Tipo L/genética , Complexos Multiproteicos/genética , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Serina-Treonina Quinases TOR/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Potenciais Evocados/fisiologia , Acoplamento Excitação-Contração/fisiologia , Regulação da Expressão Gênica , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Contração Isométrica , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Proteína Regulatória Associada a mTOR , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Membrana/deficiência , Força Muscular/fisiologia , Animais , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Calsequestrina , Regulação da Expressão Gênica , Técnicas In Vitro , Manganês/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologiaRESUMO
The decline in muscular strength with age is disproportionate to the loss in total muscle mass that causes it. Knocking out JP45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), results in decreased expression of the voltage-gated Ca(2+) channel, Ca(v)1.1; excitation-contraction uncoupling (ECU); and loss of muscle force (Delbono et al., 2007). Here, we show that Ca(v)1.1 expression, charge movement, SR Ca(2+) release, in vitro contractile force, and sustained forced running remain stable in male JP45KO mice at 12 and 18 months. They also exhibit the level of ECU reported for 3-4-month mice (Delbono et al., 2007). No further decline at later ages was recorded. Preserved ECC was not related to increased expression of any protein that directly or indirectly interacts with JP45 at the triad junction. However, maintained muscle force and physical performance were associated with ablation of JP45 expression in the brain, spontaneous and significantly diminished food intake and less tendency toward obesity when exposed to a high-fat diet compared to WT. We propose that (1) endogenously generated restriction in food intake overcomes the deleterious effects of JP45 ablation on ECC and skeletal muscle force mainly through downregulation of neuropeptide-Y expression in the hypothalamic arcuate nucleus; and (2) the JP45KO mouse constitutes an invaluable model to examine the mechanisms controlling food intake as well as skeletal muscle function with aging.
Assuntos
Envelhecimento/fisiologia , Restrição Calórica , Ingestão de Alimentos/fisiologia , Proteínas de Membrana/deficiência , Força Muscular/fisiologia , Envelhecimento/sangue , Animais , Peso Corporal/fisiologia , Canais de Cálcio Tipo L/metabolismo , Dieta Hiperlipídica , Hipocampo/metabolismo , Insulina/sangue , Leptina/sangue , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/metabolismoRESUMO
AIMS: The endoplasmic reticulum (ER) is involved in many functions, including protein folding, redox homeostasis, and Ca(2+) storage and signaling. To perform these multiple tasks, the ER is composed of distinct, specialized subregions, amongst which mitochondrial-associated ER membranes (MAM) emerge as key signaling hubs. How these multiple functions are integrated with one another in living cells remains unclear. RESULTS: Here we show that Ero1α, a key controller of oxidative folding and ER redox homeostasis, is enriched in MAM and regulates Ca(2+) fluxes. Downregulation of Ero1α by RNA interference inhibits mitochondrial Ca(2+) fluxes and modifies the activity of mitochondrial Ca(2+) uniporters. The overexpression of redox active Ero1α increases passive Ca(2+) efflux from the ER, lowering [Ca(2+)](ER) and mitochondrial Ca(2+) fluxes in response to IP3 agonists. INNOVATION: The unexpected observation that Ca(2+) fluxes are affected by either increasing or decreasing the levels of Ero1α reveals a pivotal role for this oxidase in the early secretory compartment and implies a strict control of its amounts. CONCLUSIONS: Taken together, our results indicate that the levels, subcellular localization, and activity of Ero1α coordinately regulate Ca(2+) and redox homeostasis and signaling in the early secretory compartment.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Linhagem Celular , Homeostase , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Oxirredução , Oxirredutases/genética , Ligação Proteica , Transdução de SinaisRESUMO
In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²âº released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Musculares/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Dados de Sequência Molecular , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/isolamento & purificação , Músculo Esquelético/metabolismo , NAD/metabolismo , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Distribuição TecidualRESUMO
BACKGROUND: Activation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive. RESULTS: Here we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca²âº)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca²âºconcentration [Ca²âº] difference between bulk cytosolic and the sub-plasma membrane rim. CONCLUSION: This approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane.Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation.Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.
Assuntos
Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Medições Luminescentes , Proteínas de Membrana/metabolismo , Equorina/genética , Equorina/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , Cálcio/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Transporte Proteico , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-ArrestinasRESUMO
The biogenesis of secretory IgM occurs stepwise under stringent quality control, formation of micro(2)L(2) preceding polymerization. How is efficiency of IgM secretion coupled to fidelity? We show here that ERp44, a soluble protein involved in thiol-mediated retention, interacts with ERGIC-53. Binding to this hexameric lectin contributes to ERp44 localization in the ER-golgi intermediate compartment. ERp44 and ERGIC-53 increase during B-lymphocyte differentiation, concomitantly with the onset of IgM polymerization. Both preferentially bind micro(2)L(2) and higher order intermediates. Their overexpression or silencing in non-lymphoid cells promotes or decreases secretion of IgM polymers, respectively. In IgM-secreting B-lymphoma cells, micro chains interact first with BiP and later with ERp44 and ERGIC-53. Our findings suggest that ERGIC-53 provides a platform that receives micro(2)L(2) subunits from the BiP-dependent checkpoint, assisting polymerization. In this process, ERp44 couples thiol-dependent assembly and quality control.
Assuntos
Linfócitos B/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Cadeias mu de Imunoglobulina/biossíntese , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Retículo Endoplasmático/genética , Chaperona BiP do Retículo Endoplasmático , Inativação Gênica , Complexo de Golgi/genética , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genéticaRESUMO
Formation of disulfide bonds, an essential step for the maturation and exit of secretory proteins from the endoplasmic reticulum (ER), is controlled by specific ER-resident enzymes. A pivotal element in this process is Ero1alpha, an oxidoreductin that lacks known ER retention motifs. Here we show that ERp44 mediates Ero1alpha ER localization through the formation of reversible mixed disulfides. ERp44 also prevents the secretion of an unassembled cargo protein with unpaired cysteines. We conclude that ERp44 is a key element in thiol-mediated retention. It might also favour the maturation of disulfide-linked oligomeric proteins and their quality control.