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Cell death-inducing p53-target protein 1 (CDIP1) is a proapoptotic protein that is normally expressed at low levels and is upregulated by genotoxic and endoplasmic reticulum stresses. CDIP1 has been reported to be localized to endosomes and to interact with several proteins, including B-cell receptor-associated protein 31 (BAP31) and apoptosis-linked gene 2 (ALG-2). However, the cellular and molecular mechanisms underlying CDIP1 expression-induced apoptosis remain unclear. In this study, we first demonstrated that CDIP1 was upregulated after treatment with the anticancer drug adriamycin in human breast cancer MCF-7 cells but was degraded rapidly in the lysosomal pathway. We also demonstrated that treatment with the cyclin-dependent kinase 5 (CDK5) inhibitor roscovitine led to an increase in the electrophoretic mobility of CDIP1. In addition, a phosphomimetic mutation at Ser-32 in CDIP1 resulted in an increase in CDIP1 expression-induced apoptosis. We also found that CDIP1 expression led to the induction of autophagy prior to apoptosis. Treatment of cells expressing CDIP1 with SAR405, an inhibitor of the class III phosphatidylinositol 3-kinase VPS34, caused a reduction in autophagy and promoted apoptosis. Therefore, autophagy is thought to be a defense mechanism against CDIP1 expression-induced apoptosis.
Assuntos
Apoptose , Autofagia , Neoplasias da Mama , Feminino , Humanos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Citoproteção/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células MCF-7RESUMO
BACKGROUND: Ipilimumab (Ipi) plus nivolumab (Nivo) is the recommended first-line treatment for renal cell carcinoma (RCC). This report describes a case where pancreatic metastases disappeared after only two courses of Ipi + Nivo therapy. The primary tumor was cured by surgery, and a pathological Complete Response (pCR) was observed despite multiple serious immune-related Adverse Events (irAEs). CASE PRESENTATION: A 54-year-old woman with RCC and pancreatic metastasis at stage IV, diagnosed with intermediate risk according to the International Metastatic RCC Database Consortium classification, underwent initiation of Ipi + Nivo therapy. On day 26, she developed hyperthyroidism accompanied by tachycardia, leading to the commencement of metoprolol tartrate treatment. Following the resolution of tachycardia, a second course of Ipi + Nivo therapy was administered on day 50. By day 70, the patient exhibited Grade 3 hepatic dysfunction, followed by the onset of hypothyroidism on day 75, necessitating treatment with steroids and levothyroxine. After positive treatment, a Grade 3 skin disorder emerged on day 87 while tapering steroids, prompting treatment with methylprednisolone (mPSL) pulse therapy. The skin disorder responded to steroids, allowing for tapering. However, on day 113, a recurrence of Grade 3 skin disorder occurred, necessitating another mPSL pulse. The patient responded well to treatment, exhibiting improvement in her condition. On day 131, she presented at the hospital with complaints of respiratory distress, prompting a Computed Tomography (CT) scan that revealed interstitial pneumonia. By day 272, subsequent CT imaging showed the disappearance of pancreatic metastasis and shrinkage of the primary tumor. On day 294, she underwent a laparoscopic left nephrectomy. Pathological analysis confirmed a pCR in the primary tumor, indicating successful eradication of RCC through surgical intervention. CONCLUSIONS: This case report presents a scenario where multiple severe irAEs appeared in a patient, yet metastases disappeared after only two courses of Ipi + Nivo therapy. The patient was ultimately cured by surgery and achieved a pCR. This case highlights that despite the occurrence of severe irAEs during RCC treatment with Ipi + Nivo therapy, they can be managed appropriately to maximize the therapeutic effects of checkpoint inhibitors.
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BACKGROUND: While cancer is a risk factor for developing thromboembolism, so is the use of molecularly targeted therapies. This study aimed to determine whether thromboembolism incidence differed between vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) inhibitor use in patients with unresectable advanced or recurrent colorectal cancer, and to compare the risk of thromboembolism caused by cancer and the use of molecular targeted therapy drugs. MAIN BODY: We retrospectively evaluated patients with unresectable advanced or recurrent colorectal cancer who were treated with a cytotoxic anticancer drug and a VEGF or EGFR inhibitor combination between April 2016 and October 2021. Patients were compared in terms of the regimen administered, thromboembolism occurrence during the first-line treatment period, patient background, and clinical laboratory values. Of the 179 included patients, 12 of 134 (8.9%) in the VEGF-inhibitor group and 8 of 45 (17.8%) in the EGFR-inhibitor group developed thromboembolism, with no significant difference between the groups (P = 0.11). There was no significant difference in time to thromboembolism between patients in the VEGF- inhibitor group and patients in the EGFR-inhibitor group (P = 0.206). The cutoff value determined by a receiver operating characteristic analysis for the occurrence of thromboembolism was one point. Multivariate analysis using the occurrence of thromboembolism as the response variable identified at least one risk factor for thromboembolism (odds ratio = 4.17, P = 0.006, 95% confidence interval = 1.51-11.50). Molecular targeted therapies were not identified as a risk factor. CONCLUSIONS: Although the small sample size, there was no difference in the incidence of thromboembolism between the two molecular-targeted therapies in first-line treatment of patients with unresectable advanced or recurrent colorectal cancer. Our results suggest that risk factors for thromboembolism may be more strongly influenced by cancer itself than by the use of molecularly targeted therapies.
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Environmental factors are important regulators of cell growth and proliferation. Mechanistic target of rapamycin (mTOR) is a central kinase that maintains cellular homeostasis in response to a variety of extracellular and intracellular inputs. Dysregulation of mTOR signaling is associated with many diseases, including diabetes and cancer. Calcium ion (Ca2+) is important as a second messenger in various biological processes, and its intracellular concentration is tightly regulated. Although the involvement of Ca2+ mobilization in mTOR signaling has been reported, the detailed molecular mechanisms by which mTOR signaling is regulated are not fully understood. The link between Ca2+ homeostasis and mTOR activation in pathological hypertrophy has heightened the importance in understanding Ca2+-regulated mTOR signaling as a key mechanism of mTOR regulation. In this review, we introduce recent findings on the molecular mechanisms of regulation of mTOR signaling by Ca2+-binding proteins, particularly calmodulin (CaM).
Assuntos
Serina-Treonina Quinases TOR , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte/metabolismo , Transdução de Sinais , Sirolimo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: COVID-19 has become a significant health threat and a primary healthcare concern among the most vulnerable patients with cancer. Patients with COVID-19 who have lung cancer are at great risk and need careful monitoring if they are affected. This study aimed to investigate the clinical characteristics of COVID-19-positive patients with lung cancer and the risks associated with anticancer medication. METHODS: This study was a single-center, retrospective cohort study. Patients with lung cancer who presented with COVID-19 during hospitalization were divided into two groups: those who presented with respiratory failure and those who did not. The patient's background, clinical laboratory values, and anticancer drugs used for therapy were investigated to identify risk factors for respiratory failure. RESULTS: Thirty-one patients were included in the study; 18 (58.1%) were in the respiratory failure group and 13 (41.9%) were in the group without respiratory failure. In the respiratory failure group, there was a significant difference in using immune checkpoint inhibitor (ICI) use within 90 days (p = 0.025) and the level of C-reactive protein (CRP) level (p = 0.017). The analysis of the operating characteristic of the receiver revealed a cutoff value of 2.75 mg/dL for CRP (area under the curve = 0.744, sensitivity 0.611, specificity 0.923). CONCLUSIONS: A history of ICI within 90 days and elevated CRP (≥ 2.75 mg/dL) levels are potential factors leading to respiratory failure in COVID-19-affected patients undergoing chemotherapy for lung cancer.
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We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle remain unclear. We sought to identify a novel role of the ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The small interfering RNA (siRNA) knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in the release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase, VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Coimmunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that the HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles. IMPORTANCE The ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that the HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Complexos Endossomais de Distribuição Requeridos para Transporte , Hepacivirus , Hepatite C , Proteínas Repressoras , Ubiquitina-Proteína Ligases , ATPases Vacuolares Próton-Translocadoras , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Técnicas de Silenciamento de Genes , Hepacivirus/fisiologia , Hepatite C/fisiopatologia , Hepatite C/virologia , Humanos , Sistema de Sinalização das MAP Quinases , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , VírionRESUMO
Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.
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Aminoácidos/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Ligação Proteica , Transdução de SinaisRESUMO
Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca2+-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca2+-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.
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Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Morte Celular/genética , Linhagem Celular , Humanos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Motivos EF Hand , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , UbiquitinaçãoRESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.
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Aminoácidos/metabolismo , Regulação da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Transdução de Sinais , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , CamundongosRESUMO
The penta-EF-hand (PEF) protein family includes ALG-2 (gene name, PDCD6) and its paralogs as well as classical calpain family members. ALG-2 is a prototypic PEF protein that is widely distributed in eukaryotes and interacts with a variety of proteins in a Ca2+-dependent manner. Mammalian ALG-2 and its interacting partners have various modulatory roles including roles in cell death, signal transduction, membrane repair, ER-to-Golgi vesicular transport, and RNA processing. Some ALG-2-interacting proteins are key factors that function in the endosomal sorting complex required for transport (ESCRT) system. On the other hand, mammalian calpain-7 (CAPN7) lacks the PEF domain but contains two microtubule-interacting and trafficking (MIT) domains in tandem. CAPN7 interacts with a subset of ESCRT-III proteins through the MIT domains and regulates EGF receptor downregulation. Structures and functions of ALG-2 and those of its interacting partners as well as relationships with the calpain family are reviewed in this article.
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Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calpaína/metabolismo , Evolução Biológica , Complexos Endossomais de Distribuição Requeridos para Transporte , Ativação Enzimática , Humanos , Ligação Proteica , Transporte Proteico , Relação Estrutura-AtividadeRESUMO
Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanism underlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting into MVBs in a manner dependent on its C-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB sorting and EGFR degradation mediated by ESCRT complexes.
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Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas de Membrana/genética , Microscopia de Fluorescência , Corpos Multivesiculares/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Vesículas Transportadoras/metabolismoRESUMO
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 TranscricionalRESUMO
Many non-catalytic cellular proteins exert biological functions by formation of stable or transient complexes with other proteins. Analysis of the signal-induced physical interactions is important to understand their physiological roles in cells. Here we describe a biochemical method for assessing the binding of ALG-2 (gene name, PDCD6) to its target proteins that are immunoprecipitated from cell lysates. Application of nanoluciferase (Nluc)-fused ALG-2 enables a rapid quantitative evaluation of Ca2+-dependent interactions of target proteins with ALG-2 in vitro binding assays.
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Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Luciferases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Células HEK293 , Humanos , Imunoprecipitação , Nanotecnologia , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismoRESUMO
MAP1B (microtubule-associated protein 1B) binds to microtubules and regulates microtubule dynamics. Previously, we showed calcium-dependent interaction between MAP1B and a calcium-binding protein ALG-2 (apoptosis-linked gene 2), which is involved in regulation of the protein secretion pathway. Although ALG-2 generally binds to proteins through two consensus binding motifs such as ABM-1 and ABM-2, the absence of these motifs in MAP1B suggests a unique binding mode between MAP1B and ALG-2. Here, we identified the region of mouse MAP1B responsible for binding to ALG-2, and found point mutations that abrogated binding of MAP1B to ALG-2. Furthermore, interaction between MAP1B and ALG-2 selectively prevented ALG-2 from binding to proteins with ABM-2 such as Sec31A, suggesting competition between MAP1B and ABM-2-containing proteins for binding to ALG-2. Consistently, in MAP1B knockout cells, co-localization of ALG-2 with Sec31A was increased. Moreover, overexpression of wild-type MAP1B, but not the MAP1B mutant defective in ALG-2 binding, altered localizations of ALG-2 and Sec31A into dispersed distributions, suggesting that MAP1B regulates localizations of ALG-2 and Sec31A in the cells. Finally, we found two cancer-associated mutations of human MAP1B located near ALG-2 binding sites. The introduction of the corresponding mutations in mouse MAP1B dramatically reduced the binding ability to ALG-2. Thus, these results suggest that MAP1B plays a role in regulation of ALG-2 and Sec31A localizations, and that dysregulation of calcium-dependent binding of ALG-2 to MAP1B might influence pathological conditions such as cancers.
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Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose/análise , Sítios de Ligação , Proteínas de Ligação ao Cálcio/análise , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/metabolismoRESUMO
NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization.
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Cálcio/metabolismo , Genes Reporter , Luciferases/metabolismo , Fatores de Transcrição NFATC/genética , Nanopartículas/química , Elementos de Resposta/genética , Sequências de Repetição em Tandem/genética , Animais , Sequência de Bases , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , AMP Cíclico/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Fator de Transcrição AP-1/genéticaRESUMO
Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that MAPK1-interacting and spindle-stabilizing (MISS)-like (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of secreted alkaline phosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Colágeno Tipo I/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Colágeno Tipo I/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Precursores de Proteínas/genética , Transporte Proteico/fisiologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Anexinas/genética , Anexinas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mutação/genética , Ligação Proteica , Transporte Proteico , Proteína A6 Ligante de Cálcio S100/metabolismoRESUMO
Apoptosis-linked gene 2 (ALG-2), which is a gene product of PDCD6, is a 22-kDa Ca2+ -binding protein. Accumulating evidence points to a role for ALG-2 as a Ca2+ -responsive adaptor protein. On binding to Ca2+ , ALG-2 undergoes a conformational change that facilitates its interaction with various proteins. It also forms a homodimer and heterodimer with peflin, a paralog of ALG-2. However, the differences in cellular roles for the ALG-2 homodimer and ALG-2/peflin heterodimer are unclear. In the present study, we found that Trk-fused gene (TFG) protein interacted with the ALG-2 homodimer. Immunostaining analysis revealed that TFG and ALG-2 partially overlapped at endoplasmic reticulum exit sites (ERES), a platform for COPII-mediated protein transport from the endoplasmic reticulum. Time-lapse live-cell imaging demonstrated that both green fluorescent protein-fused TFG and mCherry-fused ALG-2 are recruited to ERES after thapsigargin treatment, which raises intracellular Ca2+ levels. Furthermore, overexpression of ALG-2 induced the accumulation of TFG at ERES. TFG has an ALG-2-binding motif and deletion of the motif decreased TFG binding to ALG-2 and shortened its half-life at ERES, suggesting a critical role for ALG-2 in retaining TFG at ERES. We also demonstrated, by in vitro cross-linking assays, that ALG-2 promoted the polymerization of TFG in a Ca2+ -dependent manner. Collectively, the results suggest that ALG-2 acts as a Ca2+ -sensitive adaptor to concentrate and polymerize TFG at ERES, supporting a potential role for ALG-2 in COPII-dependent trafficking from the endoplasmic reticulum.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Imagem com Lapso de Tempo , Proteína Vermelha FluorescenteRESUMO
ALG-2 (gene name: PDCD6) is a penta-EF-hand Ca(2+)-binding protein and interacts with a variety of proteins in a Ca(2+)-dependent fashion. ALG-2 recognizes different types of identified motifs in Pro-rich regions by using different hydrophobic pockets, but other unknown modes of binding are also used for non-Pro-rich proteins. Most ALG-2-interacting proteins associate directly or indirectly with the plasma membrane or organelle membranes involving the endosomal sorting complex required for transport (ESCRT) system, coat protein complex II (COPII)-dependent ER-to-Golgi vesicular transport, and signal transduction from membrane receptors to downstream players. Binding of ALG-2 to targets may induce conformational change of the proteins. The ALG-2 dimer may also function as a Ca(2+)-dependent adaptor to bridge different partners and connect the subnetwork of interacting proteins.