RESUMEN
Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).
Asunto(s)
Matriz Extracelular/enzimología , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/metabolismo , Procesamiento Proteico-Postraduccional , Transglutaminasas/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Biocatálisis/efectos de los fármacos , Células Cultivadas , Cistina/metabolismo , Enzimas Inmovilizadas/antagonistas & inhibidores , Enzimas Inmovilizadas/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Glutatión/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/genética , Proteína Glutamina Gamma Glutamiltransferasa 2 , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Transglutaminasas/química , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.