RESUMEN
Understanding the regulation of human embryonic stem cells (hESCs) pluripotency is critical to advance the field of developmental biology and regenerative medicine. Despite the recent progress, molecular events regulating hESC pluripotency, especially the transition between naive and primed states, still remain unclear. Here we show that naive hESCs display lower levels of O-linked N-acetylglucosamine (O-GlcNAcylation) than primed hESCs. O-GlcNAcase (OGA), the key enzyme catalyzing the removal of O-GlcNAc from proteins, is highly expressed in naive hESCs and is important for naive pluripotency. Depletion of OGA accelerates naive-to-primed pluripotency transition. OGA is transcriptionally regulated by EP300 and acts as a transcription regulator of genes important for maintaining naive pluripotency. Moreover, we profile protein O-GlcNAcylation of the two pluripotency states by quantitative proteomics. Together, this study identifies OGA as an important factor of naive pluripotency in hESCs and suggests that O-GlcNAcylation has a broad effect on hESCs homeostasis.
Asunto(s)
Células Madre Embrionarias Humanas , Humanos , Acetilglucosamina/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo , Diferenciación Celular , Línea Celular , Glicosilación , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Glycans are complex biomolecules that encode rich information and regulate various biological processes, such as fertilization, host-pathogen binding, and immune recognition, through interactions with glycan-binding proteins. A key driving force for glycan-protein recognition is the interaction between the π electron density of aromatic amino acid side chains and polarized CâH groups of the pyranose (termed the CH-π interaction). However, the relatively weak binding affinity between glycans and proteins has hindered the application of glycan detection and imaging. Here, computational modeling and molecular dynamics simulations are employed to design a chemical strategy that enhances the CH-π interaction between glycans and proteins by genetically incorporating electron-rich tryptophan derivatives into a lectin PhoSL, which specifically recognizes core fucosylated N-linked glycans. This significantly enhances the binding affinity of PhoSL with the core fucose ligand and enables sensitive detection and imaging of core fucosylated glycans in vitro and in xenograft tumors in mice. Further, the study showed that this strategy is applicable to improve the binding affinity of GafD lectin for N-acetylglucosamine-containing glycans. The approach thus provides a general and effective way to manipulate glycan-protein recognition for glycoscience applications.
Asunto(s)
Polisacáridos , Polisacáridos/metabolismo , Polisacáridos/química , Animales , Ratones , Simulación de Dinámica Molecular , Lectinas/metabolismo , Lectinas/química , Unión Proteica , Humanos , Modelos Animales de EnfermedadRESUMEN
Although several covalent KRASG12C inhibitors have made great progress in the treatment of KRASG12C-mutant cancer, their clinical applications are limited by adaptive resistance, motivating novel therapeutic strategies. Through drug design and structure optimization, a series of highly potent and selective KRASG12C Proteolysis Targeting Chimeras (PROTACs) were developed by incorporating AMG510 and VHL ligand VH032. Among them, degrader YN14 significantly inhibited KRASG12C-dependent cancer cells growth with nanomolar IC50 and DC50 values, and ï¼ 95 % maximum degradation (Dmax). Molecular dynamics (MD) simulation showed that YN14 induced a stable KRASG12C: YN14: VHL ternary complex with low binding free energy (ΔG). Notably, YN14 led to tumor regression with tumor growth inhibition (TGI%) rates more than 100 % in the MIA PaCa-2 xenograft model with well-tolerated dose-schedules. We also found that KRASG12C degradation exhibited advantages in overcoming adaptive KRASG12C feedback resistance over KRASG12C inhibition. Furthermore, combination of RTKs, SHP2, or CDK9 inhibitors with YN14 exhibited synergetic efficacy in KRASG12C-mutant cancer cells. Overall, these results demonstrated that YN14 holds exciting prospects for the treatment of tumors with KRASG12C-mutation and boosted efficacy could be achieved for greater clinical applications via drug combination.
Asunto(s)
Neoplasias , Quimera Dirigida a la Proteólisis , Humanos , Proteínas Proto-Oncogénicas p21(ras) , Mutación , Citoplasma , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.
RESUMEN
Oncogenic Kras-activated pancreatic ductal adenocarcinoma (PDAC) cells highly rely on an unconventional glutamine catabolic pathway to sustain cell growth. However, little is known about how this pathway is regulated. Here we demonstrate that Kras mutation induces cellular O-linked ß-N-acetylglucosamine (O-GlcNAc), a prevalent form of protein glycosylation. Malate dehydrogenase 1 (MDH1), a key enzyme in the glutamine catabolic pathway, is positively regulated by O-GlcNAcylation on serine 189 (S189). Molecular dynamics simulations suggest that S189 glycosylation on monomeric MDH1 enhances the stability of the substrate-binding pocket and strengthens the substrate interactions by serving as a molecular glue. Depletion of O-GlcNAcylation reduces MDH1 activity, impairs glutamine metabolism, sensitizes PDAC cells to oxidative stress, decreases cell proliferation and inhibits tumor growth in nude mice. Furthermore, O-GlcNAcylation levels of MDH1 are elevated in clinical PDAC samples. Our study reveals that O-GlcNAcylation contributes to pancreatic cancer growth by regulating the metabolic activity of MDH1.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Acetilglucosamina/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Glutamina/metabolismo , Malato Deshidrogenasa/metabolismo , Ratones , Ratones Desnudos , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Serina/metabolismo , Neoplasias PancreáticasRESUMEN
SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.
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Endosomas/metabolismo , Lisosomas/metabolismo , Transducción de Señal , Acilación/genética , Endosomas/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Hep G2 , Humanos , Lisosomas/genética , Transporte de Proteínas/genéticaRESUMEN
O-linked N-acetylglucosamine (O-GlcNAc) is a prevalent protein modification that plays fundamental roles in both cell physiology and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase (OGT). The study of protein O-GlcNAc function is limited by the lack of tools to control OGT activity with spatiotemporal resolution in cells. Here, we report light control of OGT activity in cells by replacing a catalytically essential lysine residue with a genetically encoded photocaged lysine. This enables the expression of a transiently inactivated form of OGT, which can be rapidly reactivated by photo-decaging. We demonstrate the activation of OGT activity by monitoring the time-dependent increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based quantitative proteomics. We further apply this activation strategy to control the morphological contraction of fibroblasts. Furthermore, we achieved spatial activation of OGT activity predominantly in the cytosol. Thus, our approach provides a valuable chemical tool to control cellular O-GlcNAc with much needed spatiotemporal precision, which aids in a better understanding of O-GlcNAc function.
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Lisina , N-Acetilglucosaminiltransferasas , Acetilglucosamina/metabolismo , Glicoproteínas/metabolismo , Lisina/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
Ribonucleic acid (RNA) rarely exists alone in the cell. RNAs interact with a variety of proteins and form RNA-protein complexes (RP-complexes) in every step of their life cycle, from transcription to degradation. These RP-complexes play key roles in regulating a variety of physiological processes. Defects in the composition and function of RP-complexes have been associated with many diseases, including metabolic disorders, muscular atrophy, autoimmune diseases, and cancer. It is hence evident that deciphering the highly complex interaction network of RNA-binding proteins (RBPs) and their RNA targets will provide a better understanding of disease development and lead to the discovery of new targets for cancer therapy. Large-scale identification of RP-complexes at the omics level is a prerequisite for obtaining insights into the complex RNA-protein interaction network. As the first step in omics-wide decoding of RP-complexes, enrichment and purification of RP-complexes is a highly challenging task. Recently, intensive efforts have been undertaken to better enrich and identify RP-complexes. Generally, the enrichment strategies can be classified into two major categories: in vitro and in vivo. Although it has been successfully applied in many studies, the in vitro transcribed bait RNA lacks modifications or structural similarity compared with its natural counterpart. Further, since the proteins relocate and remodel after cell lysis, the use of cell lysates as a protein source may result in capturing false interacting proteins that bind non-physiologically with the bait RNA. Finally, weak interactions between the non-covalently bound proteins and RNA require mild washing to remove non-specific binding, which needs careful optimization. However, substantial sample loss is inevitable. To overcome the disadvantages of in vitro approaches, in vivo cross-linking strategies that "freeze" natural RNA-protein complexes in intact cells via covalent cross-linking have become increasingly popular. The in vivo methods allow RNA to interact with proteins in the intracellular environment. Therefore, the RP-complexes formed under physiological conditions are more biologically relevant than those obtained by in vitro methods. We herein summarize recent in vivo methodological advances in the large-scale enrichment and identification of RP-complexes, including cross-linking and immunoprecipitation (CLIP) and related methods, click chemistry-assisted methods, and organic phase separations. CLIP involves irradiating living cells with 254-nm ultraviolet (UV) light to establish covalent bonds between RNA and proteins. This enables CLIP to purify RNAs bound to a specific RBP under conditions that are stringent enough to prevent co-purification of nonspecifically bound proteins or free RNAs. Since the original study, multiple variant protocols have been derived to increase both efficiency and convenience. Photoactivatable ribonucleoside-enhanced-CLIP (PAR-CLIP) introduces a variation in the crosslinking strategy. Cells were preincubated with photoactivatable ribonucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which enables protein-RNA crosslinking with 365-nm UV-A irradiation. It increases the efficiency of cross-linking between RNA and RBPs and is particularly valuable for studying the interactions between RBPs and nascent RNA. Using a click chemistry-assisted strategy, an alkyne modified uridine analog, 5-ethynyluridine (EU), was incorporated into nascent RNAs via metabolic incorporation in living cells. Combined with UV irradiation-based cross-linking, the alkyne-functionalized RNA and the bound proteins were purified in a poly A-independent fashion by the highly selective bioorthogonal copper (I)-catalyzed azide-alkyne cycloaddition using azide-modified beads. Thus, full lists of both coding and non-coding RNAs with their interacting proteins can be purified, which is a major methodological advance. Organic phase separation methods exploiting the physicochemical difference between cross-linked RP-complexes and free RNA and proteins do not require metabolic-based alkyne labeling or polyA-based RNA capture. Each method has unique strengths and drawbacks, which makes it important to select optimal approaches for the biological question being addressed. We hope that this review points out the current limitations and provides future directions to facilitate further development of methods for large-scale investigation of RP-complexes.
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Proteínas de Unión al ARN , ARN , Sitios de Unión , Química Clic , Inmunoprecipitación , ARN/análisis , Proteínas de Unión al ARN/análisis , Rayos UltravioletaRESUMEN
N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples.
RESUMEN
RNA-protein interactions play important roles in almost every step of the lifetime of RNAs, such as RNA splicing, transporting, localization, translation and degradation. Post-translational modifications, such as O-GlcNAcylation and phosphorylation, and their "cross-talk" (OPCT) are essential to the activity and function regulation of RNA-binding proteins (RBPs). However, due to the extremely low abundance of O-GlcNAcylation and the lack of RBP-targeted enrichment strategies, large-scale simultaneous profiling of O-GlcNAcylation and phosphorylation on RBPs is still a challenging task. In the present study, we developed a tandem enrichment strategy combining metabolic labeling-based RNA tagging for selective purification of RBPs and HILIC-based enrichment for simultaneous O-GlcNAcylation and phosphorylation profiling. Benefiting from the sequence-independent RNA tagging by ethynyluridine (EU) labeling, 1115 RBPs binding to different types of RNAs were successfully enriched and identified by quantitative mass spectrometry (MS) analysis. Further HILIC enrichment on the tryptic-digested RBPs and MS analysis led to the first large-scale identification of O-GlcNAcylation and phosphorylation in the RNA-binding proteome, with 461 O-GlcNAc peptides corresponding to 300 RBPs and 671 phosphopeptides corresponding to 389 RBPs. Interestingly, â¼25% RBPs modified by two PTMs were found to be related to multiple metabolism pathways. This strategy has the advantage of high compatibility with MS and provides peptide-level evidence for the identification of O-GlcNAcylated RBPs. We expect it will support simultaneous mapping of O-GlcNAcylation and phosphorylation on RBPs and facilitate further elucidation of the crucial roles of OPCT in the function regulation of RBPs.
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Acetilglucosamina , Proteoma , Glicosilación , Fosforilación , Procesamiento Proteico-Postraduccional , ARNRESUMEN
Small extracellular vesicles (SEVs), are cell-derived, membrane-enclosed nanometer-sized vesicles that play vital roles in many biological processes. Recent years, more and more evidences proved that small EVs have close relationship with many diseases such as cancers and Alzheimer's disease. The use of phosphoproteins in SEVs as potential biomarkers is a promising new choice for early diagnosis and prognosis of cancer. However, current techniques for SEVs isolation still facing many challenges, such as highly instrument dependent, time consuming and insufficient purity. Furthermore, complex enrichment procedures and low microgram amounts of proteins available from clinical sources largely limit the throughput and the coveage depth of SEVs phosphoproteome mapping. Here, we synthesized Ti4+-modified magnetic graphene-oxide composites (GFST) and developed a "one-material" strategy for facile and efficient phosphoproteome enrichment and identification in SEVs from human serum. By taking advantage of chelation and electrostatic interactions between metal ions and phosphate groups, GFST shows excellent performance in both SEVs isolation and phosphopeptide enrichment. Close to 85% recovery is achieved within a few minutes by simple incubation with GFST and magnetic separation. Proteome profiling of the isolated serum SEVs without phosphopeptide enrichment results in 515 proteins, which is approximately one-fold more than those otained by ultracentrifugation or coprecipitation kits. Further application of GFST in one-material-based enrichment led to identification of 859 phosphosites in 530 phosphoproteins. Kinase-substrate correlation analysis reveals enriched substrates of CAMK in serum SEVs phosphoproteome. Therefore, we expect that the low instrument dependency and the limited sample requirement of this new strategy may facilitate clinical investigations in SEV-based transportation of abnormal kinases and substrates for drug target discovery and cancer monitoring.
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Vesículas Extracelulares , Proteoma , Biomarcadores , Cromatografía de Afinidad , Humanos , FosfoproteínasRESUMEN
Due to its key roles in regulating the occurrence and development of cancer, protein histidine phosphorylation has been increasingly recognized as an important form of post-translational modification in recent years. However, large-scale analysis of histidine phosphorylation is much more challenging than that of serine/threonine or tyrosine phosphorylation, mainly because of its acid lability. In this study, MoS2-Ti4+ nanomaterials were synthesized using a solvothermal method and taking advantage of the electrostatic adsorption between MoS2 nanosheets and Ti4+. The MoS2-Ti4+ nanomaterials have the advantage of the combined affinity of Ti4+ and Mo toward phosphorylation under medium acidic conditions (pH = 3), which is crucial for preventing hydrolysis and loss of histidine phosphorylation during enrichment. The feasibility of using the MoS2-Ti4+ nanomaterial for phosphopeptide enrichment was demonstrated using mixtures of ß-casein and bovine serum albumin (BSA). Further evaluation revealed that the MoS2-Ti4+ nanomaterial is capable of enriching synthetic histidine phosphopeptides from 1000 times excess tryptic-digested HeLa cell lysate. Application of the MoS2-Ti4+ nanomaterials for large-scale phosphopeptide enrichment results in the identification of 10â¯345 serine, threonine, and tyrosine phosphosites and the successful mapping of 159 histidine phosphosites in HeLa cell lysates, therefore indicating great potential for deciphering the vital biological roles of protein (histidine) phosphorylation.