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Cell Chemical Biology May 2018Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis,...
Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMN or C2'-hydroxylated prFMN under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.
Topics: Biosynthetic Pathways; Carboxy-Lyases; Escherichia coli; Escherichia coli Proteins; Flavin Mononucleotide; Hemiterpenes; Organophosphorus Compounds; Pentanols; Prenylation
PubMed: 29551348
DOI: 10.1016/j.chembiol.2018.02.007 -
RSC Advances Sep 2022Prenylation usually improves structural diversity and bioactivity in natural products. Unlike the discovered enzymatic -diprenylation of mono- and tri-cyclic aromatic...
Prenylation usually improves structural diversity and bioactivity in natural products. Unlike the discovered enzymatic -diprenylation of mono- and tri-cyclic aromatic systems, the enzymatic approach for -diprenylation of bi-cyclic hydroxynaphthalenes is new to science. Here we report an enzymatic example for dearomative C4 -diprenylation of α-hydroxynaphthalenes, by the F253G mutant of a fungal prenyltransferase CdpC3PT. Experimental evidence suggests a sequential electrophilic substitution mechanism. We also explained the alteration of catalytic properties on CdpC3PT after mutation on F253 by modeling. This study provides a valuable addition to the synthetic toolkit for compound prenylation and it also contributes to the mechanistic study of prenylating enzymes.
PubMed: 36276050
DOI: 10.1039/d2ra04837j -
Nature Chemistry Jun 2019Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras...
Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.
Topics: Adaptor Proteins, Signal Transducing; Alkynes; Animals; Cell Line; Gene Knockout Techniques; Humans; Mass Spectrometry; Mice, Knockout; Molecular Probes; Protein Prenylation; Proteins; Proteome; Proteomics
PubMed: 30936521
DOI: 10.1038/s41557-019-0237-6 -
Cell Chemical Biology Jun 2022The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that...
The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that regulates various cellular functions. We recently observed nuclear mTORC1 activity, raising an intriguing question as to how Rheb, which is known to be farnesylated and localized to intracellular membranes, regulates nuclear mTORC1. In this study, we found that active Rheb is present in the nucleus and required for nuclear mTORC1 activity. We showed that inhibition of farnesyltransferase reduced cytosolic, but not nuclear, mTORC1 activity. Furthermore, a farnesylation-deficient Rheb mutant, with preferential nuclear localization and specific lysosome tethering, enables nuclear and cytosolic mTORC1 activities, respectively. These data suggest that non-farnesylated Rheb is capable of interacting with and activating mTORC1, providing mechanistic insights into the molecular functioning of Rheb as well as regulation of the recently observed, active pool of nuclear mTORC1.
Topics: Brain; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neuropeptides; Prenylation; Ras Homolog Enriched in Brain Protein; TOR Serine-Threonine Kinases
PubMed: 35294906
DOI: 10.1016/j.chembiol.2022.02.006 -
Nature May 2022Imbalances in lipid homeostasis can have deleterious effects on health. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient...
Imbalances in lipid homeostasis can have deleterious effects on health. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption remains unclear. Here we describe a mechanism for intracellular lipid surveillance in Caenorhabditis elegans that involves transcriptional inactivation of the nuclear hormone receptor NHR-49 through its cytosolic sequestration to endocytic vesicles via geranylgeranyl conjugation to the small G protein RAB-11.1. Defective de novo isoprenoid synthesis caused by lipid depletion limits RAB-11.1 geranylgeranylation, which promotes nuclear translocation of NHR-49 and activation of rab-11.2 transcription to enhance transporter residency at the plasma membrane. Thus, we identify a critical lipid sensed by the cell, its conjugated G protein, and the nuclear receptor whose dynamic interactions enable cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Lipids; Monomeric GTP-Binding Proteins; Protein Prenylation; Receptors, Cytoplasmic and Nuclear
PubMed: 35585236
DOI: 10.1038/s41586-022-04729-7 -
Clinical Cancer Research : An Official... Apr 2015RAS proteins require membrane association for their biologic activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS... (Review)
Review
RAS proteins require membrane association for their biologic activity, making this association a logical target for anti-RAS therapeutics. Lipid modification of RAS proteins by a farnesyl isoprenoid is an obligate step in that association, and is an enzymatic process. Accordingly, farnesyltransferase inhibitors (FTI) were developed as potential anti-RAS drugs. The lack of efficacy of FTIs as anticancer drugs was widely seen as indicating that blocking RAS membrane association was a flawed approach to cancer treatment. However, a deeper understanding of RAS modification and trafficking has revealed that this was an erroneous conclusion. In the presence of FTIs, KRAS and NRAS, which are the RAS isoforms most frequently mutated in cancer, become substrates for alternative modification, can still associate with membranes, and can still function. Thus, FTIs failed not because blocking RAS membrane association is an ineffective approach, but because FTIs failed to accomplish that task. Recent findings regarding RAS isoform trafficking and the regulation of RAS subcellular localization have rekindled interest in efforts to target these processes. In particular, improved understanding of the palmitoylation/depalmitoylation cycle that regulates RAS interaction with the plasma membrane, endomembranes, and cytosol, and of the potential importance of RAS chaperones, have led to new approaches. Efforts to validate and target other enzymatically regulated posttranslational modifications are also ongoing. In this review, we revisit lessons learned, describe the current state of the art, and highlight challenging but promising directions to achieve the goal of disrupting RAS membrane association and subcellular localization for anti-RAS drug development. Clin Cancer Res; 21(8); 1819-27. ©2015 AACR. See all articles in this CCR Focus section, "Targeting RAS-Driven Cancers."
Topics: Acetyltransferases; Acylation; Alkyl and Aryl Transferases; Amino Acid Motifs; Animals; Antineoplastic Agents; Carrier Proteins; Cell Membrane; Drug Discovery; Endopeptidases; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Molecular Targeted Therapy; Neoplasms; Phosphorylation; Protein Interaction Domains and Motifs; Protein Kinase C; Protein Prenylation; Protein Transport; Proto-Oncogene Proteins p21(ras); Signal Transduction; Thiolester Hydrolases
PubMed: 25878363
DOI: 10.1158/1078-0432.CCR-14-3214 -
Journal of Molecular Biology Dec 2016Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell...
Two isoforms of the small GTPase Rap1, Rap1A and Rap1B, participate in cell adhesion; Rap1A promotes steady state adhesion, while Rap1B regulates dynamic changes in cell adhesion. These events depend on the prenylation of Rap1, which promotes its membrane localization. Here, we identify previously unsuspected differences in the regulation of prenylation of Rap1A versus Rap1B, due in part to their different phosphorylation-dependent interactions with the chaperone protein SmgGDS-607. Previous studies indicate that the activation of Gα protein-coupled receptors (GPCRs) phosphorylates S-179 and S-180 in the polybasic region (PBR) of Rap1B, which inhibits Rap1B binding to SmgGDS-607 and diminishes Rap1B prenylation and membrane localization. In this study, we investigate how phosphorylation in the PBR of multiple small GTPases, including K-Ras4B, RhoA, Rap1A, and Rap1B, affects their binding to SmgGDS, with emphasis on differences between Rap1A and Rap1B. We identify the amino acids in SmgGDS-607 necessary for binding of Rap1A and Rap1B, and present homology models examining the binding between Rap1A or Rap1B and SmgGDS-607. Unlike Rap1B, phosphorylation in the PBR of Rap1A does not detectably inhibit its prenylation or its binding to SmgGDS-607. Activation of GPCRs suppresses Rap1A prenylation, but unlike this effect on Rap1B, the GPCR-mediated suppression of Rap1A prenylation can occur independently of Rap1A phosphorylation and does not detectably diminish Rap1A membrane localization. These data demonstrate unexpected evolutionarily conserved differences in the ability of GPCRs to regulate the prenylation of Rap1B compared to Rap1A.
Topics: Amino Acid Sequence; Cell Line; Guanine Nucleotide Exchange Factors; Humans; Models, Molecular; Molecular Sequence Data; Phosphorylation; Prenylation; Protein Binding; Protein Conformation; Protein Interaction Mapping; Protein Processing, Post-Translational; Sequence Alignment; rap GTP-Binding Proteins; rap1 GTP-Binding Proteins
PubMed: 27760305
DOI: 10.1016/j.jmb.2016.10.016 -
PloS One 2017RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel...
RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery.
Topics: Animals; Cell Line; Cell Line, Tumor; Humans; Lipids; Magnetic Resonance Spectroscopy; Prenylation; Proto-Oncogene Proteins p21(ras)
PubMed: 28384226
DOI: 10.1371/journal.pone.0174706 -
Small GTPases Jul 2020Mutant RAS isoforms are the most common oncogenes affecting human cancers. After decades of effort in developing drugs targeting oncogenic RAS-driven cancers, we are... (Review)
Review
Mutant RAS isoforms are the most common oncogenes affecting human cancers. After decades of effort in developing drugs targeting oncogenic RAS-driven cancers, we are still charting an unclear path. Despite recent developments exemplified by KRAS (G12C) inhibitors, direct targeting of mutant RAS remains a difficult endeavor. Inhibiting RAS function by targeting its post-translational prenylation processing has remained an important approach, especially with recent progress on the study of isoprenylcysteine carboxylmethyltransferase (ICMT), the unique enzyme for the last step of prenylation processing of RAS isoforms and other substrates. Inhibition of ICMT has shown efficacy both and in RAS-mutant cancer models. We will discuss the roles of RAS family of proteins in human cancers and the impact of post-prenylation carboxylmethylation on RAS driven tumorigenesis. In addition, we will review what is known of the molecular and cellular impact of ICMT inhibition on cancer cells that underlie its anti-proliferative and pro-apoptosis efficacy.
Topics: Animals; Humans; Methylation; Protein Prenylation; ras Proteins
PubMed: 29261009
DOI: 10.1080/21541248.2017.1415637 -
Applied and Environmental Microbiology May 2021Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of...
Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of NRRL B-24963 revealed a two-gene locus, , encoding CDPS SazA and a unique fused enzyme (SazB) harboring two domains: phytoene synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the gene(s) in J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A to C (compounds 3 to 5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of (l-Trp-l-Trp) (cWW; compound 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C-3/C-3' prenylation(s) by SazB-PT and N-1/N-1' methylation(s) by SazB-MT. Of note, the substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT. Natural products with a 2,5-diketopiperazine (2,5-DKP) skeleton have long sparked interest in drug discovery and development. Recent advances in microbial genome sequencing have revealed that the potential of cyclodipeptide synthase (CDPS)-dependent pathways encoding new 2,5-DKPs are underexplored. In this study, we report the genome mining of a new CDPS-encoding two-gene operon and activation of this cryptic gene cluster through heterologous expression, leading to the discovery of four indole 2,5-DKP alkaloids. The (l-Trp-l-Trp) (cWW)-synthesizing CDPS SazA and the unusual prenyltransferase (PT)-methyltransferase (MT) fused enzyme SazB were characterized. Our results expand the repertoire of CDPSs and associated tailoring enzymes, setting the stage for accessing diverse prenylated alkaloids using synthetic biology strategies.
Topics: Bacterial Proteins; Indole Alkaloids; Metabolic Networks and Pathways; Microorganisms, Genetically-Modified; Peptide Synthases; Prenylation; Streptomyces
PubMed: 33741615
DOI: 10.1128/AEM.02525-20