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1.
Adv Exp Med Biol ; 1131: 183-213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646511

RESUMO

Ca2+ binding proteins (CBP) are of key importance for calcium to play its role as a pivotal second messenger. CBP bind Ca2+ in specific domains, contributing to the regulation of its concentration at the cytosol and intracellular stores. They also participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e. decoding Ca2+ signals. Since CBP are integral to normal physiological processes, possible roles for them in a variety of diseases has attracted growing interest in recent years. In addition, research on CBP has been reinforced with advances in the structural characterization of new CBP family members. In this chapter we have updated a previous review on CBP, covering in more depth potential participation in physiopathological processes and candidacy for pharmacological targets in many diseases. We review intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins, calcineurin and neuronal Ca2+ sensor proteins (NCS). We also address intracellular CBP lacking the EF-hand domain: annexins, CBP within intracellular Ca2+ stores (paying special attention to calreticulin and calsequestrin), proteins that contain a C2 domain (such as protein kinase C (PKC) or synaptotagmin) and other proteins of interest, such as regucalcin or proprotein convertase subtisilin kexins (PCSK). Finally, we summarise the latest findings on extracellular CBP, classified according to their Ca2+ binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) ɣ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca2+-dependent (C)-type lectin-like domains; (vi) Ca2+-binding pockets of family C G-protein-coupled receptors.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo
2.
Zhonghua Gan Zang Bing Za Zhi ; 27(8): 628-633, 2019 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-31594081

RESUMO

Objective: To investigate the change in expression of anti-senescence marker protein calmodulin (RGN) in liver tissues of rats with immune hepatic fibrosis, and to observe the effect of compound glutathione inosine injection (CGII) on it. Methods: Rat liver fibrosis model was induced by intraperitoneal injection of porcine serum, and CGII intervention was administered at the appropriate time. Rat liver tissues were stained with HE and Masson. RGN and protein expression at mRNA in liver tissues was detected by fluorescence quantitative PCR and immunohistochemistry. One-way Anova was used for measurement data. LDS test was used for two-way comparison, and pathological semi-quantitative results were analyzed by rank-sum test. Results: The relative expression of RGN mRNA and protein in liver tissue of fibrotic rats was 82.23 ± 15.21 and 12.52 ± 3.23, respectively, which were significantly lower than that of normal rats 176.39 ± 11.35 and 59.23 ± 9.13 (P < 0.01). The degree of liver fibrosis in fibrotic rats after CGII intervention was significantly lower than fibrotic rats. The relative expression of RGN mRNA and protein in the intervention group was 168.78 ± 21.31 and 46.42 ± 4.71, respectively, which were significantly higher than fibrosis and spontaneous recovery group. The difference was statistically significant (P < 0.01). The relative expression of RGN mRNA and protein in the spontaneous recovery group was 86.23 ± 17.16 and 14.34 ± 5.16, which was higher than model group. The difference was not statistically significant (P > 0.05). Conclusion: The expression of RGN in liver tissue of rats with hepatic fibrosis induced by porcine serum is decreased, while the expression of RGN increases with the decrease of fibrosis after CGII intervention, suggesting that the protein may play an important role in the development of liver fibrosis.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glutationa/farmacologia , Inosina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cirrose Hepática Experimental/tratamento farmacológico , Animais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Ratos , Ratos Sprague-Dawley , Suínos
3.
J Agric Food Chem ; 67(40): 11035-11043, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517486

RESUMO

Ca2+-binding proteins (CaBPs) are widely distributed as Ca2+ sensor relay proteins that regulate various cellular processes, including Ca2+ homeostasis. Diamide insecticides such as cyantraniliprole kill insects by disrupting the Ca2+ homeostasis in muscle cells. However, less attention has been paid to the roles of CaBPs in response to insecticides. In this study, two CaBP genes (BtCaBP1 and BtCaBP2) were identified in the whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and their functions in response to cyantraniliprole were investigated. After expression of BtCaBP1 and BtCaBP2 in vitro, the results of Ca2+ imaging and cytotoxicity assay revealed that the overexpression of each of the BtCaBPs stabilized Ca2+ concentration in the cytoplasm after exposure to cyantraniliprole and decreased the toxicity of cyantraniliprole against Sf9 cells. However, the knockdown of BtCaBP1 or BtCaBP2 in vivo significantly increased the toxicity of cyantraniliprole to B. tabaci. Taken together, these results provide evidence that BtCaBP1 and BtCaBP2 play a role in response to cyantraniliprole exposure through stabilization of Ca2+ concentration in whiteflies.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Pirazóis/farmacologia , ortoaminobenzoatos/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Hemípteros/classificação , Hemípteros/metabolismo , Proteínas de Insetos/genética , Filogenia
4.
Life Sci ; 233: 116746, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31401313

RESUMO

AIM: Diabetes accelerates pro-atherogenic and pro-osteogenic phenotypes of vascular smooth muscle cells (VSMCs), an important process for vascular calcification. Reticulocalbin 2 (RCN2) is a candidate gene for atherosclerosis and involved in vascular remodeling in hypertension. However, the role of RCN2 in VSMCs calcification under diabetic conditions is unclear. MATERIALS AND METHODS: Expression of RCN2 and Runt-related transcription factor 2 (Runx2) in femoropopliteal arterial plaques was compared between type 2 diabetes mellitus (DM) and non-DM patients using immunohistochemical staining (IHCS). Human aortic VSMCs (HAVSMCs) were analyzed under RCN2 gene knockdown and overexpression conditions. Alizarin red staining and intracellular calcium deposition quantification were used to observe calcification induced in vitro under normal glucose or high glucose combined with ß-glycerol phosphoric acid conditions. The cells were investigated for gene modulation of osteogenic differentiation markers using Western blotting. KEY FINDINGS: The expression of RCN2 and Runx2 in femoropopliteal artery plaques was significantly higher in DM than in non-DM patients. In addition, a significant positive correlation was observed between RCN2 and Runx2 levels. RCN2 was highly expressed when HAVSMCs were treated with high glucose and the expression levels correlated with the calcification characteristics. RCN2 upregulated osteogenic transformation markers Runx2 and Osterix in HAVSMCs and downregulated contractile phenotype markers α-SMA and SM22α. SIGNIFICANCE: The results from this study indicate RCN2 is a major factor in mediating the calcification process of HAVSMCs in diabetic conditions. Thus, RCN2 may serve as a future therapeutic target for vascular calcification in diabetes.


Assuntos
Aterosclerose/complicações , Proteínas de Ligação ao Cálcio/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Músculo Liso Vascular/citologia , Osteogênese , Calcificação Vascular/etiologia , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Músculo Liso Vascular/metabolismo , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia
5.
Chem Biol Interact ; 311: 108772, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31351049

RESUMO

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.


Assuntos
Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína 28 com Motivo Tripartido/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Proteínas dos Microfilamentos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismo , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Regulação para Cima/efeitos dos fármacos
6.
Medicine (Baltimore) ; 98(28): e16356, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31305429

RESUMO

BACKGROUND: The prognostic significance of S100A14 for survival of cancer patients remains controversial. Therefore, we conducted this meta-analysis to explore the association between S100A14 expression and cancer prognosis. METHOD: Eligible studies were identified by searching the online databases Pubmed and EMBASE up to August 2018. Odds ratios (ORs) with 95% confidence intervals (CIs) severed as the summarized statistics for clinicopathological assessments and hazard ratios (HRs) with 95% CIs were calculated to clarify the correlation between S100A14 expression and prognosis of different cancers. RESULTS: A total of 11 studies with 1651 cancer patients were enrolled. The results indicated that S100A14 expression was not significantly associated with overall survival (OS) in total various cancers (HR = 1.54, 95% CI:0.89-2.67, P = .121). Further subgroup analysis stratified by tumor type showed that elevated S100A14 expression was associated with poor OS in breast cancer (HR = 3.66, 95% CI: 1.75-7.62, P < .001) and in ovarian cancer patients (HR = 3.78, 95%CI: 1.63-8.73, P = .002). Interestingly, high S100A14 expression was correlated with poor tumor differentiation (OR = 2.51, 95% CI: 1.52-4.13, P < .001). However, there were no significant correlations between S100A14 expression and other clinicopathologic characteristics. Begg funnel plot and Egger test showed that no publication bias was detected. CONCLUSIONS: Our meta-analysis suggests that S100A14 overexpression might be a predictive biomarker for poor prognosis in patients with breast cancer and ovarian cancer. Large-scale studies are required to confirm these results.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Biomarcadores Tumorais/metabolismo , Humanos , Prognóstico
7.
Nat Commun ; 10(1): 3027, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289275

RESUMO

Fibrosis accompanying wound healing can drive the failure of many different organs. Activated fibroblasts are the principal determinants of post-injury pathological fibrosis along with physiological repair, making them a difficult therapeutic target. Although activated fibroblasts are phenotypically heterogeneous, they are not recognized as distinct functional entities. Using mice that express GFP under the FSP1 or αSMA promoter, we characterized two non-overlapping fibroblast subtypes from mouse hearts after myocardial infarction. Here, we report the identification of FSP1-GFP+ cells as a non-pericyte, non-hematopoietic fibroblast subpopulation with a predominant pro-angiogenic role, characterized by in vitro phenotypic/cellular/ultrastructural studies and in vivo granulation tissue formation assays combined with transcriptomics and proteomics. This work identifies a fibroblast subtype that is functionally distinct from the pro-fibrotic αSMA-expressing myofibroblast subtype. Our study has the potential to shift our focus towards viewing fibroblasts as molecularly and functionally heterogeneous and provides a paradigm to approach treatment for organ fibrosis.


Assuntos
Fibroblastos/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose/etiologia , Fibrose/patologia , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Quimeras de Transplante
8.
Medicine (Baltimore) ; 98(26): e15872, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31261495

RESUMO

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer accounts for ∼20% of invasive breast cancers and is associated with poor prognostics. The recent outcome of HER2+ breast cancer treatment has been vastly improved owing to the application of antibody-targeted therapies. Trastuzumab (Herceptin) is a monoclonal antibody designed to target HER2+ breast cancer cells. In addition to improved survival in the adjuvant treatment of HER2+ breast cancer, trastuzumab treatment has also been associated with cardiotoxicity side effect. However, the molecular mechanisms of trastuzumab action and trastuzumab-mediated cardiotoxicity are still not fully understood. Previous research utilized bulk transcriptomics analysis to study the underlining mechanisms, which relied on averaging molecular signals from bulk tumor samples and might have overlooked key expression features within breast cancer tumor. In contrast to previous research, we compared the single cancer cell level transcriptome profile between trastuzumab-treated and nontreated patients to reveal a more in-depth transcriptome profile. A total of 461 significantly differential expressed genes were identified, including previously defined and novel gene expression signatures. In addition, we found that trastuzumab-enhanced MGP gene expression could be used as prognostics marker for longer patient survival in breast invasive carcinoma patients, and validated our finding using TCGA (The Cancer Genome Atlas) breast cancer dataset. Moreover, our study revealed a 48-gene expression signature that is associated with cell death of cardiomyocytes, which could be used as early biomarkers for trastuzumab-mediated cardiotoxicity. This work is the first study to look at single cell level transcriptome profile of trastuzumab-treated patients, providing a new understanding of the molecular mechanism(s) of trastuzumab action and trastuzumab-induced cardiotoxicity side effects.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Transcriptoma/efeitos dos fármacos , Trastuzumab/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/epidemiologia , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/epidemiologia , Carcinoma Ductal de Mama/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Projetos Piloto , Prognóstico , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Análise de Sobrevida
9.
Life Sci ; 232: 116600, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251998

RESUMO

Neuroinflammation is one of the significant neuropathological conditions in Parkinson's disease (PD) which is due to microglial and astrocytes activation leads to progressive dopaminergic neuronal loss. To date, Current PD drugs offers only symptomatic relief with adverse effects and lack of ability to prevent the progression of neurodegeneration. Therefore, a better approach to develop a multi potent drug of natural origin would be beneficial in managing the disease. Therefore, the present study aimed to investigate the neuroprotective and anti-inflammatory effects of PHL by exploring its neuroprotective mechanism in 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP) induced PD in mice. MPTP intoxication in mice cause motor abnormalities, decreased dopamine (DA) levels, reduced tyrosine hydroxylase (TH) enzyme protein expression and inflammation which were effectively restored by PHL. Moreover gliotic specific inflammatory markers like glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor protein-1 (Iba-1), iNOS and COX-2 were found to be expressed more in MPTP intoxicated mice, Further the levels of proinflammatory cytokines like IL-ß, IL-6, and TNF-α were significantly upregulated in MPTP intoxicated mice, these deleterious responses were diminished to extend neuroprotection by PHL treatment. Our findings strongly suggest PHL as a potent therapeutic agent in treating PD.


Assuntos
Transtornos Parkinsonianos/tratamento farmacológico , Floretina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Neuroimunomodulação/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Tirosina 3-Mono-Oxigenase/metabolismo
10.
Nat Commun ; 10(1): 2709, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221966

RESUMO

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time. To test if this assumption is true, we compare the folding of a ribosome-bound, multi-domain calcium-binding protein stalled at different points in translation with the nascent chain as is it being synthesized in real-time, via optical tweezers. On stalled ribosomes, a misfolded state forms rapidly (1.5 s). However, during translation, this state is only attained after a long delay (63 s), indicating that, unexpectedly, the growing polypeptide is not equilibrated with its ensemble of accessible conformations. Slow equilibration on the ribosome can delay premature folding until adequate sequence is available and/or allow time for chaperone binding, thus promoting productive folding.


Assuntos
Modelos Moleculares , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Proteínas de Bactérias/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Peptídeos/metabolismo , Domínios Proteicos/fisiologia , Ribossomos/metabolismo , Fatores de Tempo
11.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035388

RESUMO

Transactivation of p21 (cyclin-dependent kinase inhibitor 1A, CDKN1A) is closely related to the recruitment of transcription cofactors at the p53 responsive elements (p53REs) in its promoter region. Human chromatin remodeling enzyme INO80 can be recruited to the p53REs of p21 promoter and negatively regulates p21. As one of the key subunits of the INO80 complex, YY1 has also been confirmed to bind to the p53RE sites of p21 promoter. Importantly, YY1 was recently reported to be bound and stabilized by BCCIP (BRCA2 and CDKN1A-interacting protein). Therefore, we hypothesized that the YY1/BCCIP complex plays an important role in regulating the transactivation of p21. Here we present evidence that the YY1/BCCIP complex coordinatively regulates p53RE-mediated p21 transactivation. We first confirmed the cross-interaction between YY1, BCCIP, and p53, suggesting an intrinsic link between three proteins in the regulation of p21 transcription. In dual luciferase assays, YY1 inhibited p53RE-mediated luciferase activity, whereas BCCIP revealed the opposite effect. More interestingly, the region 146-270 amino acids of YY1, which bound to BCCIP, increased p53-mediated luciferase activity, indicating the complexity of the YY1/BCCIP complex in co-regulating p21 transcription. Further in-depth research confirmed the co-occupancy of YY1/BCCIP with p53 at the p53RE-proximal region of p21. Lentiviral-mediated knockdown of BCCIP inhibited the recruitment of p53 and YY1 at the p53RE proximal region of p21; however, this phenomenon was reversed by expressing exogenous YY1, suggesting the collaborative regulation of YY1/BCCIP complex in p53RE-mediated p21 transcription. These data provide new insights into the transcriptional regulation of p21 by the YY1/BCCIP complex.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HCT116 , Humanos , Proteínas Nucleares/genética , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Fator de Transcrição YY1/genética
12.
MBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064825

RESUMO

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Adaptação Fisiológica , Transporte Biológico , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions , Citosol/química , Citosol/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade
13.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086053

RESUMO

Calcium-binding proteins (CBPs) can influence and react to Ca2+ transients and modulate the activity of proteins involved in both maintaining homeostatic conditions and protecting cells in harsh environmental conditions. Hibernation is a strategy that evolved in vertebrate and invertebrate species to survive in cold environments; it relies on molecular, cellular, and behavioral adaptations guided by the neuroendocrine system that together ensure unmatched tolerance to hypothermia, hypometabolism, and hypoxia. Therefore, hibernation is a useful model to study molecular neuroprotective adaptations to extreme conditions, and can reveal useful applications to human pathological conditions. In this review, we describe the known changes in Ca2+-signaling and the detection and activity of CBPs in the nervous system of vertebrate and invertebrate models during hibernation, focusing on cytosolic Ca2+ buffers and calmodulin. Then, we discuss these findings in the context of the neuroprotective and neural plasticity mechanisms in the central nervous system: in particular, those associated with cytoskeletal proteins. Finally, we compare the expression of CBPs in the hibernating nervous system with two different conditions of neurodegeneration, i.e., platinum-induced neurotoxicity and Alzheimer's disease, to highlight the similarities and differences and demonstrate the potential of hibernation to shed light into part of the molecular mechanisms behind neurodegenerative diseases.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Hibernação/fisiologia , Neuroproteção/fisiologia , Animais , Citoesqueleto/metabolismo , Humanos
14.
Ann Otol Rhinol Laryngol ; 128(6_suppl): 125S-133S, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31092028

RESUMO

OBJECTIVES: Early in his career, David Lim recognized the scientific impact of genetically anomalous mice exhibiting otoconia agenesis as models of drastically compromised vestibular function. While these studies focused on the mutant pallid mouse, contemporary genetic tools have produced other models with engineered functional modifications. Lim and colleagues foresaw the need to analyze vestibular epithelia from pallid mice to verify the absence of downstream consequences that might be secondary to the altered load represented by otoconial agenesis. More generally, however, such comparisons also contribute to an understanding of the susceptibility of labyrinthine sensory epithelia to more widespread cellular changes associated with what may appear as isolated modifications. METHODS: Our laboratory utilizes a model of vestibular hypofunction produced through genetic alteration, the otoferlin-null mouse, which has been shown to exhibit severely compromised stimulus-evoked neurotransmitter release in type I hair cells of the utricular striola. The present study, reminiscent of early investigations of Lim and colleagues that explored the utility of a genetically altered mouse to explore its utility as a model of vestibular hypofunction, endeavored to compare the expression of the hair cell marker oncomodulin in vestibular epithelia from wild-type and otoferlin-null mice. RESULTS: We found that levels of oncomodulin expression were much greater in type I than type II hair cells, though were similar across the 3 genotypes examined (ie, including heterozygotes). CONCLUSION: These findings support the notion that modifications resulting in a specific component of vestibular hypofunction are not accompanied by widespread morphologic and cellular changes in the vestibular sensory epithelia.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Ciliadas Vestibulares/fisiologia , Proteínas de Membrana/genética , Fenótipo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout
15.
Int J Mol Sci ; 20(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064106

RESUMO

Presynaptic Ca2+ entry occurs through voltage-gated Ca2+ (CaV) channels which are activated by membrane depolarization. Depolarization accompanies neuronal firing and elevation of Ca2+ triggers neurotransmitter release from synaptic vesicles. For synchronization of efficient neurotransmitter release, synaptic vesicles are targeted by presynaptic Ca2+ channels forming a large signaling complex in the active zone. The presynaptic CaV2 channel gene family (comprising CaV2.1, CaV2.2, and CaV2.3 isoforms) encode the pore-forming α1 subunit. The cytoplasmic regions are responsible for channel modulation by interacting with regulatory proteins. This article overviews modulation of the activity of CaV2.1 and CaV2.2 channels in the control of synaptic strength and presynaptic plasticity.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Canais de Cálcio Tipo N/genética , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Terminações Pré-Sinápticas/fisiologia , Potenciais Sinápticos
16.
Meat Sci ; 155: 50-60, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31075739

RESUMO

The aim of this study was to determine the extent to which calpastatin (CASN) variants (based on two chromatographic peaks; CASN-P1 and CASN-P2) explain variation in µ-calpain autolysis, protein degradation, and changes in the sarcoplasmic proteome observed during postmortem aging of beef. The Longissimus lumborum (LL) and Triceps brachii (TB) muscles were obtained from six crossbred steers and samples prepared from day 0, 1 and 7 postmortem (pm). The decline of CASN activity during aging was due to decrease of CASN-P2 in both muscles. The CASN-P2:µ-calpain ratio at day 0 was greater for TB, which presented lesser calpain autolysis, myofibrillar protein degradation, and fewer sarcoplasmic proteome changes during aging. Changes in abundance of Heat shock protein 70 family in the sarcoplasmic fraction were positively associated to proteolysis during aging, with greater differences in LL.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Músculo Esquelético/química , Carne Vermelha/análise , Animais , Bovinos , Proteínas de Choque Térmico HSP70/análise , Masculino , Miofibrilas , Mudanças Depois da Morte , Proteólise , Proteoma
17.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987093

RESUMO

Advanced upper urinary tract urothelial carcinoma (UTUC) is often associated with poor oncologic outcomes. The secreted protein acidic and rich in cysteine-like 1 (SPARCL1) protein, belongs to the SPARC-related family of matricellular proteins. Much literature has been published describing the role of SPARCL1 in the prognosis many cancers. In this study, methylated promoter regions in high-grade and high-stage upper urinary urothelial tumours compared with normal urothelium were analyzed and revealed that SPARCL1 was the most significantly hypermethylated gene in UTUC tissues. Then we prospectively collected UTUC samples and adjacent normal urothelium for pyrosequencing validation, identifying significant CpG site methylation in UTUC tissues. In addition, SPARCL1 RNA levels were significantly lower in UTUC samples. Multivariate Cox regression analysis from 78 patients with solitary renal pelvic or ureteral pT3N0M0 urothelial carcinomas revealed that only negative SPARCL1 expression and nonpapillary tumour architecture were independently associated with systemic recurrence (p = 0.011 and 0.008, respectively). In vitro studies revealed that the behaviour of BFTC-909 cells was less aggressive and more sensitive to radiation or chemotherapy after SPARCL1 overexpression. Thus, SPARCL1 could be considered as a prognostic marker and help decision-making in clinical practice.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Metilação de DNA/genética , Proteínas da Matriz Extracelular/genética , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologia , Urotélio/patologia , Idoso , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Estudos de Coortes , Metilação de DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Regiões Promotoras Genéticas/genética , Análise de Regressão , Neoplasias Urológicas/tratamento farmacológico , Neoplasias Urológicas/radioterapia
18.
J Neuroinflammation ; 16(1): 79, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971251

RESUMO

BACKGROUND: Microglia play crucial roles in the maintenance of brain homeostasis. Activated microglia show a biphasic influence, promoting beneficial repair and causing harmful damage via M2 and M1 microglia, respectively. It is well-known that microglia are initially activated to the M2 state and subsequently switch to the M1 state, called M2-to-M1 class switching in acute ischemic models. However, the activation process of microglia in chronic and sporadic hypertension remains poorly understood. We aimed to clarify the process using a chronic hypertension model, the deoxycorticosterone acetate (DOCA)-salt-treated Wistar rats. METHODS: After unilateral nephrectomy, the rats were randomly divided into DOCA-salt, placebo, and control groups. DOCA-salt rats received a weekly subcutaneous injection of DOCA (40 mg/kg) and were continuously provided with 1% NaCl in drinking water. Placebo rats received a weekly subcutaneous injection of vehicle and were provided with tap water. Control rats received no administration of DOCA or NaCl. To investigate the temporal expression profiles of M1- and M2-specific markers for microglia, the animals were subjected to the immunohistochemical and biochemical studies after 2, 3, or 4 weeks DOCA-salt treatment. RESULTS: Hypertension occurred after 2 weeks of DOCA and salt administration, when round-shaped microglia with slightly shortened processes were observed juxtaposed to the vessels, although the histopathological findings were normal. After 3 weeks of DOCA and salt administration, M1-state perivascular and parenchyma microglia significantly increased, when local histopathological findings began to be observed but cerebrovascular destruction did not occur. On the other hand, M2-state microglia were never observed around the vessels at this period. Interestingly, prior to M1 activation, about 55% of perivascular microglia transiently expressed Ki-67, one of the cell proliferation markers. CONCLUSIONS: We concluded that the resting perivascular microglia directly switched to the pro-inflammatory M1 state via a transient proliferative state in DOCA-salt rats. Our results suggest that the activation machinery of microglia in chronic hypertension differs from acute ischemic models. Proliferative microglia are possible initial key players in the development of hypertension-induced cerebral vessel damage. Fine-tuning of microglia proliferation and activation could constitute an innovative therapeutic strategy to prevent its development.


Assuntos
Encéfalo/patologia , Proliferação de Células/fisiologia , Hipertensão/complicações , Hipertensão/patologia , Microglia/classificação , Microglia/patologia , Animais , Antígenos CD/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Carboximetilcelulose Sódica/farmacologia , Proliferação de Células/efeitos dos fármacos , Acetato de Desoxicorticosterona/toxicidade , Modelos Animais de Doenças , Lateralidade Funcional , Hipertensão/diagnóstico por imagem , Hipertensão/etiologia , Antígeno Ki-67/metabolismo , Imagem por Ressonância Magnética , Masculino , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Mineralocorticoides/toxicidade , Nefrectomia/efeitos adversos , Ratos , Ratos Wistar , Cloreto de Sódio/toxicidade , Fatores de Tempo
19.
J Neuroinflammation ; 16(1): 78, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971286

RESUMO

BACKGROUND: Central sensitization is an important mechanism of chronic migraine (CM) and is related to the inflammatory response of microglia. The NOD-like receptor protein 3 (NLRP3) inflammasome may regulate the inflammatory process of microglia in several neurological diseases, but its role in CM is largely unknown. Therefore, the aim of this study was to identify the precise role of microglial NLRP3 in CM. METHODS: An experimental CM mouse model was established by repeated intraperitoneal (i.p) injection with nitroglycerin (NTG). We evaluated the expression levels of NLRP3 and its downstream interleukin (IL)-1ß protein in the trigeminal nucleus caudalis (TNC; which is a central area relevant to migraine pain) at different time points. To further examine the effects of the NLRP3 inflammasome pathway on central sensitization of CM, we examined MCC950, an NLRP3 inflammasome-specific inhibitor, and IL-1ra, an IL-1ß antagonist, whether altered NTG-induced mechanical hyperalgesia of the periorbital area and hind paw. The effect of MCC950 and IL-1ra on c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK) and calcitonin gene-related peptide (CGRP) expression in the TNC were also analyzed. The cell localization of NLRP3 and IL-1ß in the TNC was evaluated by immunofluorescence staining. RESULTS: Repeated NTG administration induced acute and chronic mechanical hyperalgesia and increased expression of NLRP3 and IL-1ß. Blockade of NLRP3 or IL-1ß reduced NTG-induced hyperalgesia, and this effect was accompanied by a significant inhibition of the NTG-induced increase in p-ERK, c-Fos and CGRP levels in the TNC. Immunofluorescence staining revealed that NLRP3 and IL-1ß were mainly expressed in microglia in the TNC, and the IL-1ß receptor, IL-1R, was mainly expressed in neurons in the TNC. CONCLUSIONS: These results indicate that NLRP3 activation in the TNC participates in the microglial-neuronal signal by mediating the inflammatory response. This process contributes to the central sensitization observed in CM.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Interleucina-1beta/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Sulfonas/uso terapêutico , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hiperalgesia/induzido quimicamente , Injeções Intraventriculares , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Transtornos de Enxaqueca/induzido quimicamente , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nitroglicerina/toxicidade , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Vasodilatadores/toxicidade
20.
J Neuroinflammation ; 16(1): 82, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975169

RESUMO

BACKGROUND: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. METHODS: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. RESULTS: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. CONCLUSIONS: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Neuralgia/patologia , Receptores de Lisofosfolipídeos/metabolismo , Medula Espinal/patologia , Análise de Variância , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Fatores Reguladores de Interferon/metabolismo , Lisofosfolipídeos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Medição da Dor , Limiar da Dor/fisiologia , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Receptores de Lisofosfolipídeos/antagonistas & inibidores , Receptores de Lisofosfolipídeos/genética , Fatores de Tempo
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