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1.
EMBO J ; 42(24): e114072, 2023 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-37984437

RESUMEN

Piwi-interacting RNAs (piRNAs) direct PIWI proteins to transposons to silence them, thereby preserving genome integrity and fertility. The piRNA population can be expanded in the ping-pong amplification loop. Within this process, piRNA-associated PIWI proteins (piRISC) enter a membraneless organelle called nuage to cleave their target RNA, which is stimulated by Gtsf proteins. The resulting cleavage product gets loaded into an empty PIWI protein to form a new piRISC complex. However, for piRNA amplification to occur, the new RNA substrates, Gtsf-piRISC, and empty PIWI proteins have to be in physical proximity. In this study, we show that in silkworm cells, the Gtsf1 homolog BmGtsf1L binds to piRNA-loaded BmAgo3 and localizes to granules positive for BmAgo3 and BmVreteno. Biochemical assays further revealed that conserved residues within the unstructured tail of BmGtsf1L directly interact with BmVreteno. Using a combination of AlphaFold modeling, atomistic molecular dynamics simulations, and in vitro assays, we identified a novel binding interface on the BmVreteno-eTudor domain, which is required for BmGtsf1L binding. Our study reveals that a single eTudor domain within BmVreteno provides two binding interfaces and thereby interconnects piRNA-loaded BmAgo3 and BmGtsf1L.


Asunto(s)
Bombyx , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Bombyx/genética , Bombyx/metabolismo , ARN de Interacción con Piwi , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Dominio Tudor
2.
Elife ; 112022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35229715

RESUMEN

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.


Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Hexosaminas , Animales , Vías Biosintéticas , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glicosilación , Hexosaminas/metabolismo , Ratones
3.
Nat Commun ; 12(1): 2176, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846315

RESUMEN

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Retroalimentación Fisiológica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hexosaminas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Estrés del Retículo Endoplásmico , Mutación con Ganancia de Función , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Cinética , Fosforilación , Unión Proteica , Dominios Proteicos , Serina/genética , Uridina Difosfato N-Acetilglucosamina/metabolismo
4.
Nat Commun ; 11(1): 687, 2020 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-32019926

RESUMEN

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.


Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hexosaminas/metabolismo , Retroalimentación Fisiológica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glicosilación , Hexosaminas/química , Humanos , Ligandos , Conformación Proteica , Proteostasis , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismo
5.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 5): 307-314, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29717999

RESUMEN

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods. However, this method can fail owing to the properties of the crystallized protein or the chemical composition of the crystallization trials. Here, a simple, fast and cost-efficient method employing 2,2,2-trichloroethanol (TCE) has been developed. It can be performed with a standard UV-light microscope and can be applied to cases in which detection of native tryptophan fluorescence fails. In four test cases this method had no effect on the diffraction properties of the crystals and no structural changes were observed. Further evidence is provided that TCE can be added to crystallization trials during their preparation, making this method compatible with high-throughput approaches.


Asunto(s)
Etilenclorhidrina/análogos & derivados , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Etilenclorhidrina/metabolismo , Microscopía Fluorescente/métodos , Estructura Secundaria de Proteína
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