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1.
Angew Chem Int Ed Engl ; 56(5): 1294-1297, 2017 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-27981705

RESUMEN

CSN5 is the zinc metalloprotease subunit of the COP9 signalosome (CSN), which is an important regulator of cullin-RING E3 ubiquitin ligases (CRLs). CSN5 is responsible for the cleavage of NEDD8 from CRLs, and blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state, thereby leading to auto-ubiquitination and ultimately degradation of the substrate recognition subunits. Herein, we describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion of CSN5 through an unprecedented binding mode. The best compounds inhibited CSN5 with nanomolar potency, led to degradation of the substrate recognition subunit Skp2 in cells, and reduced the viability of HCT116 cells.


Asunto(s)
Complejo del Señalosoma COP9/antagonistas & inhibidores , Indoles/metabolismo , Zinc/metabolismo , Sitios de Unión , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Dominio Catalítico , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Células HCT116 , Humanos , Indoles/química , Indoles/farmacología , Simulación del Acoplamiento Molecular , Proteína NEDD8/química , Proteína NEDD8/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Zinc/química
2.
J Immunol ; 188(6): 2794-804, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22345649

RESUMEN

Human CMV (HCMV)-encoded NK cell-evasion functions include an MHC class I homolog (UL18) with high affinity for the leukocyte inhibitory receptor-1 (CD85j, ILT2, or LILRB1) and a signal peptide (SP(UL40)) that acts by upregulating cell surface expression of HLA-E. Detailed characterization of SP(UL40) revealed that the N-terminal 14 aa residues bestowed TAP-independent upregulation of HLA-E, whereas C region sequences delayed processing of SP(UL40) by a signal peptide peptidase-type intramembrane protease. Most significantly, the consensus HLA-E-binding epitope within SP(UL40) was shown to promote cell surface expression of both HLA-E and gpUL18. UL40 was found to possess two transcription start sites, with utilization of the downstream site resulting in translation being initiated within the HLA-E-binding epitope (P2). Remarkably, this truncated SP(UL40) was functional and retained the capacity to upregulate gpUL18 but not HLA-E. Thus, our findings identify an elegant mechanism by which an HCMV signal peptide differentially regulates two distinct NK cell-evasion pathways. Moreover, we describe a natural SP(UL40) mutant that provides a clear example of an HCMV clinical virus with a defect in an NK cell-evasion function and exemplifies issues that confront the virus when adapting to immunogenetic diversity in the host.


Asunto(s)
Proteínas de la Cápside/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune/inmunología , Células Asesinas Naturales/inmunología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Northern Blotting , Western Blotting , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Separación Celular , Citomegalovirus/genética , Citomegalovirus/inmunología , Citomegalovirus/metabolismo , Infecciones por Citomegalovirus , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Células Asesinas Naturales/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genética , Proteínas Virales/inmunología , Antígenos HLA-E
3.
Nat Cell Biol ; 8(8): 894-6, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16829951

RESUMEN

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Membranas Intracelulares/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Pez Cebra/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión/genética , Línea Celular , Citosol/química , Citosol/enzimología , Citosol/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Proteínas de Pez Cebra/genética
4.
Nat Cell Biol ; 8(8): 843-8, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16829952

RESUMEN

Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Células Dendríticas/efectos de los fármacos , Interleucina-12/biosíntesis , Membranas Intracelulares/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Ácido Aspártico Endopeptidasas/genética , Proteínas Bacterianas/genética , Western Blotting , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Citometría de Flujo , Células HeLa , Humanos , Hidrólisis/efectos de los fármacos , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Fluorescente , Interferencia de ARN
5.
J Med Chem ; 65(19): 13052-13073, 2022 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-36178776

RESUMEN

Addressing resistance to third-generation EGFR TKIs such as osimertinib via the EGFRC797S mutation remains a highly unmet need in EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we present the discovery of the allosteric EGFR inhibitor 57, a novel fourth-generation inhibitor to overcome EGFRC797S-mediated resistance in patients harboring the activating EGFRL858R mutation. 57 exhibits an improved potency compared to previous allosteric EGFR inhibitors. To our knowledge, 57 is the first allosteric EGFR inhibitor that demonstrates robust tumor regression in a mutant EGFRL858R/C797S tumor model. Additionally, 57 is active in an H1975 EGFRL858R/T790M NSCLC xenograft model and shows superior efficacy in combination with osimertinib compared to the single agents. Our data highlight the potential of 57 as a single agent against EGFRL858R/C797S and EGFRL858R/T790M/C797S and as combination therapy for EGFRL858R- and EGFRL858R/T790M-driven NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Acrilamidas , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Indoles , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas
6.
Clin Cancer Res ; 28(4): 770-780, 2022 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-34782366

RESUMEN

PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.


Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Encéfalo/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico
7.
Trends Cell Biol ; 13(2): 71-8, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12559757

RESUMEN

This review summarizes recent findings and common principles for intramembrane-cleaving proteases that catalyse critical steps in cell regulation and signalling and which are involved in diseases such as Alzheimer's disease and hepatitis C virus infection.


Asunto(s)
Membrana Celular/enzimología , Endopeptidasas/metabolismo , Células Eucariotas/enzimología , Membranas Intracelulares/enzimología , Péptidos/metabolismo , Animales , Dominio Catalítico/fisiología , Humanos , Hidrólisis , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Presenilina-1
8.
ACS Chem Biol ; 14(10): 2215-2223, 2019 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-31553577

RESUMEN

Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action. A loss-of-function screen for genes required for BRD4 degradation revealed strong dependence on the E2 and E3 ubiquitin ligases as well as for members of the COP9 signalosome complex for both cereblon- and Von Hippel-Lindau-engaging BRD4 degraders. Cancer cell lines raised to resist BRD4 degraders manifested a degrader-specific mechanism of resistance, resulting from the loss of components of the ubiquitin proteasome system. In addition, degrader profiling in a cancer cell line panel revealed a differential pattern of activity of Von Hippel-Lindau- and cereblon-based degraders, highlighting the need for the identification of degradation-predictive biomarkers enabling effective patient stratification.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Azepinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Dipéptidos/farmacología , Células HEK293 , Humanos , Ftalimidas/farmacología , Prueba de Estudio Conceptual , Proteolisis , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/metabolismo
9.
Genetics ; 170(1): 139-48, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15716490

RESUMEN

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.


Asunto(s)
Ácido Aspártico Endopeptidasas/fisiología , Drosophila melanogaster/enzimología , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Técnica del Anticuerpo Fluorescente , Larva/enzimología , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Alas de Animales/enzimología , Alas de Animales/crecimiento & desarrollo
10.
Eur J Pharmacol ; 540(1-3): 10-7, 2006 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-16697367

RESUMEN

The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC50 = 1.33 microM) and the gamma-secretase potent inhibitors NVP-AHW700-NX (IC50 = 51 nM) and LY411575 (IC50 = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión/genética , Células CHO , Línea Celular , Cricetinae , Cricetulus , Dipéptidos/farmacología , Retículo Endoplásmico/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismo
11.
Nat Commun ; 7: 13166, 2016 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-27774986

RESUMEN

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin-proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.


Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Complejo del Señalosoma COP9/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Pirazoles/farmacología , Ubiquitina-Proteína Ligasas/genética , Animales , Antineoplásicos/síntesis química , Azepinas/síntesis química , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/metabolismo , Femenino , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Ratones , Ratones SCID , Terapia Molecular Dirigida , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteolisis/efectos de los fármacos , Pirazoles/síntesis química , Células THP-1 , Carga Tumoral/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Biochem J ; 384(Pt 1): 9-17, 2004 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-15373738

RESUMEN

The endoplasmic reticulum (ER) exerts a quality control over newly synthesized proteins and a variety of components have been implicated in the specific recognition of aberrant or misfolded polypeptides. We have exploited a site-specific cross-linking approach to search for novel ER components that may specifically recognize the misassembled transmembrane domains present in truncated polytopic proteins. We find that a single probe located in the transmembrane domain of a truncated opsin fragment is cross-linked to several ER proteins. These components are distinct from subunits of the Sec61 complex and represent a 'post-translocon' environment. In this study, we identify one of these post-translocon cross-linking partners as the signal peptide peptidase (SPP). We find that the interaction of truncated opsin chains with SPP is mediated by its second transmembrane domain, and propose that this interaction may contribute to the recognition of misassembled transmembrane domains during membrane protein quality control at the ER.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Opsinas de Bastones/metabolismo , Empalme Alternativo/genética , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Bovinos , Reactivos de Enlaces Cruzados/metabolismo , Dipéptidos/farmacología , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Membranas Intracelulares/química , Proteínas de la Membrana/metabolismo , Sondas Moleculares , Péptidos/química , Péptidos/metabolismo , Pliegue de Proteína , Estructura Terciaria de Proteína , Opsinas de Bastones/genética
13.
FEBS Lett ; 564(3): 213-8, 2004 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-15111098

RESUMEN

Intramembrane-cleaving proteases are members of a novel type of enzyme that hydrolyse substrate proteins within transmembrane regions. The presently known proteases that catalyse such cleavage reactions are membrane proteins of high hydrophobicity and multiple predicted transmembrane regions. A key feature is the positioning of active site residues in hydrophobic segments implying that the catalytic centre is assembled within the plane of the membrane. Nevertheless, all these proteases appear to utilise catalytic mechanisms similar to classic proteases that expose their active site domains in aqueous compartments. In the present review, we will address the mechanism of intramembrane proteolysis on the example of the signal peptide peptidase, and discuss how enzyme-catalysed hydrolysis of peptide bonds within the plane of a cellular membrane might occur.


Asunto(s)
Ácido Aspártico Endopeptidasas , Membrana Celular/metabolismo , Proteínas de la Membrana , Conformación Proteica , Animales , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Hidrólisis , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Especificidad por Sustrato
14.
J Exp Med ; 210(1): 23-30, 2013 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-23267013

RESUMEN

B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Linfocitos B/fisiología , Células Dendríticas/patología , Proteínas de la Membrana/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Ácido Aspártico Endopeptidasas/genética , Linfocitos B/patología , Supervivencia Celular , Células Dendríticas/fisiología , Etilnitrosourea/farmacología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoglobulinas/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Mutagénesis/efectos de los fármacos , Mutación , Linfocitos T/inmunología , Linfocitos T/metabolismo
15.
J Biol Chem ; 283(15): 9966-76, 2008 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-18270201

RESUMEN

N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.


Asunto(s)
Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Virus del Tumor Mamario del Ratón/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Células COS , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplásmico/genética , Glicosilación , Células HeLa , Humanos , Virus del Tumor Mamario del Ratón/genética , Ratones , Microsomas/metabolismo , Señales de Localización Nuclear/genética , Modificación Traduccional de las Proteínas/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas del Envoltorio Viral/genética
16.
Curr Pharm Des ; 13(3): 271-85, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17313361

RESUMEN

Aspartic proteases are the smallest class of human proteases with only 15 members. Over the past years, they have received considerable attention as potential targets for pharmaceutical intervention since many have been shown to play important roles in physiological and pathological processes. Despite numerous efforts, however, the only inhibitors for aspartic proteases currently on the market are directed against the HIV protease, an aspartic protease of viral origin. Nevertheless, several inhibitors including those targeting renin, BACE1 and gamma-secretase are in clinical or preclinical development, and some other aspartic proteases are discussed as potential drug target. The crystal structures of seven human aspartic proteases have now been solved and, together with a detailed kinetic understanding of their catalytic mechanism, this has greatly contributed to the design and discovery of novel inhibitors for this protease class. This review describes current aspartic protease drug targets and summarizes the drug discovery efforts in this field. In addition, it highlights recent developments which may lead to a new generation of aspartic protease inhibitors.


Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/química , Diseño de Fármacos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Diseño Asistido por Computadora , Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Molecular , Presenilinas/antagonistas & inhibidores , Presenilinas/química , Conformación Proteica , Renina/antagonistas & inhibidores , Renina/química , Relación Estructura-Actividad
17.
Anal Biochem ; 371(2): 201-7, 2007 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-17869210

RESUMEN

The dynamic modification of proteins with ubiquitin is a key regulation paradigm in eukaryotic cells that controls stability, localization, and function of the vast majority of intracellular proteins. Here we describe a robust fluorescence intensity assay for monitoring the enzymatic activity of deubiquitinating proteases, which reverse ubiquitin modifications and comprise over 100 members in humans. The assay was developed for the catalytic domain of human ubiquitin-specific protease 2 (USP2) and human ubiquitin carboxyterminal hydrolase L3 (UCH-L3), and makes use of the novel substrate ubiquitin-rhodamine110-glycine. The latter combines the advantages of a high dynamic range and beneficial optical properties. Its enzymatic behavior is characterized by the kinetic constants K(m)=1.5 microM, k(cat) = 0.53s(-1) and k(cat)/K(m) = 3.5 x 10(5)M(-1) s(-1) for USP2 and K(m) = 34 nM, k(cat)=4.72s(-1), and k(cat)/K(m) = 1.4 x 10(8)M(-1) s(-1) for UCH-L3. This new assay is suitable for inhibitor screening and characterizations, and has been established for the 384-well plate format using protease concentrations of 120 pM for USP2 and 1 pM for UCH-L3 and substrate concentrations of 100 nM for both enzymes. Due to the low protease concentrations and high sensitivity, this assay would allow the determination of inhibitory constants in the subnanomolar range.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Endopeptidasas/metabolismo , Colorantes Fluorescentes/química , Glicina/análogos & derivados , Glicina/química , Ubiquitina/química , Bioensayo/instrumentación , Bioensayo/métodos , Cisteína Endopeptidasas/química , Endopeptidasas/química , Colorantes Fluorescentes/metabolismo , Glicina/metabolismo , Humanos , Inteínas , Cinética , Modelos Biológicos , Rodaminas/química , Rodaminas/metabolismo , Espectrometría de Fluorescencia , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa
18.
J Virol ; 80(4): 1915-21, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16439547

RESUMEN

The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL)(2)-ketone, which is specific for signal peptide peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D(265)A mutant. The presence of (Z-LL)(2)-ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D(265)A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine(255) and is located in the hydrophobic center of the signal peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Virus de la Fiebre Porcina Clásica/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Porcinos , Proteínas del Núcleo Viral/química
19.
Hum Mol Genet ; 12 Spec No 2: R201-6, 2003 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-12966028

RESUMEN

Recent studies demonstrate that presenilins (PSs) and signal peptide peptidase (SPP) are members of a novel protease family of integral membrane proteins that may utilize a catalytic mechanism similar to classic aspartic proteases such as pepsin, renin and cathepsin D. The defining features of the PSs and SPP are their ability to cleave substrate polypeptides within a transmembrane region, the presence of two active site aspartate residues in adjacent membrane-spanning regions and a conserved PAL motif near their COOH-terminus. PSs appear to be the catalytic subunit of multiprotein complexes that possess gamma-secretase activity. Because this activity generates the amyloid beta peptide (Abeta) deposited in the brain of patients with Alzheimer's disease (AD), PSs are considered therapeutic targets in AD. In contrast to PSs that are not active unless part of a larger complex, SPP does not appear to require protein co-factors. Because of its requirement for hepatitis C virus maturation and a possible immune modulatory role, SPP is also considered a potential therapeutic target. Four additional PS/SPP homologs have been identified in humans; yet, their functions have not been elucidated. Herein, we will review the recent advances in our understanding of the PS/SPP family of proteases as well as discuss aspects of intramembrane cleavage that are not well understood.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Membrana Celular/enzimología , Membrana Celular/metabolismo , Hidrólisis , Presenilina-1 , Presenilina-2
20.
Mol Cell ; 10(4): 735-44, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12419218

RESUMEN

The presenilin-type aspartic protease signal peptide peptidase (SPP) can cleave signal peptides within their transmembrane region. SPP is essential for generation of signal peptide-derived HLA-E epitopes in humans and is exploited by Hepatitis C virus for processing of the viral polyprotein. Here we analyzed requirements of substrates for intramembrane cleavage by SPP. Comparing signal peptides that are substrates with those that are not revealed that helix-breaking residues within the transmembrane region are required for cleavage, and flanking regions can affect processing. Furthermore, signal peptides have to be liberated from the precursor protein by cleavage with signal peptidase in order to become substrates for SPP. We propose that signal peptides require flexibility in the lipid bilayer to exhibit an accessible peptide bond for intramembrane proteolysis.


Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Membrana Celular/enzimología , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Secuencia de Consenso , Cricetinae , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato
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