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1.
Int J Mol Sci ; 20(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315229

RESUMO

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Imagem Óptica/métodos , Fitocromo/genética , Engenharia de Proteínas/métodos , Cálcio/análise , Sinalização do Cálcio , Calmodulina/genética , Células HeLa , Humanos , Raios Infravermelhos , Fitocromo/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
Zhongguo Zhong Yao Za Zhi ; 44(13): 2777-2784, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31359690

RESUMO

According to the data of Pinellia ternate transcriptome,two calmodulin genes were cloned and named as Pt Ca M1 and PtCa M2 respectively. The results of bioinformatics analysis showed that Pt Ca Ms genes contained a 450 bp open reading frame,encoding149 amino acids.The identity of the coding sequences was 80%,and the identity of amino acids sequence was 91%. Pt Ca Ms genes contained EF-hand structure domain,belonging to the Ca M families. The Real-time PCR analysed the expression patterns of Pt Ca Ms in different tissues and different treatments. RESULTS:: showed that Pt Ca M1 and Pt Ca M2 gene were the highest expression level in tuber. Under Ca Cl2 treatment,the expressions of Pt Ca Ms were significantly higher than the control. Under EGTA,La Cl3 and TFP treatments,the expression level of Pt Ca Ms decreased gradually. In this study,the Pt Ca Ms gene were successfully cloned from P. ternate,which laid a foundation for the functional characteristic of Pt Ca Ms gene and the synthesis of alkaloids from P. ternata for further study.


Assuntos
Calmodulina/genética , Pinellia/genética , Clonagem Molecular , Genes de Plantas , Tubérculos/genética
3.
Int J Mol Sci ; 20(12)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248151

RESUMO

Pathogen-induced cell death is closely related to plant disease susceptibility and resistance. The cucumber (Cucumis sativus L.) mildew resistance locus O (CsMLO1) and calmodulin (CsCaM3) genes, as molecular components, are linked to nonhost resistance and hypersensitive cell death. In this study, we demonstrate that CsMLO1 interacts with CsCaM3 via yeast two-hybrid, firefly luciferase (LUC) complementation and bimolecular fluorescence complementation (BiFC) experiments. A subcellular localization analysis of green fluorescent protein (GFP) fusion reveals that CsCaM3 is transferred from the cytoplasm to the plasma membrane in Nicotiana benthamiana, and CsCaM3 green fluorescence is significantly attenuated via the coexpression of CsMLO1 and CsCaM3. CsMLO1 negatively regulates CsCaM3 expression in transiently transformed cucumbers, and hypersensitive cell death is disrupted by CsCaM3 and/or CsMLO1 expression under Corynespora cassiicola infection. Additionally, CsMLO1 silencing significantly enhances the expression of reactive oxygen species (ROS)-related genes (CsPO1, CsRbohD, and CsRbohF), defense marker genes (CsPR1 and CsPR3) and callose deposition-related gene (CsGSL) in infected cucumbers. These results suggest that the interaction of CsMLO1 with CsCaM3 may act as a cell death regulator associated with plant immunity and disease.


Assuntos
Calmodulina/metabolismo , Cucumis sativus/fisiologia , Resistência à Doença , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/etiologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Calmodulina/genética , Morte Celular , Resistência à Doença/genética , Resistência à Doença/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ordem dos Genes , Inativação Gênica , Vetores Genéticos , Interações Hospedeiro-Patógeno/genética , Espaço Intracelular , Fenótipo , Proteínas de Plantas/genética , Ligação Proteica
4.
Chemphyschem ; 20(14): 1860-1868, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31054266

RESUMO

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Calmodulina/genética , Extratos Celulares/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Gadolínio/química , Células HeLa , Humanos , Mutação , Conformação Proteica , Marcadores de Spin
5.
Antonie Van Leeuwenhoek ; 112(10): 1501-1521, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31140027

RESUMO

Fungi under the order Ophiostomatales (Ascomycota) are known to associate with various species of bark beetles (Coleoptera: Curculionidae: Scolytinae). In addition this group of fungi contains many taxa that can impart blue-stain on sapwood and some are important tree pathogens. A recent survey that focussed on the diversity of the Ophiostomatales in the forest ecosystems of the Czech Republic and Poland uncovered four putative new species. Phylogenetic analyses of four gene regions (ITS1-5.8S-ITS2 region, ß-tubulin, calmodulin, and translation elongation factor 1-α) indicated that these four species are members of the genus Ophiostoma. All four newly described species can be distinguished from each other and from closely related species based on DNA sequence comparisons, morphological characters, growth rates, and their insect associations. Based on this study four new taxa can be circumscribed and the following names are provided: Ophiostoma pityokteinis sp. nov., Ophiostoma rufum sp. nov., Ophiostoma solheimii sp. nov., and Ophiostoma taphrorychi sp. nov. O. rufum sp. nov. is a member of the Ophiostoma piceae species complex, while O. pityokteinis sp. nov. resides in a discrete lineage within Ophiostoma s. stricto. O. taphrorychi sp. nov. together with O. distortum formed a well-supported clade in Ophiostoma s. stricto close to O. pityokteinis sp. nov. O. solheimii sp. nov. groups within a currently undefined lineage A, which also includes Ophiostoma grandicarpum and Ophiostoma microsporum. This study highlights the need for more intensive surveys that should include additional countries of Central Europe, insect vectors and host tree species in order to elucidate Ophiostoma species diversity in this region.


Assuntos
Ophiostoma/classificação , Ophiostoma/isolamento & purificação , Filogenia , Gorgulhos/microbiologia , Animais , Calmodulina/genética , República Tcheca , DNA Espaçador Ribossômico/genética , Técnicas de Tipagem Micológica , Ophiostoma/genética , Ophiostoma/fisiologia , Fator 1 de Elongação de Peptídeos/genética , Polônia , RNA Ribossômico 5,8S/genética , Análise de Sequência de DNA , Traqueófitas/parasitologia , Tubulina (Proteína)/genética , Gorgulhos/crescimento & desenvolvimento , Madeira/parasitologia
6.
Plant Physiol Biochem ; 139: 600-612, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31030028

RESUMO

Changes in intracellular calcium (Ca2+) levels in response to developmental processes or external stimuli serve as signals in eukaryotic cells. These Ca2+ signals are likely perceived through sensor proteins that bind Ca2+ by EF-hand (a helix-loop-helix structure) motif. Calmodulins (CaMs), a group of well-characterized Ca2+ sensors, and calmodulin-like (CMLs) are implicated in a large number of diverse cellular processes, including plant development and stress responses. In this study, apple (Malus × domestica) genes encoding CaM and CML proteins that only possess EF-hand motifs with no other functional domains were analyzed. A total of 4 MdCaM and 58 MdCML genes were identified, which are spread among 16 out of the 17 apple chromosomes. Bioinformatics analyses, including protein characteristics, conserved domain, evolutionary relationships and chromosomal locations, demonstrated the conservation and divergence of MdCaMs/CMLs. In addition, expression analysis showed that MdCaMs/CMLs are expressed in more than one tissue, including shoot tips, roots, mature leaves, flowers and fruit. Furthermore, the expression of some MdCaM/CML members responded to plant hormones (abscisic acid, jasmonic acid) and salt stress, suggesting a potential role of these genes in responses to biotic and abiotic stress. Overexpression of stress-induced MdCML3 gene significantly improved the tolerance of apple calli to salinity and ABA. The identification and characterization of MdCaMs/CMLs in apple lays a foundation for future functional studies of these genes.


Assuntos
Calmodulina/genética , Genoma de Planta/genética , Malus/genética , Ácido Abscísico/farmacologia , Cálcio/metabolismo , Biologia Computacional , Ciclopentanos/farmacologia , Malus/efeitos dos fármacos , Oxilipinas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
7.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
8.
Mycologia ; 111(2): 217-224, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30896313

RESUMO

Aspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR34/L98H and TR46/Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the ß-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR46/Y121F/T289A (n = 8), TR34/L98H, and TR53. Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.


Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Azóis/administração & dosagem , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Fungicidas Industriais/administração & dosagem , Doenças das Plantas/prevenção & controle , Verduras/microbiologia , Aspergillus fumigatus/classificação , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/isolamento & purificação , Azóis/farmacologia , Calmodulina/genética , Colômbia , Fungicidas Industriais/farmacologia , Humanos , Polimorfismo Genético , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Microbiologia do Solo , Tubulina (Proteína)/genética
9.
Mycotoxin Res ; 35(3): 217-230, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30877631

RESUMO

Aspergilli section Flavi, originally isolated from air samples collected from inhabited apartments (AP), unoccupied basements (BS), and processing facilities of a grain mill (GM), were analyzed for their potential to produce aflatoxin B1 (AFB1) on solid media. The isolates were further characterized with regard to their cytotoxic, genotoxic, and pro-inflammatory properties in vitro. Aspergilli were identified based on partial calmodulin (CaM) gene sequencing; the producing capacities of isolates were analyzed by HPLC/FLD and confirmed by genes in biosynthesis (aflR, norA, omtA). In the grain mill, the Aspergilli section Flavi (up to 1.3 × 106 cfu/m3) dominated by AFB1-producing Aspergillus flavus (71%, 4.5-5254 ng/ml) which showed a serious health risk for workers. Living environments were not relevant sources of exposure. After 24 h, AFB1 (1-100 µmol/l) reduced cell viability (MTT test) in both A549 cells and THP-1 macrophage-like cells without reaching IC50. In A549 cells, the extract of the AFB1-producing A. flavus significantly decreased cell viability but not below 50%. THP-1 macrophage-like cells were more sensitive to both extracts, but IC50 was obtained only for the AFB1-producing strain (0.37 mg/ml; AFB1 2.78 µmol/l). AFB1 (1 and 10 µmol/l) induced significant DNA damage (tail intensity, alkaline comet assay) in A549 cells in contrast to Aspergilli extracts. AFB1 elevated IL-6 and IL-8, while Aspergilli extracts increased IL-1ß, TNF-α, and IL-17 release in THP-1 macrophages (ELISA). Chronic exposure to AFB1 and/or other metabolites in airborne A. flavus from occupational environments may stimulate epithelial damage of airways accompanied by lowered macrophage viability.


Assuntos
Aflatoxina B1/biossíntese , Microbiologia do Ar , Aspergillus flavus/metabolismo , Células A549 , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Calmodulina/genética , Sobrevivência Celular , Citocinas/imunologia , Dano ao DNA , Humanos , Concentração Inibidora 50 , Macrófagos/microbiologia , Células THP-1
10.
Anal Biochem ; 572: 25-32, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30825429

RESUMO

The KRAS gene is highly mutated in human cancers and the focus of current Ras drug development efforts. Recently the interface between the C-terminus of K-Ras and calmodulin (CaM) was proposed as a target site to block K-Ras driven cancer cell stemness. We therefore aimed at developing a high-throughput amenable screening assay to identify novel CaM-inhibitors as potential K-Ras stemness-signaling disruptors. A modulated time-resolved Förster resonance energy transfer (mTR-FRET)-assay was developed and benchmarked against an identically designed fluorescence anisotropy (FA)-assay. In both assays, two CaM-binding peptides were labeled with Eu(III)-chelate or fluorescein and used as single-label reporter probes that were displaced from CaM upon competitor binding. Thus, peptidic and small molecule competitors with nanomolar to micromolar affinities to CaM could be detected, including a peptide that was derived from the C-terminus of K-Ras. In order to detect CaM-residue specific covalent inhibitors, a cell lysate-based Förster resonance energy transfer (FRET)-assay was furthermore established. This assay enabled us to measure the slow, residue-specific, covalent inhibition by ophiobolin A in the presence of other endogenous proteins. In conclusion, we have developed a panel of fluorescence-assays that allows identification of conventional and covalent CaM-inhibitors as potential disruptors of K-Ras driven cancer cell stemness.


Assuntos
Calmodulina/antagonistas & inibidores , Inibidores Enzimáticos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Calmodulina/genética , Calmodulina/metabolismo , Inibidores Enzimáticos/metabolismo , Európio/química , Fluoresceína/química , Humanos , Concentração Inibidora 50 , Cinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Sesterterpenos/química , Sesterterpenos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo
11.
J Biol Chem ; 294(15): 6142-6156, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30770467

RESUMO

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.


Assuntos
Brachypodium , Cálcio , Calmodulina , Transportadores de Ânions Orgânicos , Proteínas de Plantas , Estômatos de Plantas , Substituição de Aminoácidos , Brachypodium/química , Brachypodium/genética , Brachypodium/metabolismo , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Ativação do Canal Iônico/fisiologia , Malatos/química , Malatos/metabolismo , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/química , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo
12.
J Biol Chem ; 294(15): 6094-6112, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30808708

RESUMO

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.


Assuntos
Cálcio/química , Calmodulina/química , Canais de Potássio KCNQ/química , Animais , Células CHO , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cricetulus , Cristalografia por Raios X , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
13.
Dev Cell ; 48(5): 710-725.e5, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30713075

RESUMO

Calcium is a universal signal in all eukaryotes, but the mechanism for encoding calcium signatures remains largely unknown. Calcium oscillations control pollen tube growth and fertilization in flowering plants, serving as a model for dissecting the molecular machines that mediate calcium fluctuations. We report that pollen-tube-specific cyclic nucleotide-gated channels (CNGC18, CNGC8, and CNGC7) together with calmodulin 2 (CaM2) constitute a molecular switch that either opens or closes the calcium channel depending on cellular calcium levels. Under low calcium, calcium-free calmodulin 2 (Apo-CaM2) interacts with CNGC18-CNGC8 complex, leading to activation of the influx channel and consequently increasing cytosolic calcium levels. Calcium-bound CaM2 dissociates from CNGC18/8 heterotetramer, closing the channel and initiating a downturn of cellular calcium levels. We further reconstituted the calcium oscillator in HEK293 cells, supporting the model that Ca2+-CaM-dependent regulation of CNGC channel activity provides an auto-regulatory feedback mechanism for calcium oscillations during pollen tube growth.


Assuntos
Proteínas de Arabidopsis/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Animais , Arabidopsis/crescimento & desenvolvimento , Sinalização do Cálcio/genética , Calmodulina/genética , Células HEK293 , Humanos , Ligação Proteica
14.
Methods Mol Biol ; 1929: 95-109, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710269

RESUMO

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca2+-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca2+-mobilizing agonists independent of AP-1 activation.


Assuntos
Cálcio/análise , Genes Reporter , Interleucina-2/genética , Calcineurina/genética , Calmodulina/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interleucina-2/química , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição AP-1/genética , Ativação Transcricional
15.
Methods Mol Biol ; 1929: 157-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710273

RESUMO

A multitude of Ca2+-sensor proteins containing the specific Ca2+-binding motif (helix-loop-helix, called EF-hand) are of major clinical relevance in a many human diseases. Measurements of troponin, the first intracellular Ca-sensor protein to be discovered, is nowadays the "gold standard" in the diagnosis of patients with acute coronary syndrome (ACS). Mutations have been identified in calmodulin and linked to inherited ventricular tachycardia and in patients affected by severe cardiac arrhythmias. Parvalbumin, when introduced into the diseased heart by gene therapy to increase contraction and relaxation speed, is considered to be a novel therapeutic strategy to combat heart failure. S100 proteins, the largest subgroup with the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation, and autoimmune pathologies. The intention of this review is to summarize the clinical importance of this protein family and their use as biomarkers and potential drug targets, which could help to improve the diagnosis of human diseases and identification of more selective therapeutic interventions.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Motivos EF Hand , Diagnóstico Precoce , Humanos , Família Multigênica , Parvalbuminas/química , Parvalbuminas/genética , Parvalbuminas/metabolismo , Prognóstico , Proteínas S100/química , Proteínas S100/genética , Proteínas S100/metabolismo , Troponina/química , Troponina/genética , Troponina/metabolismo
16.
Methods Mol Biol ; 1929: 207-221, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710275

RESUMO

Calmodulin (CaM) is a ubiquitous calcium-sensing protein that has one of the most highly conserved sequences among eukaryotes. CaM has been a useful tool for biologists studying calcium signaling for decades. In recent years, CaM has also been implicated in numerous cancer-associated pathways, and rare CaM mutations have been identified as a cause of human cardiac arrhythmias. Here, we present a collection of our most recent and effective protocols for the expression and purification of recombinant CaM from Escherichia coli, including various isotopic labeling schemes, primarily for nuclear magnetic resonance (NMR) spectroscopy and other biophysical applications.


Assuntos
Calmodulina/isolamento & purificação , Calmodulina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Sinalização do Cálcio , Calmodulina/genética , Cromatografia de Afinidade , Escherichia coli/genética , Expressão Gênica , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
17.
Biochim Biophys Acta Biomembr ; 1861(4): 787-797, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30639287

RESUMO

CEACAM1, a homotypic transmembrane receptor with 12 or 72 amino acid cytosolic domain isoforms, is converted from inactive cis-dimers to active trans-dimers by calcium-calmodulin (Ca2+/CaM). Previously, the weak binding of Ca2+/CaM to the human 12 AA cytosolic domain was studied using C-terminal anchored peptides. We now show the binding of 15N labeled Phe-454 cytosolic domain peptides in solution or membrane anchored using NMR demonstrates a significant role for the lipid bilayer. Although binding is increased by the mutation Phe454Ala, this mutation was previously shown to abrogate actin binding. On the other hand, Ca2+/CaM binding is abrogated by phosphorylation of nearby Thr-457, a post-translation modification required for actin binding and subsequent in vitro lumen formation. Binding of Ca2+/CaM to a membrane proximal peptide from the long 72 AA cytosolic domain anchored to lipid nanodiscs was very weak compared to lipid free conditions, suggesting membrane specific effects between the two isoforms. NMR analysis of 15N labeled Ca2+/CaM with unlabeled peptides showed the C-lobe of Ca2+/CaM is involved in peptide interactions, and hydrophobic residues such as Met-109, Val-142 and Met-144 play important roles in binding peptide. This information was incorporated into transmembrane models of CEACAM1 binding to Ca2+/CaM. The lack of Ca2+/CaM binding to phosphorylated Thr-457, a residue we have previously shown to be phosphorylated by CaMK2D, also dependent on Ca2+/CaM, suggests stepwise binding of the cytosolic domain first to Ca2+/CaM and then to actin.


Assuntos
Antígenos CD/química , Cálcio/química , Calmodulina/química , Moléculas de Adesão Celular/química , Modelos Moleculares , Nanoestruturas/química , Peptídeos/química , Antígenos CD/genética , Calmodulina/genética , Moléculas de Adesão Celular/genética , Humanos , Nanoestruturas/ultraestrutura , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética
18.
Int J Mol Sci ; 20(2)2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669290

RESUMO

Calmodulin (CaM) is the principal Ca2+ sensor in eukaryotic cells, orchestrating the activity of hundreds of proteins. Disease causing mutations at any of the three genes that encode identical CaM proteins lead to major cardiac dysfunction, revealing the importance in the regulation of excitability. In turn, some mutations at the CaM binding site of ion channels cause similar diseases. Here we provide a summary of the two sides of the partnership between CaM and ion channels, describing the diversity of consequences of mutations at the complementary CaM binding domains.


Assuntos
Calmodulina/genética , Calmodulina/metabolismo , Suscetibilidade a Doenças , Canais Iônicos/genética , Canais Iônicos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Calmodulina/química , Regulação da Expressão Gênica , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sensibilidade e Especificidade , Transdução de Sinais , Relação Estrutura-Atividade
19.
Biochem J ; 476(2): 193-209, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30530841

RESUMO

The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.


Assuntos
Cálcio/química , Calmodulina/química , Modelos Moleculares , Miocárdio/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Calmodulina/genética , Calmodulina/metabolismo , Humanos , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
20.
Mycoses ; 62(2): 144-151, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30256460

RESUMO

BACKGROUND: The incidence of fungal keratitis has increased in recent years. While the epidemiology and clinical roles of various Candida and Fusarium species have been relatively well-identified in infections of the eye, data regarding keratitis caused by Aspergillus species are scant. Accurate and rapid diagnosis is important for successful management of this infection. OBJECTIVES: To present the first molecular epidemiological data from Mexico during a 4-year period of cases admitted with Aspergillus keratitis to a tertiary care eye institution in Mexico City. PATIENTS/METHODS: A total of 25 cases of keratitis were included in the study. Aspergillus isolates were identified by sequencing the calmodulin gene. Antifungal susceptibility was tested according to CLSI. RESULTS: The aetiological agents belonged to Aspergillus flavus (n = 13), Aspergillus effusus (n = 1), Aspergillus tamarii (n = 4), Aspergillus sydowii (n = 1), Aspergillus protuberus (n = 3) and Aspergillus terreus (n = 3). All strains had low minimum inhibitory concentrations (MICs) of itraconazole and voriconazole (VCZ). Amphotericin B and natamycin showed moderate elevated MICs. CONCLUSIONS: Early diagnosis and application of topical VCZ 1% were associated with good outcome. Monitoring of local epidemiological data plays an important role in clinical practice.


Assuntos
Aspergilose/epidemiologia , Aspergillus/isolamento & purificação , Infecções Oculares Fúngicas/epidemiologia , Ceratite/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergillus/classificação , Aspergillus/efeitos dos fármacos , Aspergillus/genética , Calmodulina/genética , Infecções Oculares Fúngicas/microbiologia , Feminino , Proteínas Fúngicas/genética , Humanos , Incidência , Ceratite/microbiologia , Masculino , México/epidemiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Centros de Atenção Terciária , Adulto Jovem
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