RESUMEN
The discovery and development of structurally distinct lysine methyltransferase G9a inhibitors have been the subject of intense research in epigenetics. Structure-based optimization was conducted, starting with the previously reported seed compound 7a and lead to the identification of a highly potent G9a inhibitor, compound 7i (IC50 = 0.024 µM). X-ray crystallography for the ligand-protein interaction and kinetics study, along with surface plasmon resonance (SPR) analysis, revealed that compound 7i interacts with G9a in a unique binding mode. In addition, compound 7i caused attenuation of cellular H3K9me2 levels and induction of γ-globin mRNA expression in HUDEP-2 cells in a dose-dependent manner.
Asunto(s)
Anemia de Células Falciformes , Inhibidores Enzimáticos , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina , Humanos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Relación Estructura-Actividad , Anemia de Células Falciformes/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Epigénesis Genética/efectos de los fármacos , Estructura Molecular , Antígenos de Histocompatibilidad/metabolismo , Relación Dosis-Respuesta a Droga , Cristalografía por Rayos XRESUMEN
The relentless pursuit of novel therapeutic agents against cancer has led to the identification of multiple molecular targets, among which Sirtuin 2 (SIRT2) has garnered significant attention. This study presents an extensive SAR study of our reported trityl scaffold-based SIRT2 inhibitors. This study encompasses a range of different medicinal chemistry approaches to improve the activity of the lead compounds TH-3 and STCY1. The rationally designed and synthesized structures were confirmed using NMR and high-resolution mass spectroscopy before performing SIRT2 inhibition assay, NCI60 cytotoxicity test, and cell cycle analysis. Indeed, our strategies afforded hitherto unreported SIRT2 inhibitors with high activity, particularly 2a, 4a, 7c, and 7f. Remarkably, the presence of a lipophilic para substitution on the phenyl group of a freely rotating or a locked trityl moiety enhanced activity SIRT2 inhibition. Concomitantly, the synthesized compounds showed prominent activity against different cancer lines from the NCI60 assay. Of interest, compound 7c stands out as a potent and highly selective antiproliferative agent against leukemia and colon cancer panels. Furthermore, 7c treatment resulted in cell cycle arrest in MCF-7 cells at G2 phase and did not cause in vitro DNA cleavage.
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Antineoplásicos , Neoplasias , Humanos , Relación Estructura-Actividad , Sirtuina 2 , Histamina , Cisteamina , Ligandos , Antineoplásicos/química , Estructura Molecular , Proliferación Celular , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
The microtubule-associated protein MAP1B has been implicated in axonal growth and brain development. We found that MAP1B is highly expressed in the most aggressive and deadliest breast cancer subtype, triple-negative breast cancer (TNBC), but not in other subtypes. Expression of MAP1B was found to be highly correlated with poor prognosis. Depletion of MAP1B in TNBC cells impairs cell migration and invasion concomitant with a defect in tumorigenesis. We found that MAP1B interacts with key components for invadopodia formation, cortactin, and Tks5, the latter of which is a PtdIns(3,4)P2-binding and scaffold protein that localizes to invadopodia. We also found that Tks5 associates with microtubules and supports the association between MAP1B and α-tubulin. In accordance with their interaction, depletion of MAP1B leads to Tks5 destabilization, leading to its degradation via the autophagic pathway. Collectively, these findings suggest that MAP1B is a convergence point of the cytoskeleton to promote malignancy in TNBC and thereby a potential diagnostic and therapeutic target for TNBC.
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Proteínas Adaptadoras del Transporte Vesicular , Cortactina , Proteínas Asociadas a Microtúbulos , Neoplasias de la Mama Triple Negativas , Humanos , Carcinogénesis/genética , Transformación Celular Neoplásica , Cortactina/genética , Proteínas Asociadas a Microtúbulos/genética , Neoplasias de la Mama Triple Negativas/genética , Células MDA-MB-231 , Proteínas Adaptadoras del Transporte Vesicular/genética , Microtúbulos/metabolismo , Citoesqueleto/metabolismo , Femenino , Animales , Ratones , Ratones Endogámicos BALB C , Podosomas/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.
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Fibras Musculares de Contracción Rápida , Músculo Esquelético , Fosfolípidos , Animales , Ratones , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/química , Fosfolípidos/genética , Fosfolípidos/metabolismo , Estearatos/metabolismo , Plasmalógenos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMEN
TEAD is a transcription factor responsible for the output of the tumor suppressor Hippo pathway. The transcriptional activity of TEAD requires molecular interaction with its transcriptional coactivator, YAP. Aberrant activation of TEAD is deeply involved in tumorigenesis and is associated with poor prognosis, suggesting that inhibitors targeting the YAP-TEAD system are promising as antitumor agents. In this study, we identified NPD689, an analog of the natural product alkaloid emetine, as an inhibitor of the YAP-TEAD interaction. NPD689 suppressed the transcriptional activity of TEAD and reduced the viability of human malignant pleural mesothelioma and non-small cell lung cancer cells but not the viability of normal human mesothelial cells. Our results suggest that NPD689 is not only a new useful chemical tool for elucidating the biological role of the YAP-TEAD system but also has potential as a starting compound for developing a cancer therapeutic agent that targets the YAP-TEAD interaction.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antineoplásicos/farmacología , Emetina , Neoplasias Pulmonares/patología , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP , Factores de Transcripción de Dominio TEA/metabolismoRESUMEN
Sickle cell disease (SCD) is a heritable disorder caused by ß-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.
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Anemia de Células Falciformes , ARN Largo no Codificante , Ratones , Humanos , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , gamma-Globinas/genética , Células Eritroides/metabolismo , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Expresión Génica , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismoRESUMEN
Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes comprising multiple classes and subtypes. This paper describes the synthesis and characterization of TPPBN-1, a naphthalimide derivative conjugated with a triphenylphosphonium (TPP) cation. When 4-bromonaphthalimide (BrNaph), a previously characterized GST substrate, was conjugated to a TPP cation, the conjugate showed increased reactivity towards most alpha- and mu-class GSTs, particularly the GSTA2 subtype, compared to the parent compound, but hardly towards Pi-class GSTs. Using this probe with enhanced reactivity, the enzymatic activity of endogenous GSTA1/2 in HepG2 cells was visualized by confocal fluorescence microscopy. The results demonstrated that modification with TPP cations, which are often used as tags for targeting mitochondria, can be used to enhance the reactivity of probes for specific GST subtypes.
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Glutatión Transferasa , Naftalimidas , Naftalimidas/farmacología , Glutatión Transferasa/química , Mitocondrias , CationesRESUMEN
Ineffective hematopoiesis is a fundamental process leading to the pathogenesis of myelodysplastic syndromes (MDS). However, the pathobiological mediators of ineffective hematopoiesis in MDS remain unclear. Here, we demonstrated that overwhelming mitochondrial fragmentation in mutant hematopoietic stem cells and progenitors (HSC/P) triggers ineffective hematopoiesis in MDS. Mouse modeling of CBL exon deletion with RUNX1 mutants, previously unreported comutations in patients with MDS, recapitulated not only clinically relevant MDS phenotypes but also a distinct MDS-related gene signature. Mechanistically, dynamin-related protein 1 (DRP1)-dependent excessive mitochondrial fragmentation in HSC/Ps led to excessive reactive oxygen species production, induced inflammatory signaling activation, and promoted subsequent dysplasia formation and impairment of granulopoiesis. Mitochondrial fragmentation was generally observed in patients with MDS. Pharmacologic inhibition of DRP1 attenuated mitochondrial fragmentation and rescued ineffective hematopoiesis phenotypes in mice with MDS. These findings provide mechanistic insights into ineffective hematopoiesis and indicate that dysregulated mitochondrial dynamics could be a therapeutic target for bone marrow failure in MDS. SIGNIFICANCE: We demonstrated that excessive mitochondrial fragmentation is a fundamental pathobiological phenomenon that could trigger dysplasia formation and ineffective hematopoiesis in MDS. Our findings provide mechanistic insights into ineffective hematopoiesis and suggest dysregulated mitochondrial dynamics as a therapeutic target for treating MDS.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Células Madre Hematopoyéticas/patología , Síndromes Mielodisplásicos/patología , Animales , Modelos Animales de Enfermedad , Femenino , Hematopoyesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones MutantesRESUMEN
Epigenetics is defined as the heritable alteration of gene expression without change to the DNA sequence. Epigenetic abnormalities play a role in various diseases, including cancer. Epigenetic regulation of gene expression occurs through histone chemical modifications and DNA methylation. Lysine acetylation is one of the major histone chemical modifications essential for epigenetic gene expression. Histone acetylation is reversibly regulated by histone acetyltransferases and histone deacetylases, which are molecular targets for cancer therapy. There has been an explosion of research in epigenetic-related drug discovery, and accordingly many small molecule compounds have been developed. Notably, several small molecule inhibitors of histone deacetylases have been approved for the treatment of cancer. This review will introduce natural products, their derivative inhibitors of histone deacetylases, and their clinical development.
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Antineoplásicos/farmacología , Productos Biológicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/química , Productos Biológicos/química , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/química , HumanosRESUMEN
DDHD1 and DDHD2 are both intracellular phospholipases A1 and hydrolyze phosphatidic acid in vitro. Given that phosphatidic acid participates in neurite outgrowth, we examined whether DDHD1 and DDHD2 regulate neurite outgrowth. Depletion of DDHD1 from SH-SY5Y and PC12 cells caused elongation of neurites, whereas DDHD2 depletion prevented neurite elongation. Rescue experiments demonstrated that the enzymatic activity of DDHD1 is necessary for the prevention of neurite elongation. Depletion of DDHD1 caused enlargement of early endosomes and stimulated tubulation of recycling endosomes positive for phosphatidic acid-binding proteins syndapin2 and MICAL-L1. Knockout of DDHD1 enhanced transferrin recycling from recycling endosomes to the cell surface. Our results suggest that DDHD1 negatively controls the formation of a local phosphatidic acid-rich domain in recycling endosomes that serves as a membrane source for neurite outgrowth.
RESUMEN
Modification of the transforming growth factor ß (TGF-ß) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-ß signaling, suggesting that this mode of regulation of TGF-ß signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-ß signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-ßRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-ß signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-ß signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-ß/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-ß/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-ß/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-ß signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.
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Proteínas de Caenorhabditis elegans/metabolismo , Carcinogénesis/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Caenorhabditis elegans , Transformación Celular Neoplásica , Enzimas Desubicuitinizantes , Larva/metabolismo , Pulmón/metabolismo , Transducción de Señal/genética , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Ubiquitina Tiolesterasa/fisiología , UbiquitinaciónRESUMEN
Chronic myelomonocytic leukemia (CMML) constitutes a hematopoietic stem cell (HSC) disorder characterized by prominent monocytosis and myelodysplasia. Although genome sequencing has revealed the CMML mutation profile, the mechanism of disease development remains unclear. Here we show that aberrant histone acetylation by nucleoporin-98 (NUP98)-HBO1, a newly identified fusion in a patient with CMML, is sufficient to generate clinically relevant CMML pathogenesis. Overexpression of NUP98-HBO1 in murine HSC/progenitors (HSC/Ps) induced diverse CMML phenotypes, such as severe leukocytosis, increased CD115+ Ly6Chigh monocytes (an equivalent subpopulation to human classical CD14+ CD16- monocytes), macrocytic anemia, thrombocytopenia, megakaryocyte-lineage dysplasia, splenomegaly, and cachexia. A NUP98-HBO1-mediated transcriptional signature in human CD34+ cells was specifically activated in HSC/Ps from a CMML patient cohort. Besides critical determinants of monocytic cell fate choice in HSC/Ps, an oncogenic HOXA9 signature was significantly activated by NUP98-HBO1 fusion through aberrant histone acetylation. Increased HOXA9 gene expression level with disease progression was confirmed in our CMML cohort. Genetic disruption of NUP98-HBO1 histone acetyltransferase activity abrogated its leukemogenic potential and disease development in human cells and a mouse model. Furthermore, treatment of azacytidine was effective in our CMML mice. The recapitulation of CMML clinical phenotypes and gene expression profile by the HBO1 fusion suggests our new model as a useful platform for elucidating the central downstream mediators underlying diverse CMML-related mutations and testing multiple compounds, providing novel therapeutic potential.
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Histona Acetiltransferasas/genética , Leucemia Mielomonocítica Crónica/etiología , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/genética , Acetilación , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mielomonocítica Crónica/patología , Ratones , FenotipoRESUMEN
DDHD2/KIAA0725p is a mammalian intracellular phospholipase A1 that exhibits phospholipase and lipase activities. Mutation of the DDHD2 gene causes hereditary spastic paraplegia (SPG54), an inherited neurological disorder characterized by lower limb spasticity and weakness. Although previous studies demonstrated lipid droplet accumulation in the brains of SPG54 patients and DDHD2 knockout mice, the cause of SPG54 remains elusive. Here, we show that ablation of DDHD2 in mice induces age-dependent apoptosis of motor neurons in the spinal cord. In vitro, motor neurons and embryonic fibroblasts from DDHD2 knockout mice fail to survive and are susceptible to apoptotic stimuli. Chemical and probe-based analysis revealed a substantial decrease in cardiolipin content and an increase in reactive oxygen species generation in DDHD2 knockout cells. Reactive oxygen species production in DDHD2 knockout cells was reversed by the expression of wild-type DDHD2, but not by an active-site DDHD2 mutant, DDHD2 mutants related to hereditary spastic paraplegia, or DDHD1, another member of the intracellular phospholipase A1 family whose mutation also causes spastic paraplegia (SPG28). Our results demonstrate the protective role of DDHD2 for mitochondrial integrity and provide a clue to the pathogenic mechanism of SPG54.
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Apoptosis , Fosfolipasas A1/genética , Especies Reactivas de Oxígeno/metabolismo , Paraplejía Espástica Hereditaria/patología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Cardiolipinas/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Fosfolipasas , Fosfolipasas A1/deficiencia , Paraplejía Espástica Hereditaria/genética , Médula Espinal/metabolismo , Médula Espinal/patología , Estaurosporina/farmacologíaRESUMEN
The histone methyltransferase SET7/9 methylates not only histone but also non-histone proteins as substrates, and therefore, SET7/9 inhibitors are considered candidates for the treatment of diseases. Previously, our group identified cyproheptadine, used clinically as a serotonin receptor antagonist and histamine receptor (H1) antagonist, as a novel scaffold of the SET7/9 inhibitor. In this work, we focused on dibenzosuberene as a substructure of cyproheptadine and synthesized derivatives with various functional groups. Among them, the compound bearing a 2-hydroxy group showed the most potent activity. On the other hand, a 3-hydroxy group or another hydrophilic functional group such as acetamide decreased the activity. Structural analysis clarified a rationale for the improved potency only by tightly restricted location and type of the hydrophilic group. In addition, a SET7/9 loop, which was only partially visible in the complex with cyproheptadine, became more clearly visible in the complex with 2-hydroxycyproheptadine. These results are expected to be helpful for further structure-based development of SET7/9 inhibitors.
Asunto(s)
Ciproheptadina/química , Ciproheptadina/farmacología , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación ProteicaRESUMEN
Calpain-7 (CAPN7) is a unique intracellular cysteine protease that has a tandem repeat of microtubule interacting and trafficking (MIT) domains and lacks a penta-EF-hand domain. Although the MIT domains of CAPN7 were previously shown to interact with a subset of endosomal sorting complex required for transport (ESCRT)-III and ESCRT-III-related proteins, including charged multivesicular body protein 1 and increased sodium tolerance (IST)1, knowledge of the involvement of the protease in membrane trafficking has been limited. In the present study, compared with control cells, we found that epidermal growth factor receptor (EGFR) degradation was mildly delayed in CAPN7-knockdown HeLa cells and mouse embryonic fibroblast cells established from CAPN7 knockout (Capn7(-/-) ) mice. Re-expression of wild-type CAPN7 but not a protease-inactive mutant of CAPN7 (CAPN7(C290S) ) resulted in a recovery of the rate of EGFR degradation. We found, by immunofluorescence microscopic analysis, that monomeric GFP fused with the protease-inactive mutant of CAPN7 [monomeric green fluorescent protein (mGFP)-CAPN7(C290S) ] was mobilized to EGFR-positive endosomes upon epidermal growth factor stimulation in HeLa cells. Although mGFP-CAPN7(C290S) exhibited dominant-negative effects on EGFR degradation, a deletion mutant of MIT domains in mGFP-CAPN7(C290S) did not have such properties, suggesting that the interaction between the MIT domains and ESCRT proteins is important for the function of CAPN7. Moreover, we found that epidermal growth factor stimulation induces translocation of IST1 from the cytosol to endosomes positive in both EGFR and mGFP-CAPN7(C290S) . When IST1 was knocked down, mGFP-CAPN7(C290S) lost its co-localization with EGFR. These results demonstrate for the first time that the proteolytic activity of CAPN7 is important for the acceleration of EGFR degradation via the endosomal sorting pathway utilizing a part of the ESCRT system. STRUCTURED DIGITAL ABSTRACT: EGFR and CAPN7 colocalize by fluorescence microscopy (View interaction) EGFR, CAPN7 and IST1 colocalize by fluorescence microscopy (View interaction) EEA1 and CAPN7 colocalize by fluorescence microscopy (View interaction) CAPN7 and LAMP1 colocalize by fluorescence microscopy (View interaction).
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Calpaína/fisiología , Endosomas/enzimología , Receptores ErbB/metabolismo , Animales , Calpaína/química , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteínas Oncogénicas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ProteolisisRESUMEN
Human charged multivesicular body protein 1A (CHMP1A) displayed two bands on SDS-PAGE and differences in efficiency of complex formation with IST1. By site-directed mutagenesis and phosphate-affinity PAGE, we identified Ser(179) and Ser(182) located in the C-terminal region as major phosphorylation sites that cause a mobility shift, but interaction with IST1 was not affected by Ser-to-Ala mutations.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Proteínas Oncogénicas/química , Serina/química , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Complejos Multiproteicos/química , Mutagénesis Sitio-Dirigida , Proteínas Oncogénicas/metabolismo , Fosforilación , Relación Estructura-Actividad , Proteínas de Transporte VesicularRESUMEN
Calpain-7 is a mammalian ortholog of a fungal non-classical calpain named PalB, which is an intracellular cysteine protease and functions in fungal alkaline adaptation in association with the endosomal sorting complex required for transport (ESCRT) system. Despite our previous finding [Osako Y et al. (2010) FEBS J 277, 4412-4426] of autolytic activity, neither physiological nor non-physiological substrates of calpain-7 have yet been identified, and experimentally useful substrates that show robust evidence of intermolecular proteolytic activity of calpain-7 are required. In this study, we found limited proteolysis of C-terminally truncated ALG-2-interacting protein X (ALIX; (ALIXΔC), but not full-length ALIX, when the mutant was co-over-expressed with calpain-7 in HEK293T cells and analyzed by western blotting. The extent of ALIXΔC cleavage by calpain-7 was enhanced by co-expression with several ESCRT proteins. We investigated whether fusion of casein, a commonly used substrate for a variety of proteases including calpains, to the Bro1 domain confers the ability to serve as a substrate of calpain-7, but no specific cleavage was observed. However, when domain 1 of calpastatin, an endogenous inhibitory protein of ubiquitous classical calpains, was fused with the Bro1 domain, the fusion protein was cleaved at the C-terminal border of subdomain B (an inhibitory center for calpains) of calpastatin. These results demonstrate for the first time that calpain-7 has limited proteolytic activity and substrate preference. Moreover, the proteolytic assay system developed enabled us to perform mutational analysis of calpain-7, which revealed the importance of not only the N-terminal microtubule-interacting and trafficking (MIT) domains but also the C-terminal C2 domain-like domains for proteolytic activity.
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Calpaína/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Secuencia de Aminoácidos , Calpaína/química , Calpaína/genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutagénesis , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.
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Evolución Biológica , Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Motivos EF Hand , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Animales , Humanos , Mapas de Interacción de ProteínasRESUMEN
Some intracellular proteins involved in the endosomal sorting complex required for transport (ESCRT) system have microtubule interacting and transport (MIT) domains and bind to ESCRT-III protein family members named charged multivesicular body proteins (CHMPs) at their C-terminal regions containing MIT-interacting motifs (MIMs). While two types of MIMs (MIM1 and MIM2) have been reported, CHMP1B has MIM1 and IST1 has both MIM1 and MIM2. Previously, we demonstrated that CHMP1B and IST1 directly interacted with a tandem repeat of MIT domains of calpain-7 (CL7MIT) and that autolytic activity of calpain-7 was enhanced by IST1 in vitro but not by overexpression of IST1 in HEK293T cells. In this study, we detected enhancement of autolysis of mGFP-fused calpain-7 by coexpression with CHMP1B and observed further activation by additional coexpression of IST1 in HEK293T cells. We found that CL7MIT interacted with the second α-helical region of CHMP1B but not with the canonical C-terminal region containing MIM1 in vitro. Co-immunoprecipitation assays demonstrated that the interaction between CL7MIT and CHMP1B and between CL7MIT and IST1 became stronger when IST1 or CHMP1B was additionally coexpressed, suggesting formation of ternary complex of calpain-7, IST1 and CHMP1B. Moreover, subcellular fractionation analyses revealed increase of calpain-7 in membrane/organelle fractions by concomitant overexpression of these ESCRT-III family member proteins.
Asunto(s)
Calpaína/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Oncogénicas/metabolismo , Calpaína/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Células HEK293 , Humanos , Proteínas Oncogénicas/química , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Calpain 7, a mammalian ortholog of yeast Cpl1/Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESCRT-III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mmN-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7(C290S)-Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system.