RESUMEN
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. METHODS: The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. RESULTS: Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. CONCLUSIONS: Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.
Asunto(s)
Genes MHC Clase II , Inmunoterapia , Neoplasias , Proproteína Convertasa 9 , Proproteína Convertasas , Animales , Ratones , Antígenos de Histocompatibilidad , Lipoproteínas LDL , Neoplasias/genética , Neoplasias/terapia , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/antagonistas & inhibidores , Receptores de LDL/genética , Microambiente TumoralRESUMEN
Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencias de Aminoácidos , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Quinasa de la Caseína I/genética , Diferenciación Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Ratones , Mutación , Osteoblastos/citología , Osteoblastos/metabolismo , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/genética , Dominios Proteicos , Transporte de Proteínas , Pirrolidinas/farmacología , Vías Secretoras , Serina Endopeptidasas/genéticaRESUMEN
Renal cell carcinoma (RCC) cells have increased lipogenesis and cholesterol synthesis. Sterol regulatory element-binding protein-1 (SREBP1) is cleaved by site 1 protease (S1P) to release the transcriptionally active amino-terminal domain. PF-429242 is a potent and competitive S1P inhibitor. We here tested its activity in RCC cells. In established and primary human RCC cells, PF-429242 potently inhibited cell proliferation, migration, and invasion. The S1P inhibitor provoked apoptosis activation in RCC cells. Furthermore, shRNA-mediated S1P silencing or CRISPR/Cas9-induced S1P knockout led to RCC cell growth inhibition and apoptosis activation. Conversely, ectopic overexpression of SREBP1 or S1P augmented RCC cell proliferation and migration. Daily i.v. injection of a single dose of PF-429242 robustly inhibited RCC xenograft growth in severe combined immunodeficiency mice. Additionally, intratumoral injection of S1P shRNA lentivirus inhibited RCC xenograft growth in mice. SREBP1, S1P, and its target gene low density lipoprotein receptor (LDLR) were significantly elevated in human RCC tissues. These results suggest that targeting S1P by PF-429242 inhibited RCC cell growth in vitro and in vivo.
Asunto(s)
Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Proproteína Convertasas/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Silenciador del Gen/efectos de los fármacos , Humanos , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , Proproteína Convertasas/metabolismo , Pirrolidinas , Serina Endopeptidasas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ATF6 has two isoforms, ATF6α and ATF6ß, which are ubiquitously expressed type II transmembrane glycoproteins in the endoplasmic reticulum (ER). While the regulatory mechanisms and transcriptional roles of ATF6α in response to ER stress have been well-studied, those of its paralogue ATF6ß are less understood. Moreover, there is no specific cell-based reporter assay to monitor ATF6ß activation. Here, we developed a new cell-based reporter system that can monitor activation of endogenous ATF6ß. This system expresses a chimeric protein containing a synthetic transcription factor followed by the transmembrane domain and C-terminal luminal domain of ATF6ß. Under ER stress conditions, the chimeric protein was cleaved by regulated intramembrane proteolysis (RIP) to liberate the N-terminal synthetic transcription factor, which induced luciferase expression in the HeLa Luciferase Reporter cell line. This new stable reporter cell line will be an innovative tool to investigate RIP of ATF6ß.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Factor de Transcripción Activador 6/química , Factor de Transcripción Activador 6/genética , Línea Celular , Ditiotreitol/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Genes Reporteros , Humanos , Luciferasas de Luciérnaga/química , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Proproteína Convertasas/antagonistas & inhibidores , Dominios Proteicos , Pirrolidinas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer.
Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Neoplasias del Colon/enzimología , Perfilación de la Expresión Génica/métodos , Metalotioneína/genética , Células Madre Neoplásicas/enzimología , Regulación hacia Arriba , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Neoplásicas/química , Células Madre Neoplásicas/efectos de los fármacos , Proproteína Convertasas/antagonistas & inhibidores , Análisis de Secuencia de ARN , Secuenciación del ExomaRESUMEN
Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.
Asunto(s)
Angiotensina II/farmacología , Presión Sanguínea/efectos de los fármacos , Hipertensión/genética , Proproteína Convertasas/antagonistas & inhibidores , Pirrolidinas/farmacología , Receptores de Superficie Celular/genética , Animales , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Ratones , Receptores de Superficie Celular/metabolismo , Renina/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Serina Endopeptidasas , Receptor de ProreninaRESUMEN
Although recent therapeutic developments raise hope, melanoma remains a devastating disease with a need for new treatment targets. In other tumours prohormone convertases have been shown to be pro-tumourigenic as they are involved in processing preforms of matrix-metalloproteinases, growth factors and adhesion molecules. The aim of this study was to look for new treatment options for melanoma, by investigating the role of the prohormone convertase Paired basic Amino acid-Cleaving Enzyme 4 (PACE4/PCSK6) in melanoma cell lines and human melanoma tissue. PACE4-transfected A375 melanoma cells displayed significantly increased proliferation, MMP-2 production, gelatinase activity and migratory capacity in vitro compared with sham-transfected cells. In vivo, elevated PACE4 expression resulted in significantly increased tumour growth on immunodeficient mice. In the majority of 45 human primary melanomas and melanoma metastases ex vivo PACE4 immunoreactivity was detectable, while it was absent in in situ melanomas. These results indicate PACE4 as a regulator of melanoma cell aggressiveness.
Asunto(s)
Melanoma/enzimología , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo , Neoplasias Cutáneas/enzimología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Ratones Pelados , Ratones SCID , Terapia Molecular Dirigida , Invasividad Neoplásica , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/genética , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/uso terapéutico , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Carga TumoralRESUMEN
Planteamos un paciente de 62 años que precisa tratamiento hipolipemiante con inhibidor de la proproteína convertasa subtilisina/kexina tipo9 (PCSK9) en el que empíricamente decidimos cambiar un fármaco a otro de los dos disponibles en la actualidad, obteniendo diferente respuesta. Nuestro objetivo es transmitir nuestra experiencia y reflexionar sobre una posible opción terapéutica en los pacientes en los que excepcionalmente esto pudiera ocurrir
The case is presented of a 62-year-old patient who required lipid-lowering therapy with proprotein convertase subtilisin/kexin type9 (PCSK9). It was empirically decided to change one drug to another of the two currently available, obtaining a different response. Our objective is to present our experience and to consider a possible therapeutic option in patients in whom, exceptionally, this could happen
Asunto(s)
Humanos , Masculino , Persona de Mediana Edad , Proproteína Convertasa 9/administración & dosificación , Proproteína Convertasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Hipercolesterolemia/tratamiento farmacológico , Presión Arterial/efectos de los fármacos , Proproteína Convertasas/metabolismo , Índice de Masa Corporal , Lipoproteínas LDL/efectos de los fármacosRESUMEN
Many cancers occur from locations of inflammation due to chronic irritation and/or infection. Tumor microenvironment contains various different inflammatory cells and mediators that orchestrate diverse neoplastic processes, including proliferation, survival, adhesion and migration. In parallel, tumor cells have adapted some of the signaling molecules used by inflammatory cells, such as selectins and chemokines as well as their receptors for invasion, extravasation and subsequently metastasis. Expression and/or activation of the majority of these molecules is mediated by the proprotein convertases (PCs); proteases expressed by both tumor cells and inflammatory cells. This review analyzes the potential role of these enzymatic system in inflammation-associated cancer impacting on the malignant and metastatic potential of cancer cells, describing the possible use of PCs as a new anti-inflammatory therapeutic approach to tumor progression and metastasis.
Asunto(s)
Carcinogénesis/inmunología , Inflamación/tratamiento farmacológico , Metástasis de la Neoplasia/inmunología , Neoplasias/inmunología , Proproteína Convertasas/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Carcinogénesis/efectos de los fármacos , Quimiocinas/inmunología , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Inflamación/inmunología , Inflamación/patología , Metástasis de la Neoplasia/prevención & control , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proproteína Convertasas/antagonistas & inhibidores , Selectinas/inmunología , Selectinas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.IMPORTANCE Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.
Asunto(s)
Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Línea Celular , Dipeptidil Peptidasa 4/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/patogenicidad , Péptido Hidrolasas/metabolismo , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/metabolismo , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Receptores Fc/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Tripsina/metabolismoRESUMEN
During tumorigenesis, macrophages are recruited by tumors and orientated towards a pro-tumoral phenotype. One of the main anti-tumoral immunotherapy consists of their re-polarization in an anti-tumoral phenotype. We have demonstrated that the inhibition of proprotein convertase 1/3 combined with TLR4 activation in macrophages is a promising strategy. These macrophages display pro-inflammatory and anti-tumoral phenotypes. A hallmark is a stronger activation of the pro-inflammatory NFKB pathway. We believe that this can be explained by a modification of TLR4 expression at the cell surface or MYD88 cleavage since it exhibits a potential cleavage site for proprotein convertases. We tested these hypotheses through immunofluorescence and Western blot experiments. A proteomics study was also performed to test the sensitivity of these macrophages to IL-10. We demonstrated that these macrophages treated with LPS showed a quicker re-expression of TLR4 at the cell surface. The level of MYD88 was also higher when TLR4 was internalized. Moreover, these macrophages were resistant to the pro-tumoral effect of IL-10 and still produced pro-inflammatory factors. This established that the sensitivity to anti-inflammatory molecules and the length of TLR4 desensitization were reduced in these macrophages. Therefore, during antitumoral immunotherapy, a repeated stimulation of TLR4 may reactivate PC1/3 inhibited macrophages even in an anti-inflammatory environment.
Asunto(s)
Interleucina-10/metabolismo , Macrófagos/metabolismo , Proproteína Convertasas/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Células Cultivadas , Lipopolisacáridos/farmacología , Fenotipo , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/deficiencia , RatasRESUMEN
The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high-throughput screening format. The robust and cost-effective assay format of BPCS makes it suitable to identify novel specific small-molecule inhibitors against basic PCs for therapeutic application. Its cell-based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened 'target range' will enhance the likelihood to identify novel small-molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.
Asunto(s)
Técnicas Biosensibles/métodos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Peptidomiméticos/farmacología , Proproteína Convertasas/metabolismo , Células A549 , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Técnicas Biosensibles/normas , Descubrimiento de Drogas/normas , Inhibidores Enzimáticos/química , Genes Reporteros , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento/métodos , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Peptidomiméticos/química , Proproteína Convertasas/antagonistas & inhibidores , Sensibilidad y EspecificidadRESUMEN
No disponible
Asunto(s)
Humanos , Enfermedad Coronaria/tratamiento farmacológico , Proproteína Convertasas/antagonistas & inhibidores , Dislipidemias/tratamiento farmacológico , Resultado del Tratamiento , Combinación Ezetimiba y Simvastatina/uso terapéutico , Hipolipemiantes/uso terapéuticoRESUMEN
The antidiuretic hormone vasopressin (AVP), acting through its type 2 receptor (V2R) in the collecting duct (CD), critically controls urine concentrating capability. Here, we report that site-1 protease-derived (S1P-derived) soluble (pro)renin receptor (sPRR) participates in regulation of fluid homeostasis via targeting V2R. In cultured inner medullary collecting duct (IMCD) cells, AVP-induced V2R expression was blunted by a PRR antagonist, PRO20; a PRR-neutralizing antibody; or a S1P inhibitor, PF-429242. In parallel, sPRR release was increased by AVP and reduced by PF-429242. Administration of histidine-tagged sPRR, sPRR-His, stimulated V2R expression and also reversed the inhibitory effect of PF-429242 on the expression induced by AVP. PF-429242 treatment in C57/BL6 mice impaired urine concentrating capability, which was rescued by sPRR-His. This observation was recapitulated in mice with renal tubule-specific deletion of S1P. During the pharmacological or genetic manipulation of S1P alone or in combination with sPRR-His, the changes in urine concentration were paralleled with renal expression of V2R and aquaporin-2 (AQP2). Together, these results support that S1P-derived sPRR exerts a key role in determining renal V2R expression and, thus, urine concentrating capability.
Asunto(s)
Capacidad de Concentración Renal/fisiología , Túbulos Renales Colectores/metabolismo , ATPasas de Translocación de Protón/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Vasopresinas/metabolismo , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Acuaporina 2/genética , Células Cultivadas , Células Epiteliales , Capacidad de Concentración Renal/efectos de los fármacos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Fragmentos de Péptidos/farmacología , Cultivo Primario de Células , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Pirrolidinas/farmacología , Ratas , Receptores de Vasopresinas/genética , Renina/metabolismo , Renina/farmacología , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Urotelio/citología , ATPasas de Translocación de Protón VacuolaresRESUMEN
There are no approved therapies for Ebola virus infection. Here, to find potential therapeutic targets, we perform a screen for genes essential for Ebola virus (EBOV) infection. We identify GNPTAB, which encodes the α and ß subunits of N-acetylglucosamine-1-phosphate transferase. We show that EBOV infection of a GNPTAB knockout cell line is impaired, and that this is reversed by reconstituting GNPTAB expression. Fibroblasts from patients with mucolipidosis II, a disorder associated with mutations in GNPTAB, are refractory to EBOV, whereas cells from their healthy parents support infection. Impaired infection correlates with loss of the expression of cathepsin B, known to be essential for EBOV entry. GNPTAB activity is dependent upon proteolytic cleavage by the SKI-1/S1P protease. Inhibiting this protease with the small-molecule PF-429242 blocks EBOV entry and infection. Disruption of GNPTAB function may represent a strategy for a host-targeted therapy for EBOV.
Asunto(s)
Antivirales/farmacología , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Mucolipidosis/patología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Internalización del Virus/efectos de los fármacos , Células A549 , Animales , Antivirales/uso terapéutico , Catepsina B/metabolismo , Chlorocebus aethiops , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Fibroblastos , Técnicas de Inactivación de Genes , Fiebre Hemorrágica Ebola/virología , Humanos , Mucolipidosis/genética , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/metabolismo , Inhibidores de Proteasas/farmacología , Pirrolidinas/farmacología , Serina Endopeptidasas/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Células Vero , Secuenciación Completa del GenomaRESUMEN
The activation of viral glycoproteins by the host protease furin is an essential step in the replication of numerous pathogenic viruses. Thus, effective inhibitors of furin could serve as broad-spectrum antiviral drugs. A crystal structure of an inhibitory hexapeptide derivative in complex with furin served as template for the rational design of various types of new cyclic inhibitors. Most of the prepared derivatives are relatively potent furin inhibitors with inhibition constants in the low nanomolar or even sub-nanomolar range. For seven derivatives the crystal structures in complex with furin could be determined. In three complexes, electron density was found for the entire inhibitor. In the other cases the structures could be determined only for the P6/P5-P1 segments, which directly interact with furin. The cyclic derivatives together with two non-cyclic reference compounds were tested as inhibitors of the proteolytic activation and replication of respiratory syncytial virus in cells. Significant antiviral activity was found for both linear reference inhibitors, whereas a negligible efficacy was determined for the cyclic derivatives.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Furina/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Humanos , Compuestos Macrocíclicos/síntesis químicaRESUMEN
Overexpression of lysyl oxidase-like 2 (LOXL2) is associated with several hepatic and vascular fibrotic diseases and tumor progression in some aggressive cancers. Secreted LOXL2 promotes extracellular matrix cross-linking by catalyzing the oxidative deamination of peptidyl lysine. A great deal remains to be learned about the post-translational modifications of LOXL2, including whether such modifications modulate enzymatic and disease-promoting activities; such knowledge would inform the development of potential therapies. We discovered that upon secretion in cell culture, LOXL2 undergoes proteolytic processing of the first two of four scavenger receptor cysteine-rich domains at the N-terminus. A similar pattern of processing was also evident in tissue extracts from an invasive ductal carcinoma patient. Processing occurred at 314Arg-315Phe-316Arg-317Lys↓-318Ala-, implicating proprotein convertases. siRNA-mediated knockdown of proprotein convertases (furin, PACE4, and PC5/6), as well as incubation with their recombinant forms, showed that PACE4 is the major protease that acts on extracellular LOXL2. Unlike LOX, which requires cleavage of its propeptide for catalytic activation, cleavage of LOXL2 was not essential for tropoelastin oxidation or for cross-linking of collagen type IV in vitro. However, in the latter case, processing enhanced the extent of collagen cross-linking â¼2-fold at ≤10 nM LOXL2. These results demonstrate an important difference in the regulatory mechanisms for LOX and LOXL2 catalytic activity. Moreover, they pave the way for further studies of potential differential functions of LOXL2 isoforms in fibrosis and tumor progression.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Aminoácido Oxidorreductasas/química , Aminoácido Oxidorreductasas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Neoplasias de la Mama/enzimología , Línea Celular , Colágeno Tipo IV/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Mutagénesis Sitio-Dirigida , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Proteína-Lisina 6-Oxidasa/química , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , ARN Interferente Pequeño/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
Paired basic amino acid cleaving enzyme 4 (PACE4), a serine endoprotease of the proprotein convertases family, has been recognized as a promising target for prostate cancer. We previously reported a selective and potent peptide-based inhibitor for PACE4, named the multi-Leu peptide (Ac-LLLLRVKR-NH2 sequence), which was then modified into a more potent and stable compound named C23 with the following structure: Ac-dLeu-LLLRVK-Amba (Amba: 4-amidinobenzylamide). Despite improvements in both in vitro and in vivo profiles of C23, its selectivity for PACE4 over furin was significantly reduced. We examined other Arg-mimetics instead of Amba to regain the lost selectivity. Our results indicated that the replacement of Amba with 5-(aminomethyl)picolinimidamide increased affinity for PACE4 and restored selectivity. Our results also provide a better insight on how structural differences between S1 pockets of PACE4 and furin could be employed in the rational design of selective inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Humanos , Masculino , Simulación del Acoplamiento Molecular , Proproteína Convertasas/química , Proproteína Convertasas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Relación Estructura-ActividadRESUMEN
The serine protease, PACE4, is a proprotein convertase that plays a substantial role in malignancy of prostate cancer. Our initial selective PACE4 inhibitor (Ac-LLLLRVKR-NH2) has evolved to the current lead compound C23 (Ac-dLeu-LLLRVK-Amba), which is active both in vitro and in vivo. By screening natural residues, except Cys, in C-terminal P1' position, it was established that increasing hydrophobicity was improving cell permeability, which was directly translated into PCa cells antiproliferative activity. This cell antiproliferation enhancement seems independent from effect of P1' residue on PACE4 affinity. Replacement of P1-Amba of C23 by Acpa (( S)-2-amino-3-(4-carbamimidoylphenyl)propanoic acid) followed by addition of tryptamine in P1' resulted in compound 32 exhibiting superior PCa cells antiproliferative activity over the reference compound C23 (3-fold). This study sheds light on key factors that improve cell penetrating property and antiproliferative activity of PACE4 inhibitors.