RESUMO
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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Acrilamidas , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Humanos , Indóis , Neoplasias Pulmonares/patologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , PirimidinasRESUMO
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.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Encéfalo/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Azepinas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Células HEK293 , Humanos , Ftalimidas/farmacologia , Estudo de Prova de Conceito , Proteólise , Fatores de Transcrição/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Pirazóis/farmacologia , Ubiquitina-Proteína Ligases/genética , Animais , Antineoplásicos/síntese química , Azepinas/síntese química , Complexo do Signalossomo COP9/genética , Complexo do Signalossomo COP9/metabolismo , Feminino , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise/efeitos dos fármacos , Pirazóis/síntese química , Células THP-1 , Carga Tumoral/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas do Envelope Viral/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Células COS , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplasmático/genética , Glicosilação , Células HeLa , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Microssomos/metabolismo , Sinais de Localização Nuclear/genética , Modificação Traducional de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Corantes Fluorescentes/química , Glicina/análogos & derivados , Glicina/química , Ubiquitina/química , Bioensaio/instrumentação , Bioensaio/métodos , Cisteína Endopeptidases/química , Endopeptidases/química , Corantes Fluorescentes/metabolismo , Glicina/metabolismo , Humanos , Inteínas , Cinética , Modelos Biológicos , Rodaminas/química , Rodaminas/metabolismo , Espectrometria de Fluorescência , Especificidade por Substrato , Ubiquitina/metabolismo , Ubiquitina TiolesteraseRESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endopeptidases/metabolismo , Membranas Intracelulares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação/genética , Linhagem Celular , Citosol/química , Citosol/enzimologia , Citosol/metabolismo , Humanos , Dados de Sequência Molecular , Mutação/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Células Dendríticas/efeitos dos fármacos , Interleucina-12/biossíntese , Membranas Intracelulares/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Proteínas de Bactérias/genética , Western Blotting , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Hidrólise/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia de Fluorescência , Interferência de RNARESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Vírus da Febre Suína Clássica/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas do Core Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , Suínos , Proteínas do Core Viral/químicaRESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Opsinas de Bastonetes/metabolismo , Processamento Alternativo/genética , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Bovinos , Reagentes de Ligações Cruzadas/metabolismo , Dipeptídeos/farmacologia , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Membranas Intracelulares/química , Proteínas de Membrana/metabolismo , Sondas Moleculares , Peptídeos/química , Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Opsinas de Bastonetes/genéticaRESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Hidrólise , Presenilina-1 , Presenilina-2Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas do Core Viral/análise , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Soluções Tampão , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Hepacivirus , Humanos , Microssomos/metabolismo , Dados de Sequência Molecular , Peso Molecular , Padrões de ReferênciaRESUMO
The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/química , Antígenos HLA/química , Antígenos de Histocompatibilidade Classe I/química , Proteínas de Membrana , Complexos Multienzimáticos/química , Acetilcisteína/farmacologia , Ácidos/farmacologia , Alelos , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Relação Dose-Resposta a Droga , Epitopos/química , Citometria de Fluxo , Antígenos HLA-G , Humanos , Ligantes , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/fisiologia , Oligopeptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Serina Endopeptidases/química , Transcrição Gênica , Transfecção , Antígenos HLA-ERESUMO
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.
Assuntos
Membrana Celular/enzimologia , Endopeptidases/metabolismo , Células Eucarióticas/enzimologia , Membranas Intracelulares/enzimologia , Peptídeos/metabolismo , Animais , Domínio Catalítico/fisiologia , Humanos , Hidrólise , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Presenilina-1RESUMO
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.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/enzimologia , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/genética , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Sequência Consenso , Cricetinae , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por SubstratoRESUMO
To test how far into the protein-conducting channel of the translocon complex a nascent polypeptide domain must move before it can fold, we analyzed the folding of in vitro translated products of truncated mRNAs encoding the Semliki Forest virus capsid protease domain (Cp) during translocation into microsomes. Cp folded when the C-terminal linker connecting it to the peptidyltransferase center was 64 amino acids or longer. This means that to fold, Cp must exit the translocon channel. With an uncleaved signal sequence, about one out of four of the Cp domains could undergo folding with a C-terminal linker of only 38-66 amino acids. This suggested that the constraint imposed on folding by the translocon complex may be less stringent for signal-anchored membrane proteins.
Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Genes Reporter , Membranas Intracelulares/metabolismo , Microssomos/metabolismo , Modelos Biológicos , Modelos Moleculares , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Vírus da Floresta de SemlikiRESUMO
Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein.