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Methods in Enzymology 2022Post-translational modifications (PTMs) provide a critical means of calibrating the functional proteome and, thus, are extensively utilized by the eukaryotes to exert...
Post-translational modifications (PTMs) provide a critical means of calibrating the functional proteome and, thus, are extensively utilized by the eukaryotes to exert spatio-temporal regulation on the cellular machinery rapidly. Ubiquitination and phosphorylation are examples of the well-documented PTMs. SUMOylation, the reversible conjugation of the Small Ubiquitin-related MOdifier (SUMO) at a specific lysine residue on a target protein, bears striking similarity with ubiquitination and follows an enzymatic cascade for the attachment of SUMO to the target protein. Unlike Ubiquitination, SUMOylation can modulate the target protein's structure, stability, activity, localization, and interaction. Thus, SUMOylation regulates cellular events such as signal transduction, cell-cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Accordingly, deregulation of SUMOylation is an avenue for diseases, which makes the investigation of SUMO and its substrates within the cell essential. However, the low extent of SUMOylation has posed a significant challenge in detecting SUMO modification within the cell. Bioinformatics tools can help predict SUMOylation, and mass-spectrometric analysis can identify a pool of cellular protein SUMOylome. Nevertheless, the biochemical methods for observing the enhanced level of in vitro SUMOylation help validate protein SUMOylation, critical lysine(s) utilized in the process, and its effect on substrate protein function. This chapter provides a detailed account of biochemical methods commonly utilized to detect SUMOylated proteins that are central for understanding the biological functions and mechanism of regulation of SUMO targets.
Topics: Lysine; Proteome; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Ubiquitin; Ubiquitination
PubMed: 36220279
DOI: 10.1016/bs.mie.2022.07.017 -
The Journal of Clinical Investigation Nov 2021Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth....
Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.
Topics: AMP-Activated Protein Kinases; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Breast Neoplasms; Cell Line, Tumor; Female; Humans; Neovascularization, Pathologic; Oxidative Phosphorylation; Proto-Oncogene Proteins; Tumor Suppressor Proteins; Warburg Effect, Oncologic
PubMed: 34779410
DOI: 10.1172/JCI144871 -
The Science of the Total Environment Dec 2023Methylmercury (MeHg) readily accumulates in aquatic organisms while transferring and amplifying in the aquatic food chains. This study firstly explores the in vivo...
Methylmercury (MeHg) readily accumulates in aquatic organisms while transferring and amplifying in the aquatic food chains. This study firstly explores the in vivo accumulation sites and metabolic regulation of MeHg in the rotifer Brachionus plicatilis by aggregation-induced emission fluorogen (AIEgen) and metabolomics. Fluorescent image analysis by AIEgen showed that MeHg in B. plicatilis mainly occured in the ciliary corona, esophagus, mastax, stomach and intestine in the direct absorption group. In the other group, where B. plicatilis were indirectly supplied with MeHg via food intake, the accumulation of MeHg in the rotifer occurred in the ciliary corona, various digestive organs, and the pedal gland. However, the MeHg accumulated in the rotifer is difficult to metabolize outside the body. Metabolomics analysis showed that the significant enrichment of ABC transporters was induced by the direct exposure of rotifers to dissolved MeHg. In contrast, exposure of rotifers to MeHg via food intake appeared to influence carbon, galactose, alanine, aspartate and glutamate metabolisms. Besides, the disturbed biological pathways such as histidine metabolism, beta-alanine metabolism and pantothenate and CoA biosynthesis in rotifers may be associated with L-aspartic acid upregulation in the feeding group. The significant enrichment of ABC transporters and carbon metabolism in rotifers may be related to the accumulation of MeHg in the intestine of rotifers. In both pathways of MeHg exposure, the arginine biosynthesis and metabolism of rotifers were disturbed, which may support the hypothesis that rotifers produce more energy to resist MeHg toxicity. This study provides new insight into the accumulation and toxicity mechanisms of MeHg on marine invertebrates from the macro and micro perspectives.
Topics: Animals; Methylmercury Compounds; Rotifera; Metabolic Networks and Pathways; ATP-Binding Cassette Transporters; Carbon
PubMed: 37709075
DOI: 10.1016/j.scitotenv.2023.167063 -
Nature Communications Feb 2022When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource...
When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.
Topics: Fermentation; Gene Expression Regulation, Fungal; Glucose; Metabolic Networks and Pathways; Mitochondria; Proteome; Proteomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Yeasts
PubMed: 35145105
DOI: 10.1038/s41467-022-28467-6 -
Experimental & Molecular Medicine May 2020Sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP) is a key regulator of SREBP maturation. SCAP induces translocation of SREBP from the... (Review)
Review
Sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP) is a key regulator of SREBP maturation. SCAP induces translocation of SREBP from the endoplasmic reticulum to the Golgi apparatus, allowing it to regulate cellular triglyceride and cholesterol levels. Previous studies have shown that suppression of SREBP activation in SCAP conditional knockout mice reduced the accumulation of intracellular triglycerides, which eventually causes the development of metabolic diseases such as atherosclerosis, diabetes, hepatic steatosis, and insulin resistance. However, despite the significance of SCAP as a regulator of SREBP, its function has not been thoroughly discussed. In this review, we have summarized the function of SCAP and its regulatory proteins. Furthermore, we discuss recent studies regarding SCAP as a possible therapeutic target for hypertriglyceridemia and hyperlipidemia.
Topics: Animals; Disease Susceptibility; Energy Metabolism; Humans; Intracellular Signaling Peptides and Proteins; Intracellular Space; Lipid Metabolism; Membrane Proteins; Metabolic Syndrome; Multiprotein Complexes; Protein Binding; Signal Transduction
PubMed: 32385422
DOI: 10.1038/s12276-020-0430-0 -
Comptes Rendus Biologies Apr 2023Detection of cytosolic pathological nucleic acids is a key step for the initiation of innate immune responses. In the past decade, the stimulator of interferon genes... (Review)
Review
Detection of cytosolic pathological nucleic acids is a key step for the initiation of innate immune responses. In the past decade, the stimulator of interferon genes (STING) adaptor protein has emerged as a central platform enabling the activation of inflammatory responses in the presence of cytosolic DNAs. This has prompted a plethora of approaches aiming at modulating STING activation in order to boost or inhibit inflammatory responses. However, recent work has revealed that STING is also a direct regulator of metabolic homeostasis. In particular, STING regulates lipid metabolism directly, a function that is conserved throughout evolution. This indicates that STING targeting strategies must take into consideration potential metabolic side effects that may alter disease course, but also suggests that targeting STING may open the route to novel treatments for metabolic disorders. Here we discuss recent work describing the metabolic function of STING and the implications of these findings.
Topics: Lipid Metabolism; Membrane Proteins; Immunity, Innate; DNA
PubMed: 37254782
DOI: 10.5802/crbiol.110 -
ACS Chemical Biology Jan 2023The proteolysis targeting chimera (PROTAC) strategy results in the down-regulation of unwanted protein(s) for disease treatment. In the PROTAC process, a...
The proteolysis targeting chimera (PROTAC) strategy results in the down-regulation of unwanted protein(s) for disease treatment. In the PROTAC process, a heterobifunctional degrader forms a ternary complex with a target protein of interest (POI) and an E3 ligase, which results in ubiquitination and proteasomal degradation of the POI. While ternary complex formation is a key attribute of PROTAC degraders, modification of the PROTAC molecule to optimize ternary complex formation and protein degradation can be a labor-intensive and tedious process. In this study, we take advantage of DNA-encoded library (DEL) technology to efficiently synthesize a vast number of possible PROTAC molecules and describe a parallel screening approach that utilizes DNA barcodes as reporters of ternary complex formation and cooperative binding. We use a designed PROTAC DEL against BRD4 and CRBN to describe a dual protein affinity selection method and the direct discovery of novel, potent BRD4 PROTACs that importantly demonstrate clear SAR. Such an approach evaluates all the potential PROTACs simultaneously, avoids the interference of PROTAC solubility and permeability, and uses POI and E3 ligase proteins in an efficient manner.
Topics: Nuclear Proteins; Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitination; Proteolysis
PubMed: 36606710
DOI: 10.1021/acschembio.2c00797 -
Proteins Nov 2019The growing body of experimental and computational data describing how proteins interact with each other has emphasized the multiplicity of protein interactions and the...
The growing body of experimental and computational data describing how proteins interact with each other has emphasized the multiplicity of protein interactions and the complexity underlying protein surface usage and deformability. In this work, we propose new concepts and methods toward deciphering such complexity. We introduce the notion of interacting region to account for the multiple usage of a protein's surface residues by several partners and for the variability of protein interfaces coming from molecular flexibility. We predict interacting patches by crossing evolutionary, physicochemical and geometrical properties of the protein surface with information coming from complete cross-docking (CC-D) simulations. We show that our predictions match well interacting regions and that the different sources of information are complementary. We further propose an indicator of whether a protein has a few or many partners. Our prediction strategies are implemented in the dynJET algorithm and assessed on a new dataset of 262 protein on which we performed CC-D. The code and the data are available at: http://www.lcqb.upmc.fr/dynJET2/.
Topics: Algorithms; Animals; Binding Sites; Humans; Molecular Docking Simulation; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Interaction Maps; Proteins; Software
PubMed: 31199528
DOI: 10.1002/prot.25757 -
Proteomics Mar 2020Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance... (Review)
Review
Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance is a major contributor to the age-associated accumulation of damaged proteins that is believed to participate to the age-related decline in cellular function. In this context, quantitative proteomics approaches, including 2-D gel electrophoresis (2-DE)-based methods, represent powerful tools for monitoring the extent of protein oxidative modifications at the proteome level and for identifying the targeted proteins, also referred as to the "oxi-proteome." Previous studies have identified proteins targeted by oxidative modifications during replicative senescence of human WI-38 fibroblasts and myoblasts and have been shown to represent a restricted set within the total cellular proteome that fall in key functional categories, such as energy metabolism, protein quality control, and cellular morphology. To provide mechanistic support into the role of oxidized proteins in the development of the senescent phenotype, untargeted metabolomic profiling is also performed for young and senescent myoblasts and fibroblasts. Metabolomic profiling is indicative of energy metabolism impairment in both senescent myoblasts and fibroblasts, suggesting a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts and fibroblasts.
Topics: Aging; Animals; Cellular Senescence; Energy Metabolism; Fibroblasts; Humans; Metabolic Networks and Pathways; Myoblasts; Oxidation-Reduction; Oxidative Stress; Protein Processing, Post-Translational; Proteome; Proteomics; Proteostasis
PubMed: 31507063
DOI: 10.1002/pmic.201800421 -
Mitochondrion Jul 2020The biological function of plant mitochondrial uncoupling proteins (pUCPs) has been a matter of considerable controversy. For example, the pUCP capacity to uncouple... (Review)
Review
The biological function of plant mitochondrial uncoupling proteins (pUCPs) has been a matter of considerable controversy. For example, the pUCP capacity to uncouple respiration from ATP synthesis in vivo has never been fully acknowledged, in contrast to the mammalian UCP1 (mUCP1) role in uncoupling respiration-mediated thermogenesis. Interestingly, both pUCPs and mUCPs have been associated with stress response and metabolic perturbations. Some central questions that remain are how pUCPs and mUCPs compare in biochemical properties, molecular structure and cell biology under physiological and metabolically perturbed conditions. This review takes advantage of the large amount of data available for mUCPs to review the biochemical properties, 3D structure models and potential physiological roles of pUCPs during plant development and response to stress. The biochemical properties and structure of pUCPs are revisited in light of the recent findings that pUCPs catalyse the transport of metabolites across the mitochondrial inner membrane and the resolved mUCP2 protein structure. Additionally, transcriptional regulation and co-expression networks of UCP orthologues across species are analysed, taking advantage of publicly available curated experimental datasets. Taking these together, the biological roles of pUCPs are analysed in the context of their potential roles in thermogenesis, ROS production, cell signalling and the regulation of plant cellular bioenergetics. Finally, pUCPs biological function is discussed in the context of their potential role in protecting against environmental stresses.
Topics: Energy Metabolism; Gene Expression Regulation, Plant; Mitochondrial Uncoupling Proteins; Models, Molecular; Plant Development; Plant Proteins; Plants; Protein Conformation; Stress, Physiological
PubMed: 32439620
DOI: 10.1016/j.mito.2020.05.001