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1.
Biochim Biophys Acta Mol Cell Res ; 1870(1): 119366, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36191737

RESUMO

Intracellular Ca2+ signals are temporally controlled and spatially restricted. Signaling occurs adjacent to sites of Ca2+ entry and/or release, where Ca2+-dependent effectors and their substrates co-localize to form signaling microdomains. Here we review signaling by calcineurin, the Ca2+/calmodulin regulated protein phosphatase and target of immunosuppressant drugs, Cyclosporin A and FK506. Although well known for its activation of the adaptive immune response via NFAT dephosphorylation, systematic mapping of human calcineurin substrates and regulators reveals unexpected roles for this versatile phosphatase throughout the cell. We discuss calcineurin function, with an emphasis on where signaling occurs and mechanisms that target calcineurin and its substrates to signaling microdomains, especially binding of cognate short linear peptide motifs (SLiMs). Calcineurin is ubiquitously expressed and regulates events at the plasma membrane, other intracellular membranes, mitochondria, the nuclear pore complex and centrosomes/cilia. Based on our expanding knowledge of localized CN actions, we describe a cellular atlas of Ca2+/calcineurin signaling.


Assuntos
Calcineurina , Fatores de Transcrição NFATC , Humanos , Calcineurina/química , Calcineurina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Tacrolimo , Calmodulina/metabolismo , Transdução de Sinais
2.
Cell Rep ; 41(8): 111693, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36417851

RESUMO

Long intergenic noncoding RNAs (lincRNAs) are crucial regulators in numerous biological processes. However, the functions and mechanisms of m6A-modified lincRNAs in neuronal development remain unclear. Here, we report an m6A-modified lincRNA, Dppa2 upstream binding RNA (Dubr), abundantly expressed at the early developmental stage of dorsal root ganglion (DRG) and cerebral cortex. Silencing Dubr impairs axon elongation of DRG neurons and axon projection and migration of cortical neurons, whereas lacking m6A modification of Dubr fully loses its functions. Mechanically, Dubr interacts with m6A-binding proteins, the YTHDF1/3 complex, through its m6A motifs to protect YTHDF1/3 from degradation via the proteasome pathway. Furthermore, Tau and Calmodulin are regulated by YTHDF1/3 and m6A-modified Dubr. Overexpression of YTHDF1/3 not only rescues the reduced Tau and Calmodulin but also restores axon elongation of DRG neurons by Dubr knockdown. This study uncovers a critical role of m6A-modified lincRNA in neuronal development by regulating the degradation of RNA-binding protein.


Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Biossíntese de Proteínas , Calmodulina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neurônios/metabolismo
3.
Mol Cell Biol ; 42(11): e0017522, 2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36317924

RESUMO

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca2+/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca2+ channels, enabling Ca2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca2+ entry. We also show that Ca2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.


Assuntos
Proteínas de Ancoragem à Quinase A , Sinalização do Cálcio , Proteína ORAI1 , Molécula 1 de Interação Estromal , Humanos , Calcineurina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo
4.
Ying Yong Sheng Tai Xue Bao ; 33(10): 2725-2735, 2022 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-36384608

RESUMO

To explore whether there is an interaction between melatonin (MT) and calcium (Ca2+) in regulating heat tolerance of plants, we analyzed the response of endogenous MT and Ca2+ to heat stress, and examined the effect of MT and Ca2+ on the reactive oxygen (ROS) accumulation, antioxidant system, and transcripts of heat shock factor (HSF) and heat shock proteins (HSPs) of cucumber seedlings under high temperature stress. Seedlings were foliar sprayed with 100 µmol·L-1 MT, 10 mmol·L-1 CaCl2, 3 mmol·L-1 ethylene glycol tetraacetic acid (EGTA, Ca2+ chelating agent) +100 µmol·L-1 MT, 0.05 mmol·L-1 chlorpromazine (calmodulin antagonist, CPZ) +100 µmol·L-1 MT, 100 µmol·L-1 p-chlorophenylalanine (p-CPA, inhibitor of MT) +10 mmol·L-1 CaCl2 or deionized water (H2O), respectively. The results showed that both endogenous MT and Ca2+ in cucumber seedlings were induced by high temperature stress. The seedlings treated with exogenous MT showed significant increases in the mRNA expression of calmodulin (CaM), calcium-dependent protein kinase (CDPK5), calcineurin B-like protein (CBL3) and CBL interacting protein kinase (CIPK2) compared with the control at normal temperature. The mRNA levels of tryptophane decarboxylase (TDC), 5-hydroxytryptamine-N-acetyltransferase (SNAT) and N-acetyl-5-hydroxytryptamine methyltransferase (ASMT), key genes of MT biosynthesis and endogenous MT content were also induced by Ca2+ in cucumber seedlings. Exogenous MT and CaCl2 alleviated the heat-induced oxidative damage through increasing antioxidant ability, reducing the accumulation of reactive oxygen species (ROS), and upregulating the mRNA abundances of HSF7, HSP70.1 and HSP70.11, as evidenced by mild thermal damage symptoms, lower heat injury index and electrolyte leakage under heat stress. The positive effect of MT-induced antioxidant capacity and mRNA expression of HSPs was removed by adding EGTA and CPZ in stressed seedlings. Similarly, the mitigating role of Ca2+ in the peroxidation damage to high temperature stress was reversed by p-CPA. These results suggested that both MT and Ca2+ could induce heat tolerance of cucumber seedlings, which had crosstalk in the process of heat stress signal transduction.


Assuntos
Cucumis sativus , Melatonina , Cucumis sativus/genética , Melatonina/farmacologia , Cálcio , Antioxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Calmodulina/farmacologia , Ácido Egtázico/farmacologia , Cloreto de Cálcio/metabolismo , Cloreto de Cálcio/farmacologia , Temperatura , Estresse Fisiológico , Plântula/fisiologia , RNA Mensageiro/metabolismo , RNA Mensageiro/farmacologia
5.
Int J Mol Sci ; 23(22)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36430626

RESUMO

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565-587 and CaM2: residues 1120-1147) within the cytosolic domains of rod CNGB1. The binding of Ca2+-bound CaM to CNGB1 promotes the Ca2+-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca2+-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.


Assuntos
Calmodulina , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Calmodulina/genética , Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Nucleotídeos Cíclicos
6.
Int J Mol Sci ; 23(20)2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36293540

RESUMO

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer's disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-ß peptides (Aß) in AD. In recent works, we have proposed that Aß-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 µM Aß(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aß(1-42), respectively. Internalized Aß(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aß. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aß(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aß(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aß(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 µM Aß(1-42) oligomers. We conclude that Aß(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.


Assuntos
Cálcio , Calmodulina , Camundongos , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sinalização do Cálcio , Proteína ORAI1/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(42): e2211572119, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36215504

RESUMO

Activation of Ca2+/calmodulin-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a long accepted cellular model for learning and memory. However, how LTP and memories survive the turnover of synaptic proteins, particularly CaMKII, remains a mystery. Here, we take advantage of the finding that constitutive Ca2+-independent CaMKII activity, acquired prior to slice preparation, provides a lasting memory trace at synapses. In slice culture, this persistent CaMKII activity, in the absence of Ca2+ stimulation, remains stable over a 2-wk period, well beyond the turnover of CaMKII protein. We propose that the nascent CaMKII protein present at 2 wk acquired its activity from preexisting active CaMKII molecules, which transferred their activity to newly synthesized CaMKII molecules and thus maintain the memory in the face of protein turnover.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Calmodulina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Hipocampo/metabolismo , Aprendizagem/fisiologia , Potenciação de Longa Duração/fisiologia , Fosforilação , Sinapses/metabolismo
8.
Cell Rep ; 41(4): 111561, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36288705

RESUMO

Oral and esophageal squamous cell carcinomas (SCCs) are associated with high mortality, yet the molecular mechanisms underlying these malignancies are largely unclear. We show that DNA hypermethylation of otubain 2 (OTUB2), a previously recognized oncogene, drives tongue and esophageal SCC initiation and drug resistance. Mechanistically, OTUB2 promotes the deubiquitination and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and subsequently regulates the transcription of calmodulin-like protein 3 (CALML3). Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation (OXPHOS) and the synthesis of phosphatidylserine (PS). In mouse models, orally administered soybean-derived PS inhibits SCC initiation in cells with low OTUB2 expression and increases their sensitivity to chemotherapy. Our study indicates that the OTUB2/STAT1/CALML3/PS axis plays tumor-suppressive roles and shows the potential of PS administration as a strategy for the treatment and prevention of tongue and esophageal SCCs.


Assuntos
Calmodulina , Fosfatidilserinas , Animais , Camundongos , Calmodulina/metabolismo , Linhagem Celular Tumoral , DNA , Transdução de Sinais , Fator de Transcrição STAT1/metabolismo , Tioléster Hidrolases
9.
J Neurosci ; 42(42): 8019-8037, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36261266

RESUMO

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, we demonstrate the suppression of vapbP58S -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca2+ release channels or the inhibition Ca2+/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca2+]. We attribute the Ca2+ dyshomeostasis to delayed extrusion of cytosolic Ca2+ Suggesting that this defect in Ca2+ extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons.SIGNIFICANCE STATEMENT Whether the synchrony between the rates of ATP production and demand is lost in degenerating neurons remains poorly understood. We report that expression of a gene equivalent to an amyotrophic lateral sclerosis (ALS)-causing variant of vesicle-associated membrane protein B (VAPB) in fly neurons decouples mitochondrial ATP production from neuronal activity. Consequently, levels of ATP in mutant neurons are unable to keep up with the bioenergetic burden of neuronal activity. Reduced rate of Ca2+ extrusion, which could result from insufficient energy to power Ca2+ ATPases, results in the accumulation of residual Ca2+ in mutant neurons and leads to alterations in synaptic vesicle (SV) release and synapse development. These findings suggest that synaptic defects in a model of ALS arise from the loss of activity-induced ATP production.


Assuntos
Esclerose Amiotrófica Lateral , Masculino , Animais , Feminino , Esclerose Amiotrófica Lateral/metabolismo , Drosophila/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Neurônios Motores/metabolismo , Proteínas R-SNARE/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo
10.
FEBS Lett ; 596(22): 2974-2985, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36310389

RESUMO

Calmodulin (CaM) binds to the membrane-proximal cytosolic C-terminal domain of CaV 1.2 (residues 1520-1669, CT(1520-1669)) and causes Ca2+ -induced conformational changes that promote Ca2+ -dependent channel inactivation (CDI). We report biophysical studies that probe the structural interaction between CT(1520-1669) and CaM. The recombinantly expressed CT(1520-1669) is insoluble, but can be solubilized in the presence of Ca2+ -saturated CaM (Ca4 /CaM), but not in the presence of Ca2+ -free CaM (apoCaM). We show that half-calcified CaM (Ca2 /CaM12 ) forms a complex with CT(1520-1669) that is less soluble than CT(1520-1669) bound to Ca4 /CaM. The NMR spectrum of CT(1520-1669) reveals spectral differences caused by the binding of Ca2 /CaM12 versus Ca4 /CaM, suggesting that the binding of Ca2+ to the CaM N-lobe may induce a conformational change in CT(1520-1669).


Assuntos
Cálcio , Calmodulina , Calmodulina/metabolismo , Cálcio/metabolismo , Ligação Proteica
11.
Elife ; 112022 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-36196992

RESUMO

Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.


Assuntos
Sinalização do Cálcio , Cálcio , Animais , Camundongos , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Neurônios/fisiologia , Calmodulina/genética , Calmodulina/metabolismo
12.
J Biol Chem ; 298(11): 102579, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36220393

RESUMO

Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII "autonomous" activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents. Autonomy was reversed when the protein was incubated with DTT or thioredoxin to reduce disulfide bonds. Tryptic mapping of the activated CaMKII revealed formation of a disulfide between Cys273 and Cys290 in the regulatory domain. We determined the apparent pKa of those Cys and found that Cys273 had a low pKa while that of Cys290 was elevated. The low pKa of Cys273 facilitates oxidation of its thiol to the sulfenic acid at physiological pH. The reactive sulfenic acid then attacks the thiol of Cys290 to form the disulfide. The previously reported CaMKII mutant in which methionine residues 281 and 282 were mutated to valine (MMVV) protects mice and flies from cardiac decompensation induced by oxidative stress. Our initial hypothesis was that the MMVV mutant underwent a conformational change that prevented disulfide formation and autonomous activation. However, we found that the thiol-oxidizing agents induced autonomy in the MMVV mutant and that the mutant undergoes rapid degradation by the cell, potentially preventing accumulation of the injurious autonomous form. Together, our results highlight additional mechanistic details of CaMKII autonomous activation.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Cálcio , Camundongos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Dissulfetos/metabolismo , Calmodulina/metabolismo , Ácidos Sulfênicos , Oxirredução , Compostos de Sulfidrila , Metionina/metabolismo , Oxidantes , Estresse Oxidativo
13.
Cells ; 11(19)2022 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-36231128

RESUMO

The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related proteins participating in the actin remodeling, and their signaling pathways are unclear. We firstly examined how actin accumulates at a wound site in Dictyostelium cells. Actin assembled de novo at the wound site, independent of cortical flow. Next, we searched for actin- and signal-related proteins targeting the wound site. Fourteen of the examined proteins transiently accumulated at different times. Thirdly, we performed functional analyses using gene knockout mutants or specific inhibitors. Rac, WASP, formin, the Arp2/3 complex, profilin, and coronin contribute to the actin dynamics. Finally, we found that multiple signaling pathways related to TORC2, the Elmo/Doc complex, PIP2-derived products, PLA2, and calmodulin are involved in the actin dynamics for wound repair.


Assuntos
Actinas , Dictyostelium , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Calmodulina/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Forminas , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfolipases A2/metabolismo , Profilinas/genética , Profilinas/metabolismo , Transdução de Sinais
14.
PLoS Comput Biol ; 18(10): e1010583, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36206305

RESUMO

Calmodulin (CaM) is a calcium sensor which binds and regulates a wide range of target-proteins. This implicitly enables the concentration of calcium to influence many downstream physiological responses, including muscle contraction, learning and depression. The antipsychotic drug trifluoperazine (TFP) is a known CaM inhibitor. By binding to various sites, TFP prevents CaM from associating to target-proteins. However, the molecular and state-dependent mechanisms behind CaM inhibition by drugs such as TFP are largely unknown. Here, we build a Markov state model (MSM) from adaptively sampled molecular dynamics simulations and reveal the structural and dynamical features behind the inhibitory mechanism of TFP-binding to the C-terminal domain of CaM. We specifically identify three major TFP binding-modes from the MSM macrostates, and distinguish their effect on CaM conformation by using a systematic analysis protocol based on biophysical descriptors and tools from machine learning. The results show that depending on the binding orientation, TFP effectively stabilizes features of the calcium-unbound CaM, either affecting the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the bound domain. The conclusions drawn from this work may in the future serve to formulate a complete model of pharmacological modulation of CaM, which furthers our understanding of how these drugs affect signaling pathways as well as associated diseases.


Assuntos
Antipsicóticos , Calmodulina , Calmodulina/metabolismo , Trifluoperazina/farmacologia , Trifluoperazina/química , Trifluoperazina/metabolismo , Antipsicóticos/química , Cálcio/metabolismo , Ligação Proteica , Sítios de Ligação
15.
Cell Calcium ; 107: 102656, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36252447

RESUMO

The Ca2+-sensor protein calmodulin (CaM) is a major regulator of multiple cell functions. A unique and puzzling feature of human, and all so far investigated mammals, is the presence of three distinct CaM genes on different chromosomes, which code for identical proteins. How this case of apparent genetic redundancy evolved and why it could be to the advantage of the mammalian organisms is not well established. With a main focus on humans, this article aims to review existing literature addressing how the genes nonetheless differ in function. Clearly, the three CaM genes are differentially expressed in different tissues, during development, in response to different stimuli, and other factors including environmental conditions. As shown in hippocampal neurons, different mRNAs from the CAM genes may even localize differently within the same cell. Regulation of CaM gene expression is achieved by a variety of regulatory elements present in the three genes, including different promotor/insulator elements and 3'- and 5'-noncoding regions differing in length and sequence, as well as regulation by epigenetic factors and miRNAs. Here, we hypothesize that predicted differences in mRNA stability and translational efficiency due to divergent codon usage could play an additional regulatory role as the three genes differ markedly in their use of synonymous codons. CALM3, predicted to produce a relatively stable mRNA may be important where the transcription level is low or transiently absent, e.g. during spermatogenesis. In contrast, CALM2 with a predicted much shorter mRNA half-life, may provide better temporal control of CaM levels. Deciphering the underlying mechanisms responsible for all this complexity may help to understand why this unique multigenic arrangement may be an advantage for the optimal spatio-temporal expression of CaM in higher eukaryotes. Finally, we discuss the expression of the CaM genes in selected human pathologies, and how mutations in these genes are responsible for the appearance of serious congenital syndromes, mainly affecting the heart, and although less known, possibly also affecting the functionality of the central nervous system and other organs.


Assuntos
Calmodulina , Estabilidade de RNA , Animais , Humanos , Calmodulina/genética , Calmodulina/metabolismo , Códon , Uso do Códon , Mamíferos/metabolismo , MicroRNAs , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Microbiol Spectr ; 10(5): e0136322, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36214684

RESUMO

Toxoplasma gondii (T. gondii) is a pathogen belonging to the apicomplexan phylum, and it threatens human and animal health. Calcium ions, a critical second messenger in cells, can regulate important biological processes, including parasite invasion and egress. Calmodulin (CaM) is a small, highly conserved, Ca2+-binding protein found in all eukaryotic cells. After binding to Ca2+, CaM can be activated to interact with various proteins. However, little is known about CaM's function and its interacting proteins in T. gondii. In this study, we successfully knocked down CaM in the T. gondii parent strain TATI using a tetracycline-off system with the Toxoplasma CaM promoter. The results indicated that CaM was required for tachyzoite proliferation, invasion, and egress and that CaM depletion resulted in apicoplast loss, thus threatening parasite survival in the next lytic cycle. In the tachyzoite stage, CaM loss caused significant anomalies in the parasite's basal constriction, motility, and parasite rosette-like arrangement in the parasitophorous vacuole (PV). These phenotypic defects caused by CaM depletion indicate the importance of CaM in T. gondii. Therefore, it is important to identify the CaM-interacting proteins in T. gondii. Applying BioID technology, more than 300 CaM's proximal interacting proteins were identified from T. gondii. These CaM partners were broadly distributed throughout the parasite. Furthermore, the protein interactome and transcriptome analyses indicated the potential role of CaM in ion binding, cation binding, metal ion binding, calcium ion binding, and oxidation-reduction. Our findings shed light on the CaM function and CaM-interactome in T. gondii and other eukaryotes. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion. The implementation efficiency of T. gondii biological processes is dependent on signal transmission. Ca2+, as a second messenger, is essential for the parasite's life cycle. Calmodulin, a ubiquitous Ca2+ receptor protein, is highly conserved and mediates numerous Ca2+-dependent events in eukaryotes. Few CaM functions or regulated partners have been characterized in T. gondii tachyzoites. Here, we reported the essential functions of calmodulin in T. gondii tachyzoite and the identification of its interacting partners using BioID technology, shedding light on the CaM function and CaM-interactome in Toxoplasma gondii and other eukaryotes.


Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/genética , Calmodulina/genética , Calmodulina/metabolismo , Cálcio/metabolismo , Tecnologia , Tetraciclinas/metabolismo , Cátions/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
18.
Molecules ; 27(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36144710

RESUMO

NMDAR-dependent synaptic plasticity in the hippocampus consists of two opposing forces: long-term potentiation (LTP), which strengthens synapses and long-term depression (LTD), which weakens synapses. LTP and LTD are associated with memory formation and loss, respectively. Synaptic plasticity is controlled at a molecular level by Ca2+-mediated protein signaling. Here, Ca2+ binds the protein, calmodulin (CaM), which modulates synaptic plasticity in both directions. This is because Ca2+-bound CaM activates both LTD-and LTP-inducing proteins. Understanding how CaM responds to Ca2+ signaling and how this translates into synaptic plasticity is therefore important to understanding synaptic plasticity induction. In this paper, CaM activation by Ca2+ and calmodulin binding to downstream proteins was mathematically modeled using differential equations. Simulations were monitored with and without theoretical knockouts and, global sensitivity analyses were performed to determine how Ca2+/CaM signaling occurred at various Ca2+ signals when CaM levels were limiting. At elevated stimulations, the total CaM pool rapidly bound to its protein binding targets which regulate both LTP and LTD. This was followed by CaM becoming redistributed from low-affinity to high-affinity binding targets. Specifically, CaM was redistributed away from LTD-inducing proteins to bind the high-affinity LTP-inducing protein, calmodulin-dependent kinase II (CaMKII). In this way, CaMKII acted as a dominant affecter and repressed activation of opposing CaM-binding protein targets. The model thereby showed a novel form of CaM signaling by which the two opposing pathways crosstalk indirectly. The model also found that CaMKII can repress cAMP production by repressing CaM-regulated proteins, which catalyze cAMP production. The model also found that at low Ca2+ stimulation levels, typical of LTD induction, CaM signaling was unstable and is therefore unlikely to alone be enough to induce synaptic depression. Overall, this paper demonstrates how limiting levels of CaM may be a fundamental aspect of Ca2+ regulated signaling which allows crosstalk among proteins without requiring directly interaction.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Calmodulina , Cálcio/metabolismo , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Hipocampo/metabolismo , Plasticidade Neuronal , Fosforilação
19.
Biochem Biophys Res Commun ; 628: 155-162, 2022 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-36099691

RESUMO

BACKGROUND AND AIMS: Increased endoplasmic reticulum (ER) stress is strongly associated with the phenotypic switching of vascular smooth muscle cells (VSMCs) in atherosclerosis. Depletion of the ER Ca2+ content is one of the leading causes of increased ER stress in VSMCs. The ryanodine receptor (RyR) is a major Ca2+ release channel in the sarcoplasmic reticulum membrane. Calmodulin (CaM), which binds to RyR (CaM-RyR), stabilizes the closed state of RyR in the resting state in normal cells. Defective CaM-RyR interactions can cause abnormal Ca2+ leakage through RyR, resulting in decreased Ca2+ content, indicating that defective CaM-RyR interactions may be a cause of increased ER stress. Herein, we used a mouse VSMCs to assess whether CaM-RyR plays a pivotal role in VSMCs phenotypic switching, which is caused by ER stress, and whether dantrolene, which enhances the binding affinity of CaM to RyR, affects VSMCs phenotypic switching. METHODS AND RESULTS: Tunicamycin was used to mimic ER stress in vitro. Tunicamycin-induced ER stress caused CaM to dissociate from the RyR and translocate to the nucleus, which stimulated phenotypic switching through the activation of MEF2 and KLF5. Dantrolene suppressed tunicamycin-induced apoptosis, ER stress (restoring ER Ca2+ content), and phenotypic switching of VSMCs. Suramin, which directly unbinds CaM from RyR, promoted nuclear CaM accumulation with parallel VSMCs phenotypic switching, and dantrolene prevented these effects. CONCLUSIONS: We observed that ER stress causes CaM translocation to the nucleus and drives the phenotypic switching of VSMCs. Thus, restoration of the binding affinity of CaM to RyR may be a therapeutic target for atherosclerosis.


Assuntos
Aterosclerose , Calmodulina , Estresse do Retículo Endoplasmático , Músculo Liso Vascular , Animais , Aterosclerose/metabolismo , Calmodulina/metabolismo , Dantroleno , Estresse do Retículo Endoplasmático/fisiologia , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Suramina , Tunicamicina/farmacologia
20.
J Am Chem Soc ; 144(37): 17041-17053, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36082939

RESUMO

Paramagnetic NMR constraints are very useful to study protein interdomain motion, but their interpretation is not always straightforward. On the example of the particularly flexible complex Calmodulin/Munc13-1, we present a new approach to characterize this motion with pseudocontact shifts and residual dipolar couplings. Using molecular mechanics, we sampled the conformational space of the complex and used a genetic algorithm to find ensembles that are in agreement with the data. We used the Bayesian information criterion to determine the ideal ensemble size. This way, we were able to make an accurate, unambiguous, reproducible model of the interdomain motion of Calmodulin/Munc13-1 without prior knowledge about the domain orientation from crystallography.


Assuntos
Calmodulina , Teorema de Bayes , Calmodulina/metabolismo , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
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