RESUMEN
Cancer cells exhibit distinct metabolic activities and nutritional dependencies compared to normal cells. Thus, characterization of nutrient demands by individual tumor types may identify specific vulnerabilities that can be manipulated to target the destruction of cancer cells. We find that MYC-driven liver tumors rely on augmented tryptophan (Trp) uptake, yet Trp utilization to generate metabolites in the kynurenine (Kyn) pathway is reduced. Depriving MYC-driven tumors of Trp through a No-Trp diet not only prevents tumor growth but also restores the transcriptional profile of normal liver cells. Despite Trp starvation, protein synthesis remains unhindered in liver cancer cells. We define a crucial role for the Trp-derived metabolite indole 3-pyruvate (I3P) in liver tumor growth. I3P supplementation effectively restores the growth of liver cancer cells starved of Trp. These findings suggest that I3P is a potential therapeutic target in MYC-driven cancers. Developing methods to target this metabolite represents a potential avenue for liver cancer treatment.
Asunto(s)
Carcinogénesis , Indoles , Neoplasias Hepáticas , Proteínas Proto-Oncogénicas c-myc , Triptófano , Triptófano/metabolismo , Animales , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Indoles/metabolismo , Indoles/farmacología , Humanos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratones , Carcinogénesis/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Quinurenina/metabolismo , Ratones Endogámicos C57BL , Hígado/metabolismo , Hígado/patología , MasculinoRESUMEN
The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.
Asunto(s)
Nucléolo Celular , Proteínas de Unión al ADN , Nucleofosmina , Factores de Transcripción , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Ribosómico/metabolismo , Humanos , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.
Asunto(s)
Proteínas de Unión al ADN , Biosíntesis de Proteínas , Proteínas Ribosómicas , Subunidades Ribosómicas Pequeñas de Eucariotas , Factores de Transcripción , Nucléolo Celular/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Humanos , Animales , Ratas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.
Asunto(s)
Neoplasias del Colon , Contaminantes Ambientales , Dibenzodioxinas Policloradas , Humanos , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Vía de Señalización Wnt/genética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Ligandos , Quinurenina , Triptófano , Actinas/metabolismo , Neoplasias del Colon/genética , ARNRESUMEN
BACKGROUND: While regulated WNT activity is required for normal development and stem cell maintenance, mutations that lead to constitutive activation of the WNT pathway cause cellular transformation and drive colorectal cancer. Activation of the WNT pathway ultimately leads to the nuclear translocation of ß-catenin which, in complex with TCF/LEF factors, promotes the transcription of genes necessary for growth. The proto-oncogene MYC is one of the most critical genes activated downstream the WNT pathway in colon cancer. Here, we investigate the converse regulation of the WNT pathway by MYC. METHODS: We performed RNA-seq analyses to identify genes regulated in cells expressing MYC. We validated the regulation of genes in the WNT pathway including LEF1 by MYC using RT-qPCR, Western blotting, and ChIP-seq. We investigated the importance of LEF1 for the viability of MYC-expressing cells in in fibroblasts, epithelial cells, and colon cells. Bioinformatic analyses were utilized to define the expression of MYC-regulated genes in human colon cancer and metabolomics analyses were used to identify pathways regulated by LEF1 in MYC expressing cells. RESULTS: MYC regulates the levels of numerous WNT-related genes, including the ß-catenin co-transcription factor LEF1. MYC activates the transcription of LEF1 and is required for LEF1 expression in colon cancer cells and in primary colonic cells transformed by APC loss of function, a common mutation in colon cancer patients. LEF1 caused the retention of ß-catenin in the nucleus, leading to the activation of the WNT pathway in MYC-expressing cells. Consequently, MYC-expressing cells were sensitive to LEF1 inhibition. Moreover, we describe two examples of genes induced in MYC-expressing cells that require LEF1 activity: the peroxisome proliferator activated receptor delta (PPARδ) and the Acyl CoA dehydrogenase 9 (ACAD9). CONCLUSIONS: We demonstrated that MYC is a transcriptional regulator of LEF1 in colonic cells. Our work proposes a novel pathway by which MYC regulates proliferation through activating LEF1 expression which in turn activates the WNT pathway.
Asunto(s)
Factor de Unión 1 al Potenciador Linfoide/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Activación Transcripcional , Vía de Señalización Wnt , Acil-CoA Deshidrogenasas/genética , Línea Celular , Proliferación Celular , Neoplasias del Colon/patología , Técnicas de Silenciamiento del Gen , Humanos , Factor de Unión 1 al Potenciador Linfoide/deficiencia , PPAR delta/genética , Proto-Oncogenes Mas , beta Catenina/metabolismoRESUMEN
Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.
Asunto(s)
Neoplasias del Colon/fisiopatología , Quinurenina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Triptófano/metabolismo , Sistema de Transporte de Aminoácidos ASC/genética , Antineoplásicos/farmacología , Arilformamidasa/genética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/farmacología , Quinurenina/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Antígenos de Histocompatibilidad Menor/genética , Oximas/farmacología , Proto-Oncogenes Mas , Sulfonamidas/farmacologíaRESUMEN
MYC enhances protein synthesis by regulating genes involved in ribosome biogenesis and protein translation. Here, we show that MYC-induced protein translation is mediated by the transcription factor aryl hydrocarbon receptor (AHR), which is induced by MYC in colonic cells. AHR promotes protein synthesis by activating the transcription of genes required for ribosome biogenesis and protein translation, including OGFOD1 and NOLC1. Using surface sensing of translation (SUnSET) to measure global protein translation, we found that silencing AHR or its targets diminishes protein synthesis. Therefore, targeting AHR or its downstream pathways could provide a novel approach to limit biomass production in MYC-driven tumors.
Asunto(s)
Nucléolo Celular/metabolismo , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Hidrocarburo de Aril/fisiología , Animales , Línea Celular , Nucléolo Celular/genética , Células Cultivadas , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Receptores de Hidrocarburo de Aril/biosíntesis , Receptores de Hidrocarburo de Aril/genética , Activación TranscripcionalRESUMEN
Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels by counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insights into endosomal trafficking, illuminate the cooperativity between an ubiquitin ligase and a deubiquitinating enzyme, and establish a role for USP7 in human neurodevelopmental disease.
Asunto(s)
Trastorno del Espectro Autista/enzimología , Endosomas/metabolismo , Discapacidad Intelectual/enzimología , Proteínas de Microfilamentos/metabolismo , Ubiquitina Tiolesterasa/fisiología , Adolescente , Trastorno del Espectro Autista/genética , Niño , Preescolar , Proteínas de Unión al ADN/metabolismo , Retroalimentación Fisiológica , Femenino , Células HCT116 , Haploinsuficiencia , Humanos , Hipotálamo/metabolismo , Discapacidad Intelectual/genética , Masculino , Neuronas/enzimología , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Proteolisis , Eliminación de Secuencia , Peptidasa Específica de Ubiquitina 7 , UbiquitinaciónRESUMEN
AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.
Asunto(s)
Adenosina Monofosfato/química , Proteínas Bacterianas/metabolismo , Química Clic/métodos , Reacción de Cicloadición , Procesamiento Proteico-Postraduccional/fisiología , Secuencia de Bases , Cobre/química , Interacciones Huésped-Patógeno , Humanos , Pasteurellaceae/metabolismo , Análisis por Matrices de Proteínas , Estructura Terciaria de Proteína , Vibriosis/patología , Vibrio parahaemolyticus/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
In this issue of Molecular Cell, Yuan et al. (2014) report that the Cul3-KLHL20 E3 ubiquitin ligase regulates protein anterograde transport from the trans-Golgi network (TGN) by facilitating localized actin assembly at the TGN through K33-linked ubiquitination of coronin 7.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , HumanosRESUMEN
Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Proteínas/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/genética , Endosomas/metabolismo , Técnicas de Silenciamiento del Gen , Aparato de Golgi/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas/genética , Interferencia de ARN , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.
Asunto(s)
Adenosina Monofosfato/inmunología , Anticuerpos/química , Treonina/inmunología , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Western Blotting , Clonación Molecular , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Células HeLa , Humanos , Sueros Inmunes , Procesamiento Proteico-Postraduccional , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Treonina/química , Treonina/metabolismo , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/inmunología , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/química , Proteína de Unión al GTP rac1/inmunología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.
Asunto(s)
Proteínas Bacterianas/fisiología , Sistema de Señalización de MAP Quinasas , Acetilación , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Yersinia pestis/fisiologíaRESUMEN
Recent studies on a bacterial virulence factor, YopJ of Yersinia, have led to the realization that the acetylation of serine and threonine residues could be an important form of post-translational modification in eukaryotes. Although the identification of the machinery used for the addition and removal of acetyl groups on serine or threonine residues is in its infancy, the enzymes thus-far studied provide early insight into the mechanism of this newly discovered post-translational modification, and hint at its potential importance. For example, acetylation can compete with phosphorylation targeted to the same residues and could, therefore, alter the course of signaling pathways. What are the implications for signal transduction in eukaryotes and how widespread could acetylation of serine and threonine prove to be?
Asunto(s)
Procesamiento Proteico-Postraduccional , Serina/metabolismo , Treonina/metabolismo , Acetilación , Proteínas Bacterianas/metabolismo , Modelos Biológicos , Fosforilación , Factores de Virulencia/metabolismo , Yersinia/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Enfermedad de Alzheimer/genética , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Embrión de Mamíferos , Fibroblastos , Homeostasis , Membrana Dobles de Lípidos , Ratones , Ratones Noqueados , Mutación , Presenilina-1/genética , Presenilina-2/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Membrana Celular/enzimología , Endopeptidasas , Células HeLa , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Receptores Notch , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The multipass membrane protein APH-1, found in the gamma-secretase complex together with presenilin, nicastrin, and PEN-2, is essential for Notch signaling in Caenorhabditis elegans embryos and is required for intramembrane proteolysis of Notch and beta-amyloid precursor protein in mammalian and Drosophila cells. In C. elegans, a mutation of the conserved transmembrane Gly123 in APH-1 (mutant or28) leads to a notch/glp-1 loss-of-function phenotype. In this study, we show that the corresponding mutation in mammalian APH-1aL (G122D) disrupts the physical interaction of APH-1aL with hypoglycosylated immature nicastrin and the presenilin holoprotein as well as with mature nicastrin, presenilin, and PEN-2. The G122D mutation also reduced gamma-secretase activity in intramembrane proteolysis of membrane-tethered Notch. Moreover, we found that the conserved transmembrane Gly122, Gly126, and Gly130 in the fourth transmembrane region of mammalian APH-1aL are part of the membrane helix-helix interaction GXXXG motif and are essential for the stable association of APH-1aL with presenilin, nicastrin, and PEN-2. These findings suggest that APH-1 plays a GXXXG-dependent scaffolding role in both the initial assembly and subsequent maturation and maintenance of the active gamma-secretase complex.