RESUMEN
TMPRSS6 (matriptase-2) is a type II transmembrane serine protease involved in iron homoeostasis. At the cell surface of hepatocytes, TMPRSS6 cleaves haemojuvelin (HJV) and regulates the BMP/SMAD signalling pathway leading to production of hepcidin, a key regulator of iron absorption. Although four TMPRSS6 human isoforms and three mice Tmprss6 isoforms are annotated in databases (Ensembl and RefSeq), their relative expression or activity has not been studied. Analyses of RNA-seq data and RT-PCR from human tissues reveal that TMPRSS6 isoform 1 (TMPRSS6-1) and 3 are mostly expressed in human testis while TMPRSS6-2 and TMPRSS6-4 are the main transcripts expressed in human liver, testis and pituitary. Furthermore, we confirm the existence and analyse the relative expression of three annotated mice Tmprss6 isoforms. Using heterologous expression in HEK293 and Hep3B cells, we show that all human TMPRSS6 isoforms reach the cell surface but only TMPRSS6-1 undergoes internalization. Moreover, truncated TMPRSS6-3 or catalytically altered TMPRSS6-4 interact with HJV and prevent its cleavage by TMPRSS6-2, suggesting their potential role as dominant negative isoforms. Taken together, our results highlight the importance of understanding the precise function of each TMPRSS6 isoforms both in human and in mouse.
Asunto(s)
Homeostasis/genética , Proteínas de la Membrana/genética , Isoformas de Proteínas/genética , Serina Endopeptidasas/genética , Transcriptoma/genética , Animales , Proteínas Ligadas a GPI/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Proteína de la Hemocromatosis/genética , Humanos , Hierro/metabolismo , Ratones , Transducción de Señal/genéticaRESUMEN
Recent studies have reported that many proteases, besides the canonical α-, ß-, and γ-secretases, cleave the amyloid precursor protein (APP) and modulate ß-amyloid (Aß) peptide production. Moreover, specific APP isoforms contain Kunitz protease-inhibitory domains, which regulate the proteolytic activity of serine proteases. This prompted us to investigate the role of matriptase, a member of the type II transmembrane serine protease family, in APP processing. Using quantitative RT-PCR, we detected matriptase mRNA in several regions of the human brain with an enrichment in neurons. RNA sequencing data of human dorsolateral prefrontal cortex revealed relatively high levels of matriptase RNA in young individuals, whereas lower levels were detected in older individuals. We further demonstrate that matriptase and APP directly interact with each other and that matriptase cleaves APP at a specific arginine residue (Arg-102) both in vitro and in cells. Site-directed (Arg-to-Ala) mutagenesis of this cleavage site abolished matriptase-mediated APP processing. Moreover, we observed that a soluble, shed matriptase form cleaves endogenous APP in SH-SY5Y cells and that this cleavage significantly reduces APP processing to Aß40. In summary, this study identifies matriptase as an APP-cleaving enzyme, an activity that could have important consequences for the abundance of Aß and in Alzheimer's disease pathology.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/enzimología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Fragmentos de Péptidos/metabolismo , Serina Endopeptidasas/metabolismo , Factores de Edad , Anciano , Encéfalo/metabolismo , Cadáver , Línea Celular , Biología Computacional , Regulación Enzimológica de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Especificidad de Órganos , Corteza Prefrontal/enzimología , Corteza Prefrontal/metabolismo , Proteolisis , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Especificidad por Sustrato , Adulto JovenRESUMEN
Monosomy 5 and deletions of the chromosome 5q (-5/del(5q)) are recurrent events in de novo adult acute myeloid leukemia (AML), reaching up to 40% of cases in secondary AML. These chromosome anomalies are associated with TP53 mutations and with very poor prognosis. Using the large Leucegene genomic and transcriptomic dataset composed of 48 -5/del(5q) patient specimens and 367 control AML, we identified DELE1 - located in the common deleted region - as the most consistently downregulated gene in these leukemias. DELE1 encodes a mitochondrial protein recently characterized as the relay of mitochondrial stress to the cytosol through a newly defined OMA1-DELE1-HRI pathway which ultimately leads to the activation of ATF4, the master transcription factor of the integrated stress response. Here, we showed that the partial loss of DELE1 expression observed in -5/del(5q) patients was sufficient to significantly reduce the sensitivity to mitochondrial stress in AML cells. Overall, our results suggest that DELE1 haploinsufficiency could represent a new driver mechanism in -5/del(5q) AML.
Asunto(s)
Haploinsuficiencia , Leucemia Mieloide Aguda , Proteínas Mitocondriales , Monosomía , Adulto , Humanos , Apoptosis/genética , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , Leucemia Mieloide Aguda/genética , Proteínas Mitocondriales/genéticaRESUMEN
In this study, we leveraged machine-learning tools by evaluating expression of genes of pharmacological relevance to standard-AML chemotherapy (ara-C/daunorubicin/etoposide) in a discovery-cohort of pediatric AML patients (N = 163; NCT00136084 ) and defined a 5-gene-drug resistance score (ADE-RS5) that was predictive of outcome (high MRD1 positivity p = 0.013; lower EFS p < 0.0001 and OS p < 0.0001). ADE-RS5 was integrated with a previously defined leukemic-stemness signature (pLSC6) to classify patients into four groups. ADE-RS5, pLSC6 and integrated-score was evaluated for association with outcome in one of the largest assembly of ~3600 AML patients from 10 independent cohorts (1861 pediatric and 1773 adult AML). Patients with high ADE-RS5 had poor outcome in validation cohorts and the previously reported pLSC6 maintained strong significant association in all validation cohorts. For pLSC6/ADE-RS5-integrated-score analysis, using Group-1 (low-scores for ADE-RS5 and pLSC6) as reference, Group-4 (high-scores for ADE-RS5 and pLSC6) showed worst outcome (EFS: p < 0.0001 and OS: p < 0.0001). Groups-2/3 (one high and one low-score) showed intermediate outcome (p < 0.001). Integrated score groups remained an independent predictor of outcome in multivariable-analysis after adjusting for established prognostic factors (EFS: Group 2 vs. 1, HR = 4.68, p < 0.001, Group 3 vs. 1, HR = 3.22, p = 0.01, and Group 4 vs. 1, HR = 7.26, p < 0.001). These results highlight the significant prognostic value of transcriptomics-based scores capturing disease aggressiveness through pLSC6 and drug resistance via ADE-RS5. The pLSC6 stemness score is a significant predictor of outcome and associates with high-risk group features, the ADE-RS5 drug resistance score adds further value, reflecting the clinical utility of simultaneous testing of both for optimizing treatment strategies.
RESUMEN
Immunotherapy remains underexploited in acute myeloid leukemia (AML) compared to other hematological malignancies. Currently, gemtuzumab ozogamicin is the only therapeutic antibody approved for this disease. Here, to identify potential targets for immunotherapeutic intervention, we analyze the surface proteome of 100 genetically diverse primary human AML specimens for the identification of cell surface proteins and conduct single-cell transcriptome analyses on a subset of these specimens to assess antigen expression at the sub-population level. Through this comprehensive effort, we successfully identify numerous antigens and markers preferentially expressed by primitive AML cells. Many identified antigens are targeted by therapeutic antibodies currently under clinical evaluation for various cancer types, highlighting the potential therapeutic value of the approach. Importantly, this initiative uncovers AML heterogeneity at the surfaceome level, identifies several antigens and potential primitive cell markers characterizing AML subgroups, and positions immunotherapy as a promising approach to target AML subgroup specificities.
Asunto(s)
Inmunoterapia , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Inmunoterapia/métodos , Proteínas de la Membrana/metabolismoRESUMEN
Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.
Asunto(s)
Aminoácidos de Cadena Ramificada , Leucemia Mieloide Aguda , Receptores Acoplados a Proteínas G , Animales , Humanos , Ratones , Aminoácidos de Cadena Ramificada/uso terapéutico , Transportador de Aminoácidos Neutros Grandes 1/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , FN-kappa B/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.
Asunto(s)
Membrana Celular/enzimología , Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Membrana Celular/genética , Vesículas Cubiertas por Clatrina/enzimología , Vesículas Cubiertas por Clatrina/genética , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Proteína de la Hemocromatosis , Células Hep G2 , Hepcidinas , Homeostasis/fisiología , Humanos , Hierro/metabolismo , Proteínas de la Membrana/genética , Transporte de Proteínas/fisiología , Serina Endopeptidasas/genéticaRESUMEN
The amyloid beta peptide (Aß) is derived from the amyloid precursor protein (APP) by secretase processing. APP is also cleaved by numerous other proteases, such as the type II transmembrane serine protease matriptase, with consequences on the production of Aß. Because the APP homolog protein amyloid-like protein 1 (APLP1) shares similarities with APP, we sought to determine if matriptase also plays a role in its processing. Here, we demonstrate that matriptase directly interacts with APLP1 and that APLP1 is cleaved in cellulo by matriptase in its E1 ectodomains at arginine 124. Replacing Arg124 with Ala abolished APLP1 processing by matriptase. Using a bioluminescence resonance energy transfer (BRET) assay we found that matriptase reduces APLP1 homodimeric interactions. This study identifies matriptase as the first protease cleaving APLP1 in its dimerization domain, potentially altering the multiple functions associated with dimer formation.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Serina Endopeptidasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/fisiología , Dimerización , Células HEK293 , Humanos , Serina Endopeptidasas/fisiologíaRESUMEN
Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.
Asunto(s)
Evaluación Preclínica de Medicamentos , Hepcidinas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , Benzotiazoles/química , Sitios de Unión , Dominio Catalítico , Supervivencia Celular/efectos de los fármacos , Proteínas Ligadas a GPI/metabolismo , Proteína de la Hemocromatosis/metabolismo , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Peptidomiméticos , Proteolisis/efectos de los fármacos , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
TMPRSS6, also known as matriptase-2, is a type II transmembrane serine protease that plays a major role in iron homeostasis by acting as a negative regulator of hepcidin production through cleavage of the BMP co-receptor haemojuvelin. Iron-refractory iron deficiency anaemia (IRIDA), an iron metabolism disorder, is associated with mutations in the TMPRSS6 gene. By analysing RNA-seq data encoding TMPRSS6 isoforms and other proteins involved in hepcidin production, we uncovered significant differences in expression levels between hepatocellular carcinoma (HCC) cell lines and normal human liver samples. Most notably, TMPRSS6 and HAMP expression was found to be much lower in HepG2 and Huh7 cells when compared to human liver samples. Furthermore, we characterized the common TMPRSS6 polymorphism V736A identified in Hep3B cells, the V795I mutation found in HepG2 cells, also associated with IRIDA, and the G603R substitution recently detected in two IRIDA patients. While variant V736A is as active as wild-type TMPRSS6, mutants V795I and G603R displayed significantly reduced proteolytic activity. Our results provide important information about commonly used liver cell models and shed light on the impact of two TMPRSS6 mutations associated with IRIDA.
Asunto(s)
Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Anemia Ferropénica/genética , Anemia Ferropénica/metabolismo , Células Hep G2 , Humanos , Hierro/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Iatrogenic tumorigenesis is a major limitation for the use of human induced pluripotent stem cells (hiPSCs) in hematology. The teratoma risk comes from the persistence of hiPSCs in differentiated cell populations. Our goal was to evaluate the best system to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSCs expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in immune-deficient mice was tracked by in vivo bioimaging. We observed that systemic injection of hiPSCs produced multisite teratoma as soon as 5 weeks after injection. To eliminate hiPSCs before grafting, we tested the embryonic-specific expression of suicide genes under the control of the pmiR-302/367 promoter. This promoter was highly active in hiPSCs but not in differentiated cells. The gene/prodrug inducible Caspase-9 (iCaspase-9)/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that iCaspase-9-expressing hiPSCs died in a dose-dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly, nonspecific toxicity of AP20187 on iCaspase-9-negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+ -derived human hematopoiesis in adoptive transfers. Survivin inhibition is an alternative to the suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155 was more efficient than AP20187/iCaspase-9 for killing hiPSCs, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSC purge by survivin inhibitor fully eradicated teratoma formation in immune-deficient mice. This will be useful to improve the safety management for hiPSC-based medicine. Stem Cells Translational Medicine 2017;6:382-393.
Asunto(s)
Caspasa 9/genética , Transformación Celular Neoplásica/efectos de los fármacos , Genes Transgénicos Suicidas , Enfermedades Hematológicas/cirugía , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Imidazoles/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/trasplante , Naftoquinonas/farmacología , Medicina Regenerativa/métodos , Survivin/antagonistas & inhibidores , Tacrolimus/análogos & derivados , Teratoma/prevención & control , Animales , Caspasa 9/metabolismo , Línea Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Medición de Riesgo , Survivin/metabolismo , Tacrolimus/farmacología , Teratoma/genética , Teratoma/metabolismo , Teratoma/patología , Factores de Tiempo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Matriptase-2, a type II transmembrane serine protease (TTSP), is expressed in the liver and regulates iron homeostasis via the cleavage of hemojuvelin. Matriptase-2 emerges as an attractive target for the treatment of conditions associated with iron overload, such as hemochromatosis or beta-thalassemia. Starting from the crystal structure of its closest homolog matriptase, we constructed a homology model of matriptase-2 in order to further optimize the selectivity of serine trap peptidomimetic inhibitors for matriptase-2 vs matriptase. Careful modifications of the P4, P3 and P2 positions with the help of unnatural amino acids led to a thorough understanding of Structure-Activity Relationship and a >60-fold increase in selectivity for matriptase-2 vs matriptase. Additionally, the introduction of unnatural amino acids led to significant increases in plasma stability. Such compounds represent useful pharmacological tools to test matriptase-2 inhibition in a context of iron overload.
Asunto(s)
Aminoácidos/química , Inhibidores Enzimáticos/farmacología , Sobrecarga de Hierro/tratamiento farmacológico , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Homeostasis/efectos de los fármacos , Humanos , Hierro/metabolismo , Modelos Moleculares , Sensibilidad y Especificidad , Serina Endopeptidasas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. METHODOLOGY/PRINCIPAL FINDING: To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P') sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived. CONCLUSIONS: Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.
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Biblioteca de Péptidos , Mapeo Peptídico , Péptidos/química , Serina Proteasas/química , Secuencia de Aminoácidos , Arginina/química , Arginina/genética , Dominio Catalítico , Humanos , Lisina/química , Lisina/genética , Péptidos/síntesis química , Péptidos/genética , Conformación Proteica , Proteoma , Serina Proteasas/genética , Programas Informáticos , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
The development of effective therapies inhibiting prostate cancer progression and metastasis may substantially impact prostate cancer mortality and potentially reduce the rates of invasive treatments by enhancing the safety of active surveillance strategies. Hepsin (HPN) is a cell surface serine protease amplified in a subset of human sarcomas (7.2%), as well as in ovarian (10%), lung adeno (5.4%), lung squamous cell (4.5%), adenoid cystic (5%), breast (2.6%), uterine (1.7%) and colon (1.4%) carcinomas. While HPN is not amplified in prostate cancer, it is one of the most prominently overexpressed genes in the majority of human prostate tumors and genetic experiments in mice indicate that Hepsin promotes prostate cancer metastasis, particularly metastasis to the bone marrow. We report here the development, analysis and animal trial of the small-molecule Hepsin inhibitor HepIn-13. Long-term exposure to HepIn-13 inhibited bone, liver and lung metastasis in a murine model of metastatic prostate cancer. These findings indicate that inhibition of Hepsin with small-molecule compounds could provide an effective tool for attenuation of prostate cancer progression and metastasis.
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Adenocarcinoma/tratamiento farmacológico , Neoplasias Óseas/prevención & control , Proteínas de la Membrana/antagonistas & inhibidores , Naftalenos/farmacocinética , Naftalenos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Pirimidinas/farmacocinética , Pirimidinas/uso terapéutico , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Inhibidores de Serina Proteinasa/uso terapéutico , Adenocarcinoma/metabolismo , Adenocarcinoma/secundario , Administración Oral , Animales , Disponibilidad Biológica , Neoplasias Óseas/secundario , Células HEK293 , Semivida , Humanos , Concentración 50 Inhibidora , Masculino , Ratones , Modelos Moleculares , Naftalenos/farmacología , Neoplasias de la Próstata/metabolismo , Pirimidinas/farmacología , Inhibidores de Serina Proteinasa/farmacocinéticaRESUMEN
Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⺠blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.
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Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Hematopoyesis/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Humanos , Células Madre Pluripotentes Inducidas/citología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Tumorales CultivadasRESUMEN
Type II transmembrane serine proteases are an emerging class of proteolytic enzymes involved in tissue homeostasis and a number of human disorders such as cancer. To better define the biochemical functions of a subset of these proteases, we compared the enzymatic properties of matriptase, matriptase-2, hepsin and DESC1 using a series of internally quenched fluorogenic peptide substrates containing o-aminobenzoyl and 3-nitro-tyrosine. We based the sequence of the peptides on the P4 to P4' activation sequence of matriptase (RQAR-VVGG). Positions P4, P3, P2 and P1' were substituted with nonpolar (Ala, Leu), aromatic (Tyr), acid (Glu) and basic (Arg) amino acids, whereas P1 was fixed to Arg. Of the four type II transmembrane serine proteases studied, matriptase-2 was the most promiscuous, and matriptase was the most discriminating, with a distinct specificity for Arg residues at P4, P3 and P2. DESC1 had a preference similar to that of matriptase, but with a propensity for small nonpolar amino acids (Ala) at P1'. Hepsin shared similarities with matriptase and DESC1, but was markedly more permissive at P2. Matriptase-2 manifested broader specificities, as well as substrate inhibition, for selective internally quenched fluorescent substrates. Lastly, we found that antithrombin III has robust inhibitory properties toward matriptase, matriptase-2, hepsin and DESC1, whereas plasminogen activator inhibitor-1 and alpha(2)-antiplasmin inhibited matriptase-2, hepsin and DESC1, and to a much lesser extent, matriptase. In summary, our studies revealed that these enzymes have distinct substrate preferences.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas de la Membrana/química , Péptidos/química , Serina Endopeptidasas/química , Animales , Células Cultivadas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Cinética , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Especificidad por SustratoRESUMEN
Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.