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Bioorganic Chemistry Jun 2024Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine...
Ras GTPases and other CaaX proteins undergo multiple post-translational modifications at their carboxyl-terminus. These events initiate with prenylation of a cysteine and are followed by endoproteolytic removal of the 'aaX' tripeptide and carboxylmethylation. Some CaaX proteins are only subject to prenylation, however, due to the presence of an uncleavable sequence. In this study, uncleavable sequences were used to stage Ras isoforms in a farnesylated and uncleaved state to address the impact of CaaX proteolysis on protein localization and function. This targeted strategy is more specific than those that chemically inhibit the Rce1 CaaX protease or delete the RCE1 gene because global abrogation of CaaX proteolysis impacts the entire CaaX protein proteome and effects cannot be attributed to any specific CaaX protein of the many concurrently affected. With this targeted strategy, clear mislocalization and reduced activity of farnesylated and uncleaved Ras isoforms was observed. In addition, new peptidomimetics based on cleavable Ras CaaX sequences and the uncleavable CAHQ sequence were synthesized and tested as Rce1 inhibitors using in vitro and cell-based assays. Consistently, these non-hydrolyzable peptidomimetic Rce1 inhibitors recapitulate Ras mislocalization effects when modeled on cleavable but not uncleavable CaaX sequences. These findings indicate that a prenylated and uncleavable CaaX sequence, which can be easily applied to a wide range of mammalian CaaX proteins, can be used to probe the specific impact of CaaX proteolysis on CaaX protein properties under conditions of an otherwise normally processed CaaX protein proteome.
Topics: Humans; ras Proteins; Small Molecule Libraries; Proteolysis; Molecular Structure; Peptidomimetics; Endopeptidases
PubMed: 38583246
DOI: 10.1016/j.bioorg.2024.107316 -
Frontiers in Immunology 2024Bi-allelic pathogenic variants in the gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic...
OBJECTIVE
Bi-allelic pathogenic variants in the gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD.
METHODS
Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity.
RESULTS
Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures.
CONCLUSION
MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.
Topics: Humans; Lipopolysaccharides; THP-1 Cells; Phenotype; Mevalonate Kinase Deficiency; Oxidative Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 38550596
DOI: 10.3389/fimmu.2024.1379220 -
Aging Cell May 2024Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA)...
Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.
Topics: Lamin Type A; Humans; Progeria; Farnesyltranstransferase; Protein Prenylation; Dibenzocycloheptenes; Piperidines; Pyridines
PubMed: 38504487
DOI: 10.1111/acel.14105 -
BioRxiv : the Preprint Server For... Mar 2024Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally...
Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.
PubMed: 38496415
DOI: 10.1101/2024.03.03.583211 -
Signal Transduction and Targeted Therapy Mar 2024Posttranslational modifications increase the complexity and functional diversity of proteins in response to complex external stimuli and internal changes. Among these,... (Review)
Review
Posttranslational modifications increase the complexity and functional diversity of proteins in response to complex external stimuli and internal changes. Among these, protein lipidations which refer to lipid attachment to proteins are prominent, which primarily encompassing five types including S-palmitoylation, N-myristoylation, S-prenylation, glycosylphosphatidylinositol (GPI) anchor and cholesterylation. Lipid attachment to proteins plays an essential role in the regulation of protein trafficking, localisation, stability, conformation, interactions and signal transduction by enhancing hydrophobicity. Accumulating evidence from genetic, structural, and biomedical studies has consistently shown that protein lipidation is pivotal in the regulation of broad physiological functions and is inextricably linked to a variety of diseases. Decades of dedicated research have driven the development of a wide range of drugs targeting protein lipidation, and several agents have been developed and tested in preclinical and clinical studies, some of which, such as asciminib and lonafarnib are FDA-approved for therapeutic use, indicating that targeting protein lipidations represents a promising therapeutic strategy. Here, we comprehensively review the known regulatory enzymes and catalytic mechanisms of various protein lipidation types, outline the impact of protein lipidations on physiology and disease, and highlight potential therapeutic targets and clinical research progress, aiming to provide a comprehensive reference for future protein lipidation research.
Topics: Lipid Metabolism; Proteins; Protein Processing, Post-Translational; Signal Transduction; Lipids
PubMed: 38485938
DOI: 10.1038/s41392-024-01759-7 -
Physiological Reports Mar 2024Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein...
Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.
Topics: Rats; Male; Animals; Mechanistic Target of Rapamycin Complex 1; Mevalonic Acid; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Hindlimb Suspension; Signal Transduction; Muscle, Skeletal; Muscular Atrophy
PubMed: 38453353
DOI: 10.14814/phy2.15969 -
MBio Apr 2024is a widespread intracellular protozoan pathogen infecting virtually all warm-blooded animals. This parasite acquires host-derived resources to support its replication...
is a widespread intracellular protozoan pathogen infecting virtually all warm-blooded animals. This parasite acquires host-derived resources to support its replication inside a membrane-bound parasitophorous vacuole within infected host cells. Previous research has discovered that actively endocytoses host proteins and transports them to a lysosome-equivalent structure for digestion. However, few molecular determinants required for trafficking of host-derived material within the parasite were known. A recent study (Q.-Q. Wang, M. Sun, T. Tang, D.-H. Lai, et al., mBio 14:e01309-23, 2023, https://doi.org/10.1128/mbio.01309-23) identified a critical role for membrane anchoring of proteins via prenylation in the trafficking of endocytosed host proteins by , including an essential ortholog of Rab1B. The authors also found that TgRab1 is crucial for protein trafficking of the rhoptry secretory organelles, indicating a dual role in endocytic and exocytic protein trafficking. This study sets the stage for further dissecting endomembrane trafficking in , along with potentially exploiting protein prenylation as a target for therapeutic development.
Topics: Animals; Toxoplasma; Protein Prenylation; Proteins; Organelles; Protein Transport
PubMed: 38407123
DOI: 10.1128/mbio.00283-24 -
Journal of Lipid Research Mar 2024LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a...
LDL-C lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is now a validated therapeutic strategy that lowers serum LDL-C and reduces coronary events. Ironically, the most widely used medicine to lower cholesterol, statins, has been shown to increase circulating PCSK9 levels, which limits their efficacy. Here, we show that geranylgeranyl isoprenoids and hepatic Rap1a regulate both basal and statin-induced expression of PCSK9 and contribute to LDL-C homeostasis. Rap1a prenylation and activity is inhibited upon statin treatment, and statin-mediated PCSK9 induction is dependent on geranylgeranyl synthesis and hepatic Rap1a. Accordingly, treatment of mice with a small-molecule activator of Rap1a lowered PCSK9 protein and plasma cholesterol and inhibited statin-mediated PCSK9 induction in hepatocytes. The mechanism involves inhibition of the downstream RhoA-ROCK pathway and regulation of PCSK9 at the post-transcriptional level. These data further identify Rap1a as a novel regulator of PCSK9 protein and show that blocking Rap1a prenylation through lowering geranylgeranyl levels contributes to statin-mediated induction of PCSK9.
Topics: Mice; Animals; Proprotein Convertase 9; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Cholesterol, LDL; Antibodies, Monoclonal; Cholesterol
PubMed: 38309417
DOI: 10.1016/j.jlr.2024.100515 -
International Journal of Molecular... Jan 2024In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of... (Review)
Review
Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites.
In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.
Topics: Melanins; Lipoylation; Calcium; Membranes; Proteins
PubMed: 38279221
DOI: 10.3390/ijms25021224 -
PLoS Pathogens Jan 2024A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic...
A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.
Topics: Humans; Animals; Cattle; Parasites; Phosphates; Terpenes; Pentanols; Diterpenes; Fosfomycin; Hemiterpenes
PubMed: 38277417
DOI: 10.1371/journal.ppat.1011557