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1.
Mem Inst Oswaldo Cruz ; 115: e200142, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33053076

RESUMO

BACKGROUND: Calpains are present in almost all organisms and comprise a family of calcium-dependent cysteine peptidases implicated in crucial cellular functions. Trypanosoma cruzi, the causative agent of Chagas disease, presents an expansion on this gene family with unexplored biological properties. OBJECTIVES: Here, we searched for calpains in the T. cruzi genome, evaluated the mRNA levels, calpain activity and the protein expression and determined the cellular localisation in all three parasite life cycle forms. METHODS/FINDINGS: Sixty-three calpain sequences were identified in T. cruzi CL Brener genome, with fourteen domain arrangements. The comparison of calpain mRNA abundance by quantitative polymerase chain reaction (qPCR) revealed seven up-regulated sequences in amastigotes and/or bloodstream trypomastigotes and five in epimastigotes. Western Blotting analysis revealed seven different molecules in the three parasite forms, and one amastigote-specific, while no proteolytic activity could be detected. Flow cytometry assays revealed a higher amount of intracellular calpains in amastigotes and/or trypomastigotes in comparison to epimastigotes. Finally, ultrastructural analysis revealed the presence of calpains in the cytoplasm, vesicular and plasma membranes of the three parasite forms, and in the paraflagellar rod in trypomastigotes. CONCLUSION: Calpains are differentially expressed and localised in the T. cruzi life cycle forms. This study adds data on the calpain occurrence and expression pattern in T. cruzi.


Assuntos
Calpaína/genética , Trypanosoma cruzi/genética , Animais , Western Blotting , Calpaína/metabolismo , Doença de Chagas , Estágios do Ciclo de Vida , RNA Mensageiro/genética
2.
Am J Chin Med ; 48(5): 1179-1202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668972

RESUMO

Over-expression of calpains in tumor tissues can be associated with cancer progression. Thus, inhibition of calpain activity using specific inhibitors has become a novel approach to control tumor growth. In this study, the anticancer potential of cryptotanshinone in combination with calpain inhibitor had been investigated in colon cancer cells and tumor xenograft. Cryptotanshinone elicited an initial endoplasmic reticular (ER) stress response, whereas prolonged stress would result in the promotion of apoptosis. It was then discovered that cryptotanshinone could cause rapid and sustained increase in cytosolic calcium in colon cancer cells accompanied by early GRP78 overexpression, which could be attenuated by pre-treatment of the calcium chelator BAPTA-AM. Cryptotanshinone also facilitated an early increase in calpain activity, which could be blocked by BAPTA-AM or the calpain inhibitor PD150606. A dynamic interaction between GRP78 and calpain during the action of cryptotanshinone was unveiled. This together with the altered NF-[Formula: see text]B signaling could be abolished by calpain inhibitor. GRP78 knockdown increased the sensitivity of cancer cells to cryptotanshinone-evoked apoptosis and reduction of cancer cell colony formation. Such sensitization of drug action had been confirmed to be p53-dependent by using p53-mutated (HT-29) and p53-deficient (HCT116 p53-∕-) cells. The synergistic antitumor effect of cryptotanshinone and calpain inhibitor was further exhibited in vivo. Taken together, findings in this study exemplify a new chemotherapeutic regimen comprising cryptotanshinone and calpain inhibitor by regulation of calpain and calcium homeostasis. This has provided us with new insights in the search of a potential target-specific neoadjuvant therapy against colon cancer.


Assuntos
Antineoplásicos Fitogênicos , Apoptose/efeitos dos fármacos , Apoptose/genética , Cálcio/metabolismo , Calpaína/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Homeostase/efeitos dos fármacos , Fenantrenos/farmacologia , Fenantrenos/uso terapêutico , Fitoterapia , Proteína Supressora de Tumor p53/metabolismo , Animais , Calpaína/genética , Neoplasias do Colo/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos Nus , Células Tumorais Cultivadas
3.
Vascular ; 28(5): 655-663, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32375599

RESUMO

OBJECTIVES: Arterial stiffness is recognized as an important predictor of cardiovascular disease morbidity and mortality, independent of traditional cardiovascular disease risk factors. Given that arterial tissue is not easily accessible, most gene expression studies on arterial stiffness have been conducted on animals or on patients who have undergone by-pass surgeries. In order to obtain a deeper understanding of early changes of arterial stiffness, this study compared transcriptome profiles between healthy adults with higher and lower arterial stiffness. METHODS: The sample included 20 healthy female adults without cardiovascular disease. Arterial stiffness was measured by carotid-femoral pulse wave velocity, the "gold-standard" measure of central arterial stiffness. Peripheral blood samples collected to PAXgene™ RNA tubes were used for RNA sequencing (RNA-seq). The potential confounding effects of age, body mass index, and mean arterial pressure were controlled for in RNA-seq analysis. To validate RNA-seq results, quantitative real-time PCR (qRT-PCR) was performed for six selected genes. RESULTS: The findings demonstrated that genes including CAPN9, IL32, ERAP2, RAB6B, MYBPH, and miRNA626 were down-regulated, and that MOCS1 gene was up-regulated among the people with higher arterial stiffness. Real-time PCR showed that the changes of CAPN9, IL32, ERAP2, and RAB6B were in concordance with RNA-seq data, and confirmed the validity of the gene expression profiles obtained by RNA-seq analysis. CONCLUSIONS: Previous studies have suggested the potential roles of CAPN9, IL32, and ERAP2 in structural changes of the arterial wall through up-regulation of metalloproteinases. However, the current study showed that CAPN9, IL32, and ERAP2 were down-regulated in the individuals with higher arterial stiffness, compared with those with lower arterial stiffness. The unexpected directions of expression of these genes may indicate an effort to maintain vascular homeostasis during increased arterial stiffness among healthy individuals. Further studies are guaranteed to investigate the roles of CAPN9, IL32, and ERAP2 in regulating arterial stiffness in people with and without cardiovascular disease.


Assuntos
Aminopeptidases/genética , Calpaína/genética , Perfilação da Expressão Gênica , Interleucinas/genética , RNA-Seq , Transcriptoma , Rigidez Vascular/genética , Adolescente , Adulto , Pressão Arterial , Velocidade da Onda de Pulso Carótido-Femoral , Regulação para Baixo , Feminino , Redes Reguladoras de Genes , Humanos , Pessoa de Meia-Idade , Adulto Jovem
4.
Am J Physiol Cell Physiol ; 318(6): C1226-C1237, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348180

RESUMO

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.


Assuntos
Calpaína/deficiência , Membrana Celular/enzimologia , Músculo Esquelético/enzimologia , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Distrofia Muscular Animal/enzimologia , Animais , Proteínas de Bactérias/farmacologia , Sinalização do Cálcio , Calpaína/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Modelos Animais de Doenças , Disferlina/deficiência , Disferlina/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Saponinas/farmacologia , Índice de Gravidade de Doença , Estreptolisinas/farmacologia
5.
Oncol Rep ; 43(6): 2093-2104, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236617

RESUMO

Human epidermal growth factor receptor 2 (HER2) is composed of an extracellular domain (ECD), a lipophilic transmembrane region and an intracellular domain (ICD). The most commonly used method to determine the status of HER2 is immunohistochemistry. However, false­negative results are sometimes given, which causes some patients to lose the opportunity for anti­HER2 therapy. We found that calpain­10 may prohibit HER2­ECD into peripheral blood resulting in a HER2­negative result by the immunohistochemical method. We enrolled 289 patients into our experiment to assess the relationship between sHER2­ECD and calpain­10. The results showed that there was a positive correlation between sHER2­ECD and calpain­10. Moreover, we also investigated the prognostic values of sHER2­ECD and calpain­10 in breast cancer patients. According to the follow­up results, positive sHER2­ECD and tissue calpain­10 were indicative of a poor prognosis in breast cancer patients. Subsequently, we further validated the relationship between the two molecules in in vitro experiments. In the in vitro experiments, the level of HER2­ECD in the culture medium was increased or decreased with a decrease or increase in calpain­10 by transfection technology, showing an inverse association. The results indicated that sHER2­ECD and tissue calpain­10 levels were powerful factors to assess the status of HER2. In combination with tissue HER2 detection, the occurrence of false­negative HER2 was reduced, providing patients with additional treatment opportunities. In conclusion, sHER2­ECD and tissue calpain­10 may be used as new prognostic indices for breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Calpaína/metabolismo , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Calpaína/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Domínios Proteicos , Regulação para Cima
6.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140411, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32200007

RESUMO

Calpain-3 (CAPN3), a 94-kDa member of the calpain protease family, is abundant in skeletal muscle. Mutations in the CAPN3 gene cause limb girdle muscular dystrophy type 2A, indicating that CAPN3 plays important roles in muscle physiology. CAPN3 has several unique features. A crystallographic study revealed that its C-terminal penta-EF-hand domains form a homodimer, suggesting that CAPN3 functions as a homodimeric protease. To analyze complex formation of CAPN3 in a more convenient manner, we performed blue native polyacrylamide gel electrophoresis and found that the observed molecular weight of native CAPN3, as well as recombinant CAPN3, was larger than 240 kDa. Further analysis by cross-linking and sequential immunoprecipitation revealed that CAPN3 in fact forms a homotrimer. Trimer formation was abolished by the deletion of the PEF domain, but not the CAPN3-specific insertion sequences NS, IS1, and IS2. The PEF domain alone formed a homodimer, as reported, but addition of the adjacent CBSW domain to its N-terminus reinforced the trimer-forming property. Collectively, these results suggest that CAPN3 forms a homotrimer in which the PEF domain's dimer-forming ability is influenced by other domains.


Assuntos
Calpaína/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Animais , Calpaína/química , Calpaína/genética , Linhagem Celular , Motivos EF Hand , Feminino , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutagênese Insercional , Mutação , Domínios Proteicos
7.
Ann Clin Lab Sci ; 50(1): 24-30, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32161009

RESUMO

OBJECTIVE: Calpain 6 (CAPN6) is one of the calcium-dependent intracellular nonlysosomal proteases that are dysregulated in uterine leiomyomas (UtLMs). However, its function and mechanism in UtLMs is still unknown. METHODS: The correlation between CAPN6 expression and UtLMs was analyzed by the Gene Expression Omnibus (GEO). The expression of CAPN6 and Rac1 was detected by quantitative real-time PCR (qPCR) and western blot analysis. Cell proliferation ability was analyzed by a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by flow cytometry. RESULTS: CAPN6 was overexpressed in UtLMs compared with uterine smooth muscle cells (UtSMCs). The downregulation of CAPN6 resulted in decreased cell proliferation and increased apoptosis of UtLMs. Furthermore, mechanical investigations revealed that these inhibitory effects were correlated with Rac1/PAK1 signaling pathways. Silencing the expression of CAPN6 resulted in decreased Rac1 and phospho-PAK1. On the other hand, upregulated Rac1 expression could reverse the reduced phosphorylation of PAK1 induced by CAPN6 silencing. CONCLUSIONS: This data suggests that CAPN6 regulates UtLMs proliferation and apoptosis while being mediated through the Rac1/PAK1 signaling pathway.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Calpaína/metabolismo , Proliferação de Células , Leiomioma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Biomarcadores Tumorais/genética , Calpaína/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leiomioma/metabolismo , Leiomioma/cirurgia , Proteínas Associadas aos Microtúbulos/genética , Miócitos de Músculo Liso/metabolismo , Prognóstico , Células Tumorais Cultivadas , Proteínas rac1 de Ligação ao GTP/genética
8.
Fish Shellfish Immunol ; 98: 19-24, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31899359

RESUMO

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.


Assuntos
Calpaína/genética , Calpaína/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Dourada/genética , Dourada/imunologia , Imunidade Adaptativa/genética , Sequência de Aminoácidos , Animais , Calpaína/química , DNA Complementar/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária
9.
Cancer Sci ; 111(1): 72-83, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31691433

RESUMO

Capn4, also known as CapnS1, is a member of the calpain family, which plays a crucial role in maintaining the activity and function of calpain. We previously reported that Capn4 also plays an essential role in the migration of nasopharyngeal carcinoma (NPC) cells through regulation of (MMP-2) by nuclear factor-kappa B activation. Epstein-Barr virus latent membrane protein 1 (LMP1) is closely related to the malignant functions of NPC; however, the relationship between LMP1 and Capn4 in NPC remain unclear. Immunohistochemical studies showed that the level of LMP1 and Capn4 expression was high in both primary and metastatic NPC tissues, with a significantly positive correlation. We further found that LMP1 was able to upregulate the Capn4 promoter in a dose-dependent way through the C-terminal activation region (CTAR)1 and CTAR2 domains to activate AP-1. Moreover, we also found that LMP1 activated AP-1 through ERK/JNK phosphorylation. These findings indicate that Capn4 coordination with LMP1 promotes actin rearrangement and, ultimately, cellular migration. These results show that Capn4 coordination with LMP1 enhances NPC migration by increasing actin rearrangement involving ERK/JNK/AP-1 signaling. Therapeutically, additional and more specific LMP1 and Capn4 targeted inhibitors could be exploited to treat NPC.


Assuntos
Calpaína/genética , Sistema de Sinalização das MAP Quinases/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Metástase Neoplásica/genética , Fator de Transcrição AP-1/genética , Proteínas da Matriz Viral/genética , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Regulação Neoplásica da Expressão Gênica/genética , Herpesvirus Humano 4/patogenicidade , Humanos , NF-kappa B/genética , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/virologia , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Metástase Neoplásica/patologia , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Regulação para Cima/genética
10.
Life Sci ; 244: 117153, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830479

RESUMO

AIMS: Increased activity of calpain-1 and matrix metalloproteinase (MMP)-2 was observed in different models of arterial hypertension and contribute to thicken the left ventricle (LV) walls and to hypertrophy cardiac myocytes. MMP-2 activity may be regulated by calpain-1 via bioactive molecules activation such as transforming growth factor (TGF)-ß in cardiovascular diseases. This study analyzed whether calpain-1 causes cardiac hypertrophy and dysfunction by modulating the expression and activity of MMP-2 in renovascular hypertension. MAIN METHODS: Male Wistar rats were submitted to two kidneys, one clip (2K1C) model of hypertension or sham surgery and were treated with verapamil (VRP, 8 mg/kg/bid) by gavage from the second to tenth week post-surgery. Systolic blood pressure (SBP) was weekly assessed by tail-cuff plethysmography and morphological and functional parameters of LV were analyzed by echocardiography. MMP-2 activity was analyzed by in situ and gelatin zymography, while calpain-1 activity by caseinolytic assay. MMP-2, calpain-1, TGF-ß and MMP-14/TIMP-2 levels were identified in the LV by western blots. Fluorescence assays were performed to evaluate oxidative stress, MMP-2 and calpain-1 levels. KEY FINDINGS: SBP increased in 2K1C rats and was unaltered by VRP. However, VRP notably ameliorated hypertension-induced increase in LV thickness. VRP decreased hypertension-induced enhances in calpain-1 and MMP-2 activities, oxidative stress and mature TGF-ß levels. Treatment with VRP also decreased the accentuated MMP-14/TIMP-2 levels in 2K1C. SIGNIFICANCE: Treatment with VRP decreases calpain-1 and MMP-2 activities and also reduces TGF-ß and MMP-14/TIMP-2 levels in the LV of hypertensive rats, thus contributing to ameliorate cardiac hypertrophy.


Assuntos
Calpaína/metabolismo , Cardiomegalia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão/complicações , Metaloproteinase 2 da Matriz/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Verapamil/farmacologia , Animais , Calpaína/genética , Cardiomegalia/etiologia , Masculino , Metaloproteinase 2 da Matriz/genética , Ratos , Ratos Wistar , Vasodilatadores/farmacologia
11.
Zhen Ci Yan Jiu ; 45(12): 968-72, 2020 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-33415854

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) on motor function, calpain and calpastatin expression in rats with spinal cord injury, so as to explore the mechanism of EA underlying improvement of acute spinal cord injury. METHODS: Thirty male SD rats were randomly divided into sham operation group, model group and EA group, with 10 rats in each group. The acute moderate spinal cord injury model was established by using a NYU spinal cord impactor. EA was applied to "Jizhong"(GV6) and "Mingmen" (GV4) for 30 min, once daily for 28 d. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function. Histopathological changes of the injured spinal cord were observed after sectioning and Nissl staining, and the expression levels of calpain1, calpain2 and calpastatin mRNA and protein in the spinal cord tissues were detected by using quantitative real-time PCR and Western blot, respectively. RESULTS: The BBB score of the model group was significantly lower than that of the sham operation group (P<0.01), and was significantly higher in the EA group than that of the model group on 14th and 28th day (P<0.01). Compared with the sham operation group, the number of neurons in the model group decreased, and Nissl body stained cells decreased or even disappeared, which was evidently milder in the EA group. Compared with the sham operation group, the expression levels of calpain1 mRNA and protein in the spinal cord of the model group were significantly increased (P<0.01), while the expression levels of calpastatin mRNA and protein were significantly reduced (P<0.01). Following EA intervention, in contrast to the model group, the expression levels of calpain1 mRNA and protein in the EA group were significant down-regulated (P<0.01), calpastatin mRNA and protein expression levels were significantly up-regulated(P<0.01). There was no significant difference in calpain2 mRNA and protein expression among the 3 groups(P>0.05). CONCLUSION: EA can improve the locomotor function of rats with spinal cord injury, which may be related to its effect in inhibiting the activity of calpain in the injured spinal cord.


Assuntos
Eletroacupuntura , Traumatismos da Medula Espinal , Animais , Calpaína/genética , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/terapia
12.
Acta Myol ; 38(3): 163-171, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31788660

RESUMO

Limb-girdle muscular dystrophy (LGMD) type 2A (calpainopathy) is an autosomal recessive disease caused by mutation in the CAPN3 gene. The aim of this study was to examine genetic and phenotypic features of Serbian patients with calpainopathy. The study comprised 19 patients with genetically confirmed calpainopathy diagnosed at the Neurology Clinic, Clinical Center of Serbia and the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia during a ten-year period. Eighteen patients in this cohort had c.550delA mutation, with nine of them being homozygous. In majority of the patients, disease started in childhood or early adulthood. The disease affected shoulder girdle - upper arm and pelvic girdle - thigh muscles with similar frequency, with muscles of lower extremities being more severely impaired. Facial and bulbar muscles were spared. All patients in this cohort, except two, remained ambulant. None of the patients had cardiomyopathy, while 21% showed mild conduction defects. Respiratory function was mildly impaired in 21% of patients. Standard muscle histopathology showed myopathic and dystrophic pattern. In conclusion, the majority of Serbian LGMD2A patients have the same mutation and similar phenotype.


Assuntos
Calpaína/genética , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Adolescente , Adulto , Idade de Início , Alelos , Biópsia , Criança , Feminino , Genótipo , Humanos , Imagem por Ressonância Magnética , Masculino , Distrofia Muscular do Cíngulo dos Membros/diagnóstico por imagem , Distrofia Muscular do Cíngulo dos Membros/epidemiologia , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Mutação , Fenótipo , Sérvia/epidemiologia
13.
Int J Mol Sci ; 20(23)2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31801218

RESUMO

Lithium is the mainstay in the maintenance of bipolar disorder (BD) and the most efficacious pharmacological treatment in suicide prevention. Nevertheless, its use is hampered by a high interindividual variability and important side effects. Genetic and epigenetic factors have been suggested to modulate lithium response, but findings so far have not allowed identifying molecular targets with predictive value. In this study we used next generation sequencing to measure genome-wide miRNA expression in lymphoblastoid cell lines from BD patients excellent responders (ER, n = 12) and non-responders (NR, n = 12) to lithium. These data were integrated with microarray genome-wide expression data to identify pairs of miRNA/mRNA inversely and significantly correlated. Significant pairs were prioritized based on strength of association and in-silico miRNA target prediction analyses to select candidates for validation with qRT-PCR. Thirty-one miRNAs were differentially expressed in ER vs. NR and inversely correlated with 418 genes differentially expressed between the two groups. A total of 331 of these correlations were also predicted by in-silico algorithms. miR-320a and miR-155-3p, as well as three of their targeted genes (CAPNS1 (Calpain Small Subunit 1) and RGS16 (Regulator of G Protein Signaling 16) for miR-320, SP4 (Sp4 Transcription Factor) for miR-155-3p) were validated. These miRNAs and mRNAs were previously implicated in psychiatric disorders (miR-320a and SP4), key processes of the central nervous system (CAPNS1, RGS16, SP4) or pathways involved in mental illnesses (miR-155-3p). Using an integrated approach, we identified miRNAs and their targeted genes potentially involved in lithium response in BD.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Lítio/uso terapêutico , MicroRNAs/genética , Psicotrópicos/uso terapêutico , RNA Mensageiro/genética , Adulto , Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Transtorno Bipolar/fisiopatologia , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Humano , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , MicroRNAs/classificação , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Fator de Transcrição Sp4/genética , Fator de Transcrição Sp4/metabolismo , Resultado do Tratamento
14.
Sci Rep ; 9(1): 15771, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673071

RESUMO

Angiogenesis is involved in both normal physiological and pathological conditions. Vascular endothelial growth factor (VEGF) is a major factor for promoting angiogenesis. The current anti-VEGF therapies have limited efficacy and significant adverse effects. To find novel targets of VEGFA for angiogenesis inhibition, we performed yeast two-hybrid screening and identified calpain-6 as a novel VEGFA-interaction partner and confirmed the endogenous VEGFA-calpain-6 interaction in mammalian placenta. A domain mapping study revealed that the Gly321-Asp500 domain in calpain-6 is required for the interaction with the C-terminus of the VEGFA protein. The functional significance of the VEGFA-calpain-6 interaction was explored by assessing its effect on angiogenesis in vitro. Whereas forced overexpression of calpain-6 increased the secretion of the VEGF protein and tube formation, knockdown of calpain-6 expression abrogated the calpain-6-mediated VEGF secretion and tube formation in HUVECs. Consistent with the domain mapping result, overexpressing calpain-6 without the VEGFA-interacting domain III (Gly321-Asp500) failed to increase the secretion of VEGF protein. Our results identify calpain-6, an unconventional non-proteolytic calpain, as a novel VEGFA-interacting protein and demonstrate that their interaction is necessary to enhance VEGF secretion. Thus, calpain-6 might be a potential molecular target for angiogenesis inhibition in many diseases.


Assuntos
Calpaína/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Calpaína/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Domínios Proteicos , Fator A de Crescimento do Endotélio Vascular/genética
15.
Front Immunol ; 10: 2481, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695698

RESUMO

Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.


Assuntos
Calpaína/imunologia , Citrulinação/imunologia , Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Lâmina Nuclear/imunologia , Animais , Calpaína/genética , Citrulinação/genética , Armadilhas Extracelulares/genética , Humanos , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Lâmina Nuclear/genética , Proteína-Arginina Desiminase do Tipo 4/genética , Proteína-Arginina Desiminase do Tipo 4/imunologia
16.
Sci Transl Med ; 11(520)2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776291

RESUMO

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.


Assuntos
Calpaína/genética , Calpaína/uso terapêutico , Cardiotoxicidade/etiologia , Conectina/genética , Terapia Genética/efeitos adversos , Proteínas Musculares/genética , Proteínas Musculares/uso terapêutico , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/terapia , Processamento de RNA/genética , Animais , Sítios de Ligação , Biomarcadores/sangue , Cardiotoxicidade/sangue , Conectina/química , Dependovirus/genética , Disferlina/deficiência , Disferlina/metabolismo , Estabilidade Enzimática , Regulação da Expressão Gênica , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/sangue , Distrofia Muscular do Cíngulo dos Membros/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Primatas , Domínios Proteicos , Proteólise , Especificidade da Espécie , Distribuição Tecidual , Transgenes
17.
FASEB J ; 33(12): 13131-13144, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31638431

RESUMO

Despite the high and preferential expression of p38γ MAPK in the myocardium, little is known about its function in the heart. The aim of the current study was to elucidate the physiologic and biochemical roles of p38γ in the heart. Expression and subcellular localization of p38 isoforms was determined in mouse hearts. Comparisons of the cardiac function and structure of wild-type and p38γ knockout (KO) mice at baseline and after abdominal aortic banding demonstrated that KO mice developed less ventricular hypertrophy and that contractile function is better preserved. To identify potential substrates of p38γ, we generated an analog-sensitive mutant to affinity tag endogenous myocardial proteins. Among other proteins, this technique identified calpastatin as a direct p38γ substrate. Moreover, phosphorylation of calpastatin by p38γ impaired its ability to inhibit the protease, calpain. We have identified p38γ as an important determinant of the progression of pathologic cardiac hypertrophy after aortic banding in mice. In addition, we have identified calpastatin, among other substrates, as a novel direct target of p38γ that may contribute to the protection observed in p38γKO mice.-Loonat, A. A., Martin, E. D., Sarafraz-Shekary, N., Tilgner, K., Hertz, N. T., Levin, R., Shokat, K. M., Burlingame, A. L., Arabacilar, P., Uddin, S., Thomas, M., Marber, M. S., Clark, J. E. p38γ MAPK contributes to left ventricular remodeling after pathologic stress and disinhibits calpain through phosphorylation of calpastatin.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Remodelação Ventricular/fisiologia , Animais , Calpaína/genética , Ecocardiografia , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Proteína Quinase 12 Ativada por Mitógeno/genética , Fosforilação , Isoformas de Proteínas , Espectrometria de Massas em Tandem , Remodelação Ventricular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Rinsho Shinkeigaku ; 59(11): 740-745, 2019 Nov 08.
Artigo em Japonês | MEDLINE | ID: mdl-31656265

RESUMO

A 33-year-old man presented with slowly progressive weakness in the lower extremities over 8 years. At the age of 16 years, the elevation of serum creatine kinase level was detected. Physical examination revealed scapular winging, exaggerated lumbar lordosis and tendoachilles contracture. Gowers sign was positive and proximal dominant limb weakness was noted. Hypertrophy was observed in the upper limbs such as the biceps brachii and forearm flexor muscles. Muscle biopsy showed distinct differences in size of muscle fibers and regenerating and necrotic muscle fibers. A histological study revealed decreased calpain3 expression. Gene analysis of CAPN3 revealed two known gene mutations, leading to a diagnosis of calpainopathy (limb girdle muscular dystrophy 2A; LGMD2A). We here report our patient to discuss findings of upper limb hypertrophy, which are frequently missed compared to the lower limb, but important clinical findings.


Assuntos
Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Extremidade Superior , Adulto , Biópsia , Calpaína/deficiência , Calpaína/genética , Diagnóstico Diferencial , Imagem de Difusão por Ressonância Magnética , Humanos , Hipertrofia , Masculino , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculo Esquelético/diagnóstico por imagem , Distrofia Muscular do Cíngulo dos Membros/classificação , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , Tomografia Computadorizada por Raios X
19.
BMB Rep ; 52(10): 619-624, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31619317

RESUMO

The primary cilium is a microtubule-based structure projecting from a cell. Although the primary cilium shows no motility, it can recognize environmental stimuli. Thus, ciliary defects cause severe abnormalities called ciliopathies. Ciliogenesis is a very complex process and involves a myriad of components and regulators. In order to excavate the novel positive regulators of ciliogenesis, we performed mRNA microarray using starved NIH/3T3 cells. We selected 62 murine genes with corresponding human orthologs, with significantly upregulated expression at 24 h after serum withdrawal. Finally, calpain-6 was selected as a positive regulator of ciliogenesis. We found that calpain-6 deficiency reduced the percentage of ciliated cells and impaired sonic hedgehog signaling. It has been speculated that this defect might be associated with decreased levels of α-tubulin acetylation at lysine 40. This is the first study to report a novel role of calpain-6 in the formation of primary cilia. [BMB Reports 2019; 52(10): 619-624].


Assuntos
Calpaína/metabolismo , Cílios/metabolismo , Acetilação , Animais , Calpaína/genética , Proteínas Hedgehog/metabolismo , Camundongos , Análise em Microsséries , Microtúbulos/metabolismo , Células NIH 3T3 , Transdução de Sinais , Tubulina (Proteína)/metabolismo
20.
Ann Clin Transl Neurol ; 6(11): 2328-2333, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31612648

RESUMO

CAPN3 mutations cause a limb girdle muscular dystrophy. Functional characterization of novel mutations facilitates diagnosis of future cases. We have identified a novel (c.1992 + 2T>G) CAPN3 mutation that disrupts the donor splice site of intron 17 splicing out exon 17, with mRNA levels severely reduced or undetectable. The mutation induces a strong change in the 3D structure of the mRNA which supports no-go mRNA decay as the probable mechanism for RNA degradation. The mutation was identified in two unrelated Roma individuals showing a common ancestral origin and founder effect. This is the first Roma CAPN3 mutation to be reported.


Assuntos
Calpaína/genética , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Adolescente , Criança , Feminino , Efeito Fundador , Humanos , Íntrons/genética , Masculino , Mutação , Processamento de RNA , Estabilidade de RNA/genética , Roma/genética
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