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1.
FASEB J ; 35(10): e21901, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34569665

RESUMO

Chagasic cardiomyopathy (CCC) is one of the main causes of heart failure and sudden death in Latin America. To date, there is no available medication to prevent or reverse the onset of cardiac symptoms. CCC occurs in a scenario of disrupted calcium dynamics and enhanced oxidative stress, which combined, may favor the hyper activation of calcium/calmodulin (Ca2+ /CaM)-calcium/calmodulin-dependent protein kinase II (CaMKII) (Ca2+ /CaM-CaMKII) pathway, which is fundamental for heart physiology and it is implicated in other cardiac diseases. Here, we evaluated the association between Ca2+ /CaM-CaMKII in the electro-mechanical (dys)function of the heart in the early stage of chronic experimental Trypanosoma cruzi infection. We observed that in vitro and ex vivo inhibition of Ca2+ /CaM-CaMKII reversed the arrhythmic profile of isolated hearts and isolated left-ventricles cardiomyocytes. The benefits of the limited Ca2+ /CaM-CaMKII activation to cardiomyocytes' electrical properties are partially related to the restoration of Ca2+ dynamics in a damaged cellular environment created after T. cruzi infection. Moreover, Ca2+ /CaM-CaMKII inhibition prevented the onset of arrhythmic contractions on isolated heart preparations of chagasic mice and restored the responsiveness to the increase in the left-ventricle pre-load. Taken together, our data provide the first experimental evidence for the potential of targeting Ca2+ /CaM-CaMKII pathway as a novel therapeutic target to treat CCC.


Assuntos
Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Cardiomiopatia Chagásica/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Arritmias Cardíacas/parasitologia , Cardiomiopatia Chagásica/parasitologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C
2.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502077

RESUMO

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell-cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.


Assuntos
Calmodulina/genética , Canalopatias/genética , Conexinas/genética , Doenças Genéticas Inatas/genética , Animais , Calmodulina/metabolismo , Comunicação Celular , Canalopatias/metabolismo , Conexinas/metabolismo , Doenças Genéticas Inatas/metabolismo , Humanos , Mutação
3.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
4.
Int J Mol Sci ; 22(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445734

RESUMO

Mutant huntingtin (m-HTT) proteins and calmodulin (CaM) co-localize in the cerebral cortex with significant effects on the intracellular calcium levels by altering the specific calcium-mediated signals. Furthermore, the mutant huntingtin proteins show great affinity for CaM that can lead to a further stabilization of the mutant huntingtin aggregates. In this context, the present study focuses on describing the interactions between CaM and two huntingtin mutants from a biophysical point of view, by using classical Molecular Dynamics techniques. The huntingtin models consist of a wild-type structure, one mutant with 45 glutamine residues and the second mutant with nine additional key-point mutations from glutamine residues into proline residues (9P(EM) model). Our docking scores and binding free energy calculations show higher binding affinities of all HTT models for the C-lobe end of the CaM protein. In terms of dynamic evolution, the 9P(EM) model triggered great structural changes into the CaM protein's structure and shows the highest fluctuation rates due to its structural transitions at the helical level from α-helices to turns and random coils. Moreover, our proposed 9P(EM) model suggests much lower interaction energies when compared to the 45Qs-HTT mutant model, this finding being in good agreement with the 9P(EM)'s antagonistic effect hypothesis on highly toxic protein-protein interactions.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Proteína Huntingtina/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteína Huntingtina/genética , Mutação
5.
Plant Mol Biol ; 107(1-2): 1-20, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34398355

RESUMO

KEY MESSAGE: Analyses of the function of Arabidopsis Calmodulin7 (CAM7) in concert with multiple regulatory proteins involved in various signal transduction processes. Calmodulin (CaM) plays various regulatory roles in multiple signaling pathways in eukaryotes. Arabidopsis CALMODULIN 7 (CAM7) is a unique member of the CAM family that works as a transcription factor in light signaling pathways. CAM7 works in concert with CONSTITUTIVE PHOTOMORPHOGENIC 1 and ELONGATED HYPOCOTYL 5, and plays an important role in seedling development. Further, it is involved in the regulation of the activity of various Ca2+-gated channels such as cyclic nucleotide gated channel 6 (CNGC6), CNGC14 and auto-inhibited Ca2+ ATPase 8. Recent studies further indicate that CAM7 is also an integral part of multiple signaling pathways including hormone, immunity and stress. Here, we review the recent advances in understanding the multifaceted role of CAM7. We highlight the open-ended questions, and also discuss the diverse aspects of CAM7 characterization that need to be addressed for comprehensive understanding of its cellular functions.


Assuntos
Calmodulina/metabolismo , Desenvolvimento Vegetal , Estresse Fisiológico , Sequência de Aminoácidos , Apoptose , Calmodulina/química , Morfogênese , Reguladores de Crescimento de Plantas/metabolismo
6.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34198993

RESUMO

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.


Assuntos
Aminoácidos/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Ligação Proteica , Transdução de Sinais
7.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209674

RESUMO

Pre-mRNA processing factor 4B (PRP4) has previously been shown to induce epithelial-mesenchymal transition (EMT) and drug resistance in cancer cell lines. As melanin plays an important photoprotective role in the risk of sun-induced skin cancers, we have investigated whether PRP4 can induce drug resistance and regulate melanin biosynthesis in a murine melanoma (B16F10) cell line. Cells were incubated with a crucial melanogenesis stimulator, alpha-melanocyte-stimulating hormone, followed by transfection with PRP4. This resulted in the inhibition of the production of melanin via the downregulation of adenylyl cyclase-cyclic adenosine 3',5'-monophosphate (AC)-(cAMP)-tyrosinase synthesis signaling pathway. Inhibition of melanin production by PRP4 leads to the promotion of carcinogenesis and induced drug resistance in B16F10 cells. Additionally, PRP4 overexpression upregulated the expression of ß-arrestin 1 and desensitized the extracellular calcium-sensing receptor (CaSR), which in turn, inhibited the influx of extracellular Ca2+ ions. The decreased influx of Ca2+ was confirmed by a decreased expression level of calmodulin. We have demonstrated that transient receptor potential cation channel subfamily C member 1 was involved in the influx of CaSR-induced Ca2+ via a decreasing level of its expression. Furthermore, PRP4 overexpression downregulated the expression of AC, decreased the synthesis of cAMP, and modulated the actin cytoskeleton by inhibiting the expression of Ras homolog family member A (RhoA). Our investigation suggests that PRP4 inhibits the production of melanin in B16F10 cells, blocks the influx of Ca2+ through desensitization of CaSR, and modulates the actin cytoskeleton through downregulating the AC-cAMP pathway; taken together, these observations collectively lead to the promotion of skin carcinogenesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Melaninas/biossíntese , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Animais , Antineoplásicos , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Transporte , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Espaço Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Melanoma Experimental , Camundongos , Ligação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia
8.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073116

RESUMO

The plant-specific mildew resistance locus O (MLO) proteins, which contain seven transmembrane domains and a conserved calmodulin-binding domain, play important roles in many plant developmental processes. However, their mechanisms that regulate plant development remain unclear. Here, we report the functional characterization of the MLO4 protein in Arabidopsis roots. The MLO4 was identified as interacting with CML12 in a screening for the interaction between the proteins from Arabidopsis MLO and calmodulin/calmodulin-like (CaM/CML) families using yeast two hybrid (Y2H) assays. Then, the interaction between MLO4 and CML12 was further verified by Luciferase Complementation Imaging (LCI) and Bimolecular Fluorescence Complementation (BiFC) assays. Genetic analysis showed that the mlo4, cml12, and mlo4 cml12 mutants displayed similar defects in root gravity response. These results imply that the MLO4 might play an important role in root gravity response through interaction with CML12. Moreover, our results also demonstrated that the interaction between the MLO and CaM/CML families might be conservative.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Calmodulina/metabolismo , Gravitropismo , Doenças das Plantas/genética , Raízes de Plantas , Arabidopsis/genética , Resistência à Doença/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Ligação Proteica
9.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066691

RESUMO

Calmodulin (CaM) is a highly-expressed Ca2+ binding protein known to bind hundreds of protein targets. Its binding selectivity to many of these targets is partially attributed to the protein's flexible alpha helical linker that connects its N- and C-domains. It is not well established how its linker mediates CaM's binding to regulatory targets yet. Insights into this would be invaluable to understanding its regulation of diverse cellular signaling pathways. Therefore, we utilized Martini coarse-grained (CG) molecular dynamics simulations to probe CaM/target assembly for a model system: CaM binding to the calcineurin (CaN) regulatory domain. The simulations were conducted assuming a 'wild-type' calmodulin with normal flexibility of its linker, as well as a labile, highly-flexible linker variant to emulate structural changes that could be induced, for instance, by post-translational modifications. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 µs of simulation time; of these, 1.7% sampled the fully-bound state observed in the experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be ka= 8.7 × 108 M-1 s-1, which is similar to the diffusion-limited, experimentally-determined rate of 2.2 × 108 M-1 s-1. Furthermore, our simulations recapitulated its well-known inverse relationship between the association rate and the solution ionic strength. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states which are controlled by the linker flexibility. Therefore, our simulations suggest that variations in the CaM linker's propensity for alpha helical secondary structure can modulate the kinetics of target binding.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Animais , Simulação de Dinâmica Molecular , Concentração Osmolar , Ligação Proteica , Domínios Proteicos , Ratos
10.
Nat Commun ; 12(1): 3451, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103486

RESUMO

Several cell-surface receptors for neurotoxic forms of amyloid-ß (Aß) have been described, but their molecular interactions with Aß assemblies and their relative contributions to mediating Alzheimer's disease pathology have remained uncertain. Here, we used super-resolution microscopy to directly visualize Aß-receptor interactions at the nanometer scale. We report that one documented Aß receptor, PrPC, specifically inhibits the polymerization of Aß fibrils by binding to the rapidly growing end of each fibril, thereby blocking polarized elongation at that end. PrPC binds neurotoxic oligomers and protofibrils in a similar fashion, suggesting that it may recognize a common, end-specific, structural motif on all of these assemblies. Finally, two other Aß receptors, FcγRIIb and LilrB2, affect Aß fibril growth in a manner similar to PrPC. Our results suggest that receptors may trap Aß oligomers and protofibrils on the neuronal surface by binding to a common molecular determinant on these assemblies, thereby initiating a neurotoxic signal.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Neurotoxinas/química , Multimerização Proteica , Receptores de Superfície Celular/metabolismo , Animais , Benzotiazóis/metabolismo , Calmodulina/metabolismo , Humanos , Cinética , Camundongos , Modelos Biológicos , Polimerização , Príons/metabolismo , Ligação Proteica , Receptores de IgG/metabolismo , Receptores Imunológicos/metabolismo
11.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067061

RESUMO

Intraneuronal amyloid ß (Aß) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer's disease (AD)-affected brains, intraneuronal Aß oligomers can derive from Aß peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aß neurotoxicity in AD. However, the identification of primary Aß-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aß peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aß concentrations in the low nanomolar range. In turn, the concentration of Aß-CaM complexes within neurons will increase as a function of time after the induction of Aß production, and free Aß will rise sharply when accumulated Aß exceeds all available CaM. Thus, Aß-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aß; a point that has been overlooked until now. In this review, we address the implications of Aß-CaM complexation in the formation of neurotoxic Aß oligomers, in the alteration of intracellular calcium homeostasis induced by Aß, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aß.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Calmodulina/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Animais , Humanos
12.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074030

RESUMO

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Hibridização In Situ , Lantânio/farmacologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
13.
J Agric Food Chem ; 69(24): 6870-6878, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34101455

RESUMO

Lanthanum (La), calcium (Ca), and cadmium (Cd) have similar physical and chemical properties because of their similar ionic radius. Although the interactions between La, Ca, and Cd have been frequently reported in plants, few studies have investigated the interactive effects of La and Ca on the growth and Cd accumulation in plants. Therefore, we investigated the interactive effects of La and Ca on the growth and Cd accumulation in wheat under Cd exposure by a hydroponic experiment. The results indicated that wheat growth was significantly affected by La-Cd and La-Ca interactions. The accumulation of Cd in wheat was significantly affected by La-Ca and La-Cd interactions and La-Ca-Cd interplay. Correlation analysis indicated that Ca deficiency stimulated La to promote wheat growth and mitigate Cd toxicity. Simultaneously, a low Ca supply stimulated La to decrease Cd accumulation in wheat and induce TaNramp5 expression. In addition, Cd accumulation in wheat was significantly affected by the W7-La interaction and W7-La-Ca interplay. All of the results suggested that La, Ca, and Cd probably share the same binding sites in calmodulin (TaCaM) and La could affect Cd accumulation in wheat by interacting with TaCaM and then downregulating the expression of TaNramp5.


Assuntos
Cádmio , Poluentes do Solo , Cádmio/análise , Cádmio/toxicidade , Cálcio , Calmodulina/genética , Calmodulina/metabolismo , Lantânio , Poluentes do Solo/análise , Triticum/genética , Triticum/metabolismo
14.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33988695

RESUMO

Induction of long-term potentiation (LTP) in excitatory neurons triggers a large transient increase in the volume of dendritic spines followed by decays to sustained size expansion, a process termed structural LTP (sLTP) that contributes to the cellular basis of learning and memory. Although mechanisms regulating the early and sustained phases of sLTP have been studied intensively, how the acute spine enlargement immediately after LTP stimulation is achieved remains elusive. Here, we report that endophilin A1 orchestrates membrane dynamics with actin polymerization to initiate spine enlargement in NMDAR-mediated LTP. Upon LTP induction, Ca2+/calmodulin enhances binding of endophilin A1 to both membrane and p140Cap, a cytoskeletal regulator. Consequently, endophilin A1 rapidly localizes to the plasma membrane and recruits p140Cap to promote local actin polymerization, leading to spine head expansion. Moreover, its molecular functions in activity-induced rapid spine growth are required for LTP and long-term memory. Thus, endophilin A1 serves as a calmodulin effector to drive acute structural plasticity necessary for learning and memory.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Calmodulina/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia
15.
J Surg Res ; 266: 245-253, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34034059

RESUMO

BACKGROUND: Myocardial dysfunction is an important adverse factor of hemorrhagic shock that induces refractory hypotension, and post-hemorrhagic shock mesenteric lymph (PHSML) return is involved in this adverse effect. This study investigated whether mesenteric lymph drainage (MLD) improves PHSML return-induced cardiac contractile dysfunction via the restoration of cardiomyocyte calcium sensitivity. MATERIALS AND METHODS: A hemorrhage shock model was established by using a controlled hemorrhage through the femoral artery that maintained a mean arterial pressure of 40 ± 2 mmHg for 3 h. MLD and mesenteric lymph duct ligation (MLDL) were performed from 1 to 3 h during hypotension. The papillary muscles of the heart were collected for measurement of calmodulin expression and for determining contractile responses to either isoprenaline or calcium. RESULTS: The results showed that either MLD or MLDL reversed the hemorrhagic shock-induced downregulation of calmodulin expression, a marker protein of cardiomyocyte calcium sensitization, in papillary muscles. MLD also improved the decreased contractile response and ±df/dt of the papillary muscle strip to gradient isoprenaline or calcium caused by hemorrhagic shock. CONCLUSION: These findings indicate that increased cardiac contractibility may be associated with the restoration of calcium sensitivity produced by PHSML drainage.


Assuntos
Calmodulina/metabolismo , Cardiomiopatias/prevenção & controle , Vasos Linfáticos/cirurgia , Músculos Papilares/fisiologia , Choque Hemorrágico/complicações , Animais , Cardiomiopatias/etiologia , Masculino , Ratos Wistar , Choque Hemorrágico/metabolismo
16.
Int J Mol Sci ; 22(7)2021 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801703

RESUMO

Salt stress is a major increasing threat to global agriculture. Pongamia (Millettia pinnata), a semi-mangrove, is a good model to study the molecular mechanism of plant adaptation to the saline environment. Calcium signaling pathways play critical roles in the model plants such as Arabidopsis in responding to salt stress, but little is known about their function in Pongamia. Here, we have isolated and characterized a salt-responsive MpCML40, a calmodulin-like (CML) gene from Pongamia. MpCML40 protein has 140 amino acids and is homologous with Arabidopsis AtCML40. MpCML40 contains four EF-hand motifs and a bipartite NLS (Nuclear Localization Signal) and localizes both at the plasma membrane and in the nucleus. MpCML40 was highly induced after salt treatment, especially in Pongamia roots. Heterologous expression of MpCML40 in yeast cells improved their salt tolerance. The 35S::MpCML40 transgenic Arabidopsis highly enhanced seed germination rate and root length under salt and osmotic stresses. The transgenic plants had a higher level of proline and a lower level of MDA (malondialdehyde) under normal and stress conditions, which suggested that heterologous expression of MpCML40 contributed to proline accumulation to improve salt tolerance and protect plants from the ROS (reactive oxygen species) destructive effects. Furthermore, we did not observe any measurable discrepancies in the development and growth between the transgenic plants and wild-type plants under normal growth conditions. Our results suggest that MpCML40 is an important positive regulator in response to salt stress and of potential application in producing salt-tolerant crops.


Assuntos
Sinalização do Cálcio , Calmodulina/metabolismo , Millettia/metabolismo , Sinais de Localização Nuclear , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Calmodulina/genética , Regulação da Expressão Gênica de Plantas , Malondialdeído/química , Millettia/genética , Fases de Leitura Aberta , Osmose , Fenótipo , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas , Plantas Geneticamente Modificadas , Prolina/química , Estresse Salino , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/metabolismo , Sementes/metabolismo
17.
J Chem Phys ; 154(12): 124104, 2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33810667

RESUMO

It is challenging to parameterize the force field for calcium ions (Ca2+) in calcium-binding proteins because of their unique coordination chemistry that involves the surrounding atoms required for stability. In this work, we observed a wide variation in Ca2+ binding loop conformations of the Ca2+-binding protein calmodulin, which adopts the most populated ternary structures determined from the molecular dynamics simulations, followed by ab initio quantum mechanical (QM) calculations on all 12 amino acids in the loop that coordinate Ca2+ in aqueous solution. Ca2+ charges were derived by fitting to the electrostatic potential in the context of a classical or polarizable force field (PFF). We discovered that the atomic radius of Ca2+ in conventional force fields is too large for the QM calculation to capture the variation in the coordination geometry of Ca2+ in its ionic form, leading to unphysical charges. Specifically, we found that the fitted atomic charges of Ca2+ in the context of PFF depend on the coordinating geometry of electronegative atoms from the amino acids in the loop. Although nearby water molecules do not influence the atomic charge of Ca2+, they are crucial for compensating for the coordination of Ca2+ due to the conformational flexibility in the EF-hand loop. Our method advances the development of force fields for metal ions and protein binding sites in dynamic environments.


Assuntos
Cálcio/química , Cálcio/metabolismo , Calmodulina/metabolismo , Animais , Sítios de Ligação , Calmodulina/química , Bovinos , Motivos EF Hand , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Teoria Quântica , Eletricidade Estática , Água/química
18.
Methods Mol Biol ; 2263: 83-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33877594

RESUMO

To understand cellular processes such as biochemical pathways and signaling networks, we need to understand binding and reaction rates of often competing reactions, their dependence on cellular concentrations of participating molecules, and the regulation of these rates through allostery, posttranslational modifications, or other mechanisms. To do so, we break these systems down into their elementary steps, which are almost invariably either unimolecular or bimolecular reactions that frequently occur on sub-second, often sub-millisecond, time scales. Rapid mixing techniques, which generally achieve mixing in less than 2 ms, are generally suitable for the study of such reactions. The application of these techniques to the study of enzyme mechanisms is described in several excellent texts (Cornish-Bowden, Fundamentals of enzyme kinetics, 1995; Gutfreund, Kinetics for the life sciences. Receptors, transmitters and catalysis, 1995); flow techniques are used to study individual steps by monitoring the approach to equilibrium (the pre-steady state) under single turnover conditions.The individual steps in complex biochemical reaction schemes determine how fast systems can respond to incoming signals and adapt to changed conditions [1, 2]. This chapter is concerned with in vitro techniques that have been developed to study fast reactions in solution, and we present the study of various interactions of calmodulin as an example. The kinetic information obtained with these techniques is indispensable for understanding the dynamics of biochemical processes and complements the static structural and thermodynamic information available from X-ray crystallography, NMR, and equilibrium binding studies.


Assuntos
Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Algoritmos , Animais , Catálise , Fluorescência , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Conformação Proteica , Transdução de Sinais , Espectrometria de Fluorescência , Termodinâmica
19.
Int Arch Allergy Immunol ; 182(9): 800-806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33882500

RESUMO

BACKGROUND: The ciliary beat of the airway epithelium, including the sinonasal epithelium, has a significant role in frontline defense and is thought to be controlled by the level of intracellular Ca2+. Involvement of calmodulin and adenylate/guanylate cyclases in the regulation of ciliary beats has been reported, and here we investigated the interrelation between these components of the ciliary beat regulatory pathway. METHODS: The inferior turbinates were collected from 29 patients with chronic hypertrophic rhinitis/rhinosinusitis during endoscopic sinonasal surgery. The turbinate mucosa was cut into thin strips, and mucociliary movement was observed under a phase-contrast light microscope equipped with a high-speed digital video camera. RESULTS: The ciliary beat frequency (CBF) was significantly increased by stimulation with 100 µM CALP3 (calmodulin agonist), which was completely suppressed by adding 100 µM SQ22536 (adenylate cyclase inhibitor) and 10 µM ODQ (guanylate cyclase inhibitor) together and by adding 1 µM KT5720 (protein kinase A inhibitor) and 1 µM KT5823 (protein kinase G inhibitor) together. The CBF was significantly increased by stimulation with 10 µM forskolin (adenylate cyclase activator) and 10 µM BAY41-2272 (guanylate cyclase activator) and by stimulation with 100 µM 8-bromo-cAMP (cAMP analog) and 100 µM 8-bromo-cGMP (cGMP analog), which was not changed by adding 1 µM calmidazolium (calmodulin antagonist). CONCLUSIONS: These results confirmed that the regulatory pathway of ciliary beats in the human nasal mucosa involves calmodulin, adenylate/guanylate cyclases, and protein kinases A/G and indicate that adenylate/guanylate cyclases and protein kinases A/G act downstream of calmodulin, but not vice versa, and that these cyclases relay calmodulin signaling.


Assuntos
Adenilil Ciclases/metabolismo , Calmodulina/metabolismo , Cílios/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Mucosa Nasal/metabolismo , Cálcio/metabolismo , GMP Cíclico/análogos & derivados , Endoscopia , Humanos , Depuração Mucociliar , Rinite/etiologia , Rinite/metabolismo , Rinite/patologia , Rinite/terapia , Transdução de Sinais , Sinusite/etiologia , Sinusite/metabolismo , Sinusite/patologia , Sinusite/terapia
20.
Anim Sci J ; 92(1): e13552, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33890345

RESUMO

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation "full-type and nonfull-type patterns" can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca2+ and calmodulin antagonists "W-7 and W-5 (W-5; a less potent antagonist)" on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42 mM CaCl2 for 120-180 min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca2+ -dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca2+ -sensing molecule which can function in the full-type hyperactivation.


Assuntos
Cálcio/metabolismo , Calmodulina/fisiologia , AMP Cíclico/farmacologia , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Calmodulina/farmacologia , Bovinos , Células Cultivadas , Masculino , Camundongos , Sulfonamidas/farmacologia , Suínos
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