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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 -
The Journal of Biological Chemistry Mar 2024Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved...
Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved immunoreceptor tyrosine-based activation motif and prenylation motifs within MIEN1, we identified potent anticancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and epithelial-mesenchymal transition pathways, concurrently suppressing epidermal growth factor-induced nuclear factor kappa B nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and CD spectra indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high intraperitoneal (ip) peptide doses of 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anticancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.
Topics: Animals; Mice; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Intracellular Signaling Peptides and Proteins; Molecular Docking Simulation; Neoplasm Proteins; Signal Transduction; Humans; Cell Line; Neoplasms
PubMed: 38272230
DOI: 10.1016/j.jbc.2024.105680 -
JHEP Reports : Innovation in Hepatology Jan 2024Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen...
BACKGROUND & AIMS
Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly.
METHODS
Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study.
RESULTS
C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif.
CONCLUSIONS
Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV.
IMPACT AND IMPLICATIONS
Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.
PubMed: 38192534
DOI: 10.1016/j.jhepr.2023.100961 -
Journal of Cell Science Jan 2024RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and...
RhoU is an atypical member of the Rho family of small G-proteins, which has N- and C-terminal extensions compared to the classic Rho GTPases RhoA, Rac1 and Cdc42, and associates with membranes through C-terminal palmitoylation rather than prenylation. RhoU mRNA expression is upregulated in prostate cancer and is considered a marker for disease progression. Here, we show that RhoU overexpression in prostate cancer cells increases cell migration and invasion. To identify RhoU targets that contribute to its function, we found that RhoU homodimerizes in cells. We map the region involved in this interaction to the C-terminal extension and show that C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension reduces RhoU-induced activation of p21-activated kinases (PAKs), which are known downstream targets for RhoU, and induces cell morphological changes consistent with inhibiting RhoU function. Our results show for the first time that the activity of a Rho family member is stimulated by self-association, and this is important for its activity.
Topics: Humans; Male; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Prostatic Neoplasms; rho GTP-Binding Proteins
PubMed: 38180080
DOI: 10.1242/jcs.261645 -
Life Science Alliance Mar 2024IFN-stimulated gene 2',3' cyclic nucleotide 3' phosphodiesterase (CNP) comprises two isoforms: the short CNP1 and the long CNP2, featuring an additional N-terminal...
IFN-stimulated gene 2',3' cyclic nucleotide 3' phosphodiesterase (CNP) comprises two isoforms: the short CNP1 and the long CNP2, featuring an additional N-terminal segment of 20 amino acids (N20aa) proposed as a mitochondrial targeting sequence. Notably, CNP1 can be produced by cleaving the N20aa segment from CNP2. Although previous investigations have recognized the HIV-1 particle assembly impairment capability of CNP2, the antiviral activity of CNP1 remains ambiguous. Our study clarifies that CNP1, as opposed to CNP2, serves as the primary isoform exerting anti-HIV-1 activity. Both CNP1 and CNP2 can localize to the cell membrane, but the N20aa segment of CNP2 impedes CNP2-HIV-1 Gag interaction. Cleavage of the N20aa segment from CNP2 results in the formation of a functional, truncated form known as CNP1. Intriguingly, this posttranslational processing of CNP2 N20aa occurs within the cytoplasmic matrix rather than the mitochondria. Regulated by CTII motif prenylation, CNP1 proteins translocate to the cell membrane and engage with HIV-1 Gag. In conclusion, our findings underscore the pivotal role of posttranslational modification in governing the inhibitory potential of CNP in HIV-1 replication.
Topics: 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; HIV-1; Protein Isoforms; Mitochondria
PubMed: 38167610
DOI: 10.26508/lsa.202302188 -
Genes Dec 2023Protein prenylation mediated by the () gene plays a crucial role in plant growth, development, and environmental response by adding a 15-carbon farnesyl group or one to...
Protein prenylation mediated by the () gene plays a crucial role in plant growth, development, and environmental response by adding a 15-carbon farnesyl group or one to two 20-carbon geranylgeranyl groups onto one to two cysteine residues at the C-terminus of the target protein. However, the homologous genes and their functions of in rapeseed are unclear. In this study, bioinformatics analysis and gene cloning demonstrated the existence of two homologous genes of in the L. genome, namely, and . Evolutionary analysis revealed that originated from the L. genome, while originated from the L. genome. Genetic transformation analysis revealed that the overexpression of in plants exhibited earlier flowering initiation, a prolonged flowering period, increased plant height, and longer main inflorescence length compared to the wild type. Contrarily, the downregulation of expression in plants led to delayed flowering initiation, shortened flowering period, decreased plant height, and reduced main inflorescence length compared to the wild type. These findings indicate that the gene positively regulates flowering time, plant height, and main inflorescence length. This provides a new gene for the genetic improvement of flowering time and plant architecture in rapeseed.
Topics: Brassica napus; Inflorescence; Genes, Plant; Arabidopsis; Carbon
PubMed: 38137028
DOI: 10.3390/genes14122206 -
Advanced Science (Weinheim,... Feb 2024Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing...
Post-translational prenylations, found in eukaryotic primary metabolites and bacterial secondary metabolites, play crucial roles in biomolecular interactions. Employing genome mining methods combined with AlphaFold2-based predictions of protein interactions, PalQ , a prenyltransferase responsible for the tryptophan prenylation of RiPPs produced by Paenibacillus alvei, is identified. PalQ differs from cyanobactin prenyltransferases because of its evolutionary relationship to isoprene synthases, which enables PalQ to transfer extended prenyl chains to the indole C3 position. This prenylation introduces structural diversity to the tryptophan side chain and also leads to conformational dynamics in the peptide backbone, attributed to the cis/trans isomerization that arises from the formation of a pyrrolidine ring. Additionally, PalQ exhibited pronounced positional selectivity for the C-terminal tryptophan. Such enzymatic characteristics offer a toolkit for peptide therapeutic lipidation.
Topics: Dimethylallyltranstransferase; Tryptophan; Prenylation; Protein Processing, Post-Translational; Peptides
PubMed: 38059776
DOI: 10.1002/advs.202307372 -
Brain Research Bulletin Jan 2024Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with unknown causes, which mainly affects motor neurons in the anterior horn of the spinal...
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with unknown causes, which mainly affects motor neurons in the anterior horn of the spinal cord, brain stem, and cerebral cortex, also known as motor neuron disease. An important pathological feature of ALS is the formation of aggregates of mutant SOD1 protein, CTF25 of TDP-43, or other abnormal proteins in motor neurons, which require autophagy for degradation. Protein prenylation is known to participate in membrane association and proper localization of proteins. RABGGTB is the β subunit of GGTase II (one of the prenyltransferases) that can regulate autophagy via Rab7 geranylgeranylation. In this study, we overexpressed RABGGTB via lentiviral transfection in NSC34-hSOD1G93A and TDP-43 cells. Overexpression of RABGGTB improved ALS cell proliferation by facilitating autophagosome-lysosome fusion. Furthermore, the abnormal aggregation of SOD1 protein was reduced. This indicates that protein prenylation is important for the proliferation and autophagy of cells autophagy. Enhanced autophagy has been observed in two of the most widely used ALS cell models. These findings indicate the widespread applicability of prenylation in ALS. In summary, overexpression of RABGGTB improved the geranylgeranylation of the Rab7 protein and had a positive effect on cells. These findings provide insights into the development of a novel therapeutic strategy for ALS.
Topics: Humans; Amyotrophic Lateral Sclerosis; Superoxide Dismutase-1; Neurodegenerative Diseases; Motor Neurons; Spinal Cord; DNA-Binding Proteins
PubMed: 38042502
DOI: 10.1016/j.brainresbull.2023.110833 -
Clinical Genetics Mar 2024The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and...
The evolutionarily conserved mevalonate pathway plays an important role in the synthesis of cholesterol and isoprenoid compounds. Mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) enzymes regulate key rate-limiting steps in this pathway by sequentially phosphorylating mevalonic acid to yield downstream metabolites that regulate protein prenylation and cell signaling. Biallelic pathogenic variants in MVK cause a spectrum of rare autoinflammatory disorders that encompass milder forms of hyper-IgD syndrome (HIDS) at one end and the more severe mevalonic aciduria on the other. In contrast, pathogenic variants reported in PMVK are heterozygous and associated with porokeratosis, a skin disorder with no systemic manifestations. Recently, biallelic variants in PMVK were reported as a cause for an autoinflammatory disorder for the first time in two unrelated patients. In this study, we describe a child with recurrent arthritis and a HIDS-like phenotype harboring a novel homozygous variant c.398 C>T (p.Ala133Val) in PMVK. Mononuclear cells isolated from the patient showed significantly elevated production of interleukin 1β, a key cytokine that shapes the inflammatory response in HIDS. Protein modeling studies suggested potential defects in PMVK enzyme activity. These results posit a further expanding of the genotypic spectrum of autoinflammatory disease to include biallelic PMVK variants.
Topics: Child; Humans; Genotype; Mevalonate Kinase Deficiency; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Phosphotransferases (Phosphate Group Acceptor)
PubMed: 38018277
DOI: 10.1111/cge.14451