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PLoS Biology Nov 2023The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome...
The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1's evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats.
Topics: Animals; Humans; Chiroptera; SARS-CoV-2; Phylogeny; COVID-19; 2',5'-Oligoadenylate Synthetase
PubMed: 38015855
DOI: 10.1371/journal.pbio.3002398 -
Brain Sciences Oct 2023Currently, there is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder. Many biomarkers have been proposed, but...
BACKGROUND
Currently, there is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder. Many biomarkers have been proposed, but because ALS is a clinically heterogeneous disease with an unclear etiology, biomarker discovery for ALS has been challenging due to the lack of specificity of these biomarkers. In recent years, the role of autophagy in the development and treatment of ALS has become a research hotspot. In our previous studies, we found that the expression of RabGGTase (low RABGGTB expression and no change in RABGGTA) is lower in the lumbar and thoracic regions of spinal cord motoneurons in SOD1G93A mice compared with WT (wild-type) mice groups, and upregulation of RABGGTB promoted prenylation modification of Rab7, which promoted autophagy to protect neurons by degrading SOD1. Given that RabGGTase is associated with autophagy and autophagy is associated with inflammation, and based on the above findings, since peripheral blood mononuclear cells are readily available from patients with ALS, we proposed to investigate the expression of RabGGTase in peripheral inflammatory cells.
METHODS
Information and venous blood were collected from 86 patients diagnosed with ALS between January 2021 and August 2023. Flow cytometry was used to detect the expression of RABGGTB in monocytes from peripheral blood samples collected from patients with ALS and healthy controls. Extracted peripheral blood mononuclear cells (PBMCs) were differentiated in vitro into macrophages, and then the expression of RABGGTB was detected by immunofluorescence. RABGGTB levels in patients with ALS were analyzed to determine their impact on disease progression.
RESULTS
Using flow cytometry in monocytes and immunofluorescence in macrophages, we found that RABGGTB expression in the ALS group was significantly higher than in the control group. Age, sex, original location, disease course, C-reactive protein (CRP), and interleukin-6 (IL-6) did not correlate with the ALS functional rating scale-revised (ALSFRS-R), whereas the RABGGTB level was significantly correlated with the ALSFRS-R. In addition, multivariate analysis revealed a significant correlation between RABGGTB and ALSFRS-R score. Further analysis revealed a significant correlation between RABGGTB expression levels and disease progression levels (ΔFS).
CONCLUSIONS
The RABGGTB level was significantly increased in patients with ALS compared with healthy controls. An elevated RABGGTB level in patients with ALS is associated with the rate of progression in ALS, suggesting that elevated RABGGTB levels in patients with ALS may serve as an indicator for tracking ALS progression.
PubMed: 38002490
DOI: 10.3390/brainsci13111531 -
Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.Insect Molecular Biology Apr 2024Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and...
Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.
Topics: Humans; Animals; Bees; Farnesyltranstransferase; Pesticides; Phylogeny; Zoledronic Acid
PubMed: 37962063
DOI: 10.1111/imb.12885 -
BioRxiv : the Preprint Server For... Oct 2023Low-density lipoprotein cholesterol (LDL-C) lowering is the main goal of atherosclerotic cardiovascular disease prevention, and proprotein convertase subtilisin/kexin...
Low-density lipoprotein cholesterol (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.
PubMed: 37961667
DOI: 10.1101/2023.10.23.563509 -
RSC Chemical Biology Nov 2023Protein lipidation is a post-translational modification that confers hydrophobicity on protein substrates to control their cellular localization, mediate protein...
Protein lipidation is a post-translational modification that confers hydrophobicity on protein substrates to control their cellular localization, mediate protein trafficking, and regulate protein function. In particular, protein prenylation is a C-terminal modification on proteins bearing canonical motifs catalyzed by prenyltransferases. Prenylated proteins have been of interest due to their numerous associations with various diseases. Chemical proteomic approaches have been pursued over the last decade to define prenylated proteomes (prenylome) and probe their responses to perturbations in various cellular systems. Here, we describe the discovery of prenylation of a non-canonical prenylated protein, ALDH9A1, which lacks any apparent prenylation motif. This enzyme was initially identified through chemical proteomic profiling of prenylomes in various cell lines. Metabolic labeling with an isoprenoid probe using overexpressed ALDH9A1 revealed that this enzyme can be prenylated inside cells but does not respond to inhibition by prenyltransferase inhibitors. Site-directed mutagenesis of the key residues involved in ALDH9A1 activity indicates that the catalytic C288 bears the isoprenoid modification likely through an NAD-dependent mechanism. Furthermore, the isoprenoid modification is also susceptible to hydrolysis, indicating a reversible modification. We hypothesize that this modification originates from endogenous farnesal or geranygeranial, the established degradation products of prenylated proteins and results in a thioester form that accumulates. This novel reversible prenoyl modification on ALDH9A1 expands the current paradigm of protein prenylation by illustrating a potentially new type of protein-lipid modification that may also serve as a novel mechanism for controlling enzyme function.
PubMed: 37920391
DOI: 10.1039/d3cb00089c -
International Journal of Molecular... Oct 2023Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by...
Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) patient-derived fibroblasts and (2) zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.
Topics: Animals; Humans; Adult; Choroideremia; Zebrafish; Retina; Mutation; Retinal Dystrophies; Plasmids; Adaptor Proteins, Signal Transducing
PubMed: 37894906
DOI: 10.3390/ijms242015225 -
Cancer Drug Resistance (Alhambra,... 2023This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and...
This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.
PubMed: 37842233
DOI: 10.20517/cdr.2023.20 -
BioRxiv : the Preprint Server For... Sep 2023The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur...
The C-terminal CaaX sequence (cysteine-aliphatic-aliphatic-any of several amino acids) is subject to isoprenylation on the conserved cysteine and is estimated to occur in 1-2% of proteins within yeast and human proteomes. Recently, non-canonical CaaX sequences in addition to shorter and longer length CaX and CaaaX sequences have been identified that can be prenylated. Much of the characterization of prenyltransferases has relied on the yeast system because of its genetic tractability and availability of reporter proteins, such as the -factor mating pheromone, Ras GTPase, and Ydj1 Hsp40 chaperone. To compare the properties of yeast and human prenyltransferases, including the recently expanded target specificity of yeast farnesyltransferase, we have developed yeast strains that express human farnesyltransferase or geranylgeranyltransferase-I in lieu of their yeast counterparts. The humanized yeast strains display robust prenyltransferase activity that functionally replaces yeast prenyltransferase activity in a wide array of tests, including the prenylation of a wide variety of canonical and non-canonical human CaaX sequences, virus encoded CaaX sequences, non-canonical length sequences, and heterologously expressed human proteins HRas and DNAJA2. These results reveal highly overlapping substrate specificity for yeast and human farnesyltransferase, and mostly overlapping substrate specificity for GGTase-I. This yeast system is a valuable tool for further defining the prenylome of humans and other organisms, identifying proteins for which prenylation status has not yet been determined.
PubMed: 37786692
DOI: 10.1101/2023.09.19.558494 -
Antioxidants (Basel, Switzerland) Aug 2023Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM...
Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy, affecting the photoreceptors, retinal pigment epithelium (RPE) and choroid, with no approved therapy. CHM is caused by mutations in the gene, which encodes the ubiquitously expressed Rab escort protein 1 (REP1). REP1 is involved in prenylation, a post-translational modification of Rab proteins, and plays an essential role in intracellular trafficking. In this study, we examined oxidative and endoplasmic reticulum (ER) stress pathways in zebrafish and patient fibroblasts, and screened a number of neuroprotectants for their ability to reduce stress. The expression of the oxidative stress markers , and , and the ER stress markers , and , were dysregulated in fish. The expression of was also reduced in fibroblasts, along with reduced and increased expression. The lack of REP1 is associated with defects in vesicular trafficking, photoreceptor outer segment phagocytosis and melanosome transport, leading to increased levels of stress within the retina and RPE. Drugs targeting oxidative and ER stress pathways represent novel therapeutic avenues.
PubMed: 37759997
DOI: 10.3390/antiox12091694 -
Molecular Cancer Therapeutics Jan 2024Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer... (Review)
Review
Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.
Topics: Humans; Farnesyltranstransferase; Enzyme Inhibitors; Terpenes; Protein Prenylation; Neoplasms
PubMed: 37756579
DOI: 10.1158/1535-7163.MCT-23-0358