RESUMEN
The integral membrane protease ZMPSTE24 plays an important role in the lamin A maturation pathway. ZMPSTE24 is the only known enzyme to cleave the last 15 residues from the C-terminus of prelamin A, including a farnesylated and carboxyl methylated cysteine. Mutations in ZMPSTE24 lead to progeroid diseases with abnormal prelamin A accumulation in the nucleus. Ste24 is the yeast functional homolog of ZMPSTE24 and similarly cleaves the a-factor pheromone precursor during its posttranslational maturation. To complement established qualitative techniques used to detect the upstream enzymatic cleavage by ZMPSTE24 and Ste24, including gel-shift assays and mass spectrometry analyses, we developed an enzymatic in vitro FRET-based assay to quantitatively measure the upstream cleavage activities of these two enzymes. This assay uses either purified enzyme or enzyme in crude membrane preparations and a 33-amino acid a-factor analog peptide that is a substrate for both Ste24 and ZMPSTE24. This peptide contains a fluorophore (2-aminobenzoic acid-Abz) at its N-terminus and a quencher moiety (dinitrophenol-DNP) positioned four residues downstream from the cleavage site. Upon cleavage, a fluorescent signal is generated in real time at 420 nm that is proportional to cleavage of the peptide and these kinetic data are used to quantify activity. This assay should provide a useful tool for kinetic analysis and for studying the catalytic mechanism of both ZMPSTE24 and Ste24.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas de la Membrana/química , Metaloendopeptidasas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Humanos , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.
Asunto(s)
Lamina Tipo A/metabolismo , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Mutación Missense/genética , Progeria/genética , Alelos , Secuencias de Aminoácidos , Vías Biosintéticas , Humanos , Lamina Tipo A/biosíntesis , Proteínas de la Membrana/química , Metaloendopeptidasas/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Canales de Translocación SEC/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.
Asunto(s)
Alquinos/química , Azidas/química , Factor de Apareamiento/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Terpenos/química , Alquinos/síntesis química , Alquinos/metabolismo , Azidas/síntesis química , Azidas/metabolismo , Línea Celular , Factor de Apareamiento/síntesis química , Factor de Apareamiento/metabolismo , Prenilación de Proteína , Proteolisis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/síntesis química , Proteínas de Saccharomyces cerevisiae/metabolismo , Terpenos/síntesis química , Terpenos/metabolismoRESUMEN
Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.
Asunto(s)
Dimetilaliltranstransferasa/antagonistas & inhibidores , Dimetilaliltranstransferasa/química , Colorantes Fluorescentes/química , Sondas Moleculares/química , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Dimetilaliltranstransferasa/genética , Difosfonatos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/química , Farnesiltransferasa/genética , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/química , Geraniltranstransferasa/genética , Humanos , Imidazoles/farmacología , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Especificidad por Sustrato , Ácido ZoledrónicoRESUMEN
Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.