RESUMEN
Many intracellular proteins are modified by N-acetylglucosamine, a post-translational modification termed O-GlcNAc. This modification is found on serine and threonine side chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.
Asunto(s)
Acetilglucosamina/genética , Lisofosfolípidos/farmacología , Fosfatasa de Miosina de Cadena Ligera/genética , Esfingosina/análogos & derivados , Actinas/fisiología , Animales , Citoesqueleto/efectos de los fármacos , Fibroblastos , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Ratones , Contracción Muscular/efectos de los fármacos , Células 3T3 NIH , Fosforilación , Procesamiento Proteico-Postraduccional , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato/agonistas , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Receptores de Esfingosina-1-Fosfato/efectos de los fármacosRESUMEN
The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.
Asunto(s)
Acetilglucosamina/metabolismo , Hexosaminas/biosíntesis , N-Acetilglucosaminiltransferasas/metabolismo , Estrés Fisiológico , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Técnicas de Inactivación de Genes , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/deficiencia , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glicosilación , RatonesRESUMEN
Glycans form one of the four classes of biomolecules, are found in every living system and present a huge structural and functional diversity. As an illustration of this diversity, it has been reported that more than 50% of the human proteome is glycosylated and that 2% of the human genome is dedicated to glycosylation processes. Glycans are involved in many biological processes such as signalization, cell-cell or host pathogen interactions, immunity, etc. However, fundamental processes associated with glycans are not yet fully understood and the development of glycobiology is relatively recent compared to the study of genes or proteins. Approximately 25 years ago, the studies of Bertozzi's and Reutter's groups paved the way for metabolic oligosaccharide engineering (MOE), a strategy which consists in the use of modified sugar analogs which are taken up into the cells, metabolized, incorporated into glycoconjugates, and finally detected in a specific manner. This groundbreaking strategy has been widely used during the last few decades and the concomitant development of new bioorthogonal ligation reactions has allowed many advances in the field. Typically, MOE has been used to either visualize glycans or identify different classes of glycoproteins. The present review aims to highlight recent studies that lie somewhat outside of these more traditional approaches and that are pushing the boundaries of MOE applications.
RESUMEN
Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two-step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Interestingly, the rate-determining enzyme of the HBP, glutamine fructose-6-phosphate amidotransferase (GFAT), is feedback inhibited by UDP-GlcNAc. This raises the possibility that a build-up of UDP-MCRs would block the biosynthesis of UDP-GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP-MCRs. We find that MCRs with larger substitutions at the N-acetyl position do not inhibit GFAT, whereas those with modifications of the 2- or 6-hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.
Asunto(s)
Alquinos/metabolismo , Azidas/metabolismo , Vías Biosintéticas , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hexosaminas/metabolismo , Uridina Difosfato/metabolismo , Alquinos/química , Azidas/química , Química Clic , Glicosilación , Humanos , Proteínas Recombinantes/metabolismo , Uridina Difosfato/químicaRESUMEN
O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.
Asunto(s)
Acetilglucosamina/metabolismo , Apoptosis , Caspasas/metabolismo , Acetilglucosamina/química , Animales , Caspasas/química , Glicosilación , Humanos , Cinética , Células MCF-7 , Ratones , Células 3T3 NIHRESUMEN
Since the pioneering work by Reutter and co-workers that demonstrated structural flexibility in the carbohydrate biosynthesis and glycosylation pathways, many different labs have used unnatural monosaccharide analogues to perform glycan engineering on the surface of living cells. A subset of these unnatural monosaccharides contain bioorthogonal groups that enable the selective installation of visualization or enrichment tags. These metabolic chemical reporters (MCRs) have proven to be powerful for the unbiased identification of glycoproteins; however, they do have certain limitations. For example, they are incorporated substoichiometrically into glycans, and most MCRs are not selective for one class (e.g., O-GlcNAcylation) of glycoprotein. Here, we explore the relationship between the biosynthesis of MCR donor sugars in cells and the labeling levels of four different N-acetylglucosamine- and N-acetylgalactosamine-based MCRs. We found that the buildup of the different donor sugars correlated well with the overall labeling levels but less so with intracellular labeling of proteins by O-GlcNAcylation.
Asunto(s)
Acetilglucosamina/metabolismo , Materiales Biomiméticos/metabolismo , Glicoproteínas/metabolismo , Monosacáridos/metabolismo , Polisacáridos/metabolismo , Coloración y Etiquetado/métodos , Acetilglucosamina/química , Animales , Materiales Biomiméticos/química , Conformación de Carbohidratos , Glicoproteínas/química , Glicosilación , Células HEK293 , Células HeLa , Humanos , Ingeniería Metabólica/métodos , Ratones , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Monosacáridos/química , Células 3T3 NIH , Especificidad de Órganos , Polisacáridos/químicaRESUMEN
Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.
Asunto(s)
Acetilglucosamina/metabolismo , Desoxiglucosa/análogos & derivados , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Cromatografía Liquida , Desoxiglucosa/metabolismo , Ratones , Células 3T3 NIH , Espectrometría de Masas en TándemRESUMEN
A large array of posttranslational modifications can dramatically change the properties of proteins and influence different aspects of their biological function such as enzymatic activity, binding interactions, and proteostasis. Despite the significant knowledge that has been gained about the function of posttranslational modifications using traditional biological techniques, the analysis of the site-specific effects of a particular modification, the identification of the full complement of modified proteins in the proteome, and the detection of new types of modifications remains challenging. Over the years, chemical methods have contributed significantly in both of these areas of research. This review highlights several posttranslational modifications where chemistry-based approaches have made significant contributions to our ability to both prepare homogeneously modified proteins and identify and characterize particular modifications in complex biological settings. As the number and chemical diversity of documented posttranslational modifications continues to rise, we believe that chemical strategies will be essential to advance the field in years to come.
Asunto(s)
Bioquímica/métodos , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Acetilación , Animales , Técnicas de Química Analítica/métodos , Técnicas de Química Sintética/métodos , Glicosilación , Humanos , Lípidos/análisis , Metilación , Fosforilación , Proteínas/química , Proteómica/métodosRESUMEN
The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.