Protein Prenylation Research Articles

Understanding the Antilymphoma Activity of Annona macroprophyllata Donn and Its Acyclic Terpenoids: In Vivo, In Vitro, and In Silico Studies.

Annona macroprophyllata Donn (A. macroprophyllata) is used in traditional Mexican medicine for the treatment of cancer, diabetes, inflammation, and pain. In this work, we evaluated the antitumor activity of three acyclic terpenoids obtained from A. macroprophyllata to assess their potential as antilymphoma agents. We identified the terpenoids farnesyl acetate (FA), phytol (PT) and geranylgeraniol (Gg) using gas chromatography–mass spectroscopy (GC-MS) and spectroscopic (1H, and 13C NMR) methods applied to petroleum ether extract of leaves from A. macroprophyllata (PEAm). We investigated antitumor potential in Balb/c mice inoculated with U-937 cells by assessing brine shrimp lethality (BSL), and cytotoxic activity in these cells. In addition, to assess the potential toxicity of PEAm, FA, PT and Gg in humans, we tested their acute oral toxicity in mice. Our results showed that the three terpenoids exhibited considerable antilymphoma and cytotoxic activity. In terms of lethality, we determined a median lethal dose (LD50) for thirteen isolated products of PEAm. Gg, PT and AF all exhibited a higher lethality with values of 1.41 ± 0.42, 3.03 ± 0.33 and 5.82 ± 0.58 µg mL−1, respectively. To assess cytotoxic activity against U-937 cells, we calculated the mean cytotoxic concentration (CC50) and found that FA and PT were closer in respect to the control drug methotrexate (MTX, 0.243 ± 0.007 µM). In terms of antilymphoma activity, we found that FA, PT and Gg considerably inhibited lymph node growth, with median effective doses (ED50) of 5.89 ± 0.39, 6.71 ± 0.31 and 7.22 ± 0.51 mg kg−1 in females and 5.09 ± 0.66, 5.83 ± 0.50 and 6.98 ± 0.57mg kg −1 in males, respectively. Regarding acute oral toxicity, we classified all three terpenoids as category IV, indicating a high safety margin for human administration. Finally, in a molecular docking study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, we found binding of terpenoids to some amino acids of the catalytic site, suggesting an effect upon activity with a resulting decrease in the synthesis of intermediates involved in the prenylation of proteins involved in cancer progression. Our findings suggest that the acyclic terpenoids FA, PT, and Gg may serve as scaffolds for the development of new treatments for non-Hodgkin’s lymphoma.

ANGPTL3 deficiency associates with the expansion of regulatory T cells with reduced lipid content

Background and aimsAngiopoietin-like 3 (ANGPTL3) regulates lipid and glucose metabolism. Loss-of-function mutations in its gene, leading to ANGPTL3 deficiency, cause in humans the familial combined hypolipidemia type 2 (FHBL2) phenotype, characterized by very low concentrations of circulating lipoproteins and reduced risk of atherosclerotic cardiovascular disease. Whether this condition is accompanied by immune dysfunctions is unknown. Regulatory T cells (Tregs) are CD4 T lymphocytes endowed with immune suppressive and atheroprotective functions and sensitive to metabolic signals. By investigating FHBL2, we explored the hypothesis that Tregs expand in response to extreme hypolipidemia, through a modulation of the Treg-intrinsic lipid metabolism. MethodsTreg frequency, phenotype, and intracellular lipid content were assessed ex vivo from FHBL2 subjects and age- and sex-matched controls, through multiparameter flow cytometry. The response of CD4 T cells from healthy controls to marked hypolipidemia was tested in vitro in low-lipid culture conditions. ResultsThe ex vivo analysis revealed that FHBL2 subjects showed higher percentages of Tregs with a phenotype undistinguishable from controls and with a lower lipid content, which directly correlated with the concentrations of circulating lipoproteins. In vitro, lipid restriction induced the upregulation of genes of the mevalonate pathway, including those involved in isoprenoid biosynthesis, and concurrently increased the expression of the Treg markers FOXP3 and Helios. The latter event was found to be prenylation-dependent, and likely related to increased IL-2 production and signaling. ConclusionsOur study demonstrates that FHBL2 is characterized by high Treg frequencies, a feature which may concur to the reduced atherosclerotic risk in this condition. Mechanistically, hypolipidemia may directly favor Treg expansion, through the induction of the mevalonate pathway and the prenylation of key signaling proteins.

Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation.

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.

Increased core body temperature exacerbates defective protein prenylation in mouse models of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1β, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.

The Heptaprenyl Diphosphate Synthase (Coq1) Is the Target of a Lipophilic Bisphosphonate That Protects Mice against Toxoplasma gondii Infection.

ABSTRACTPrenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer “polyprenyl” diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis.

In Vivo Prenylomic Profiling in the Brain of a Transgenic Mouse Model of Alzheimer's Disease Reveals Increased Prenylation of a Key Set of Proteins.

Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimer's disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed the identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to nontransgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.

Deciphering Regulatory Proteins of Prenylated Protein via the FRET Technique Using Nitroso-Based Ene-Ligation and Sequential Azidation and Click Reaction.

We report here the selective incorporations of nitroso species into a wide range of proteins targeting lysine residue(s). The corresponding azo functionalities were formed in a highly selective manner with excellent yields, displaying rather good stability under physiological conditions. Furthermore, the azodation proceeded smoothly in high yields on targeted peptides. Fluorescent and/or dual fluorescent labeling of varied proteins following this protocol have been determined efficiently and selectively. With this established protocol, we aim to determine its usage in the evaluation of the interaction of prenylated proteins with their interacted enzyme(s) via FRET assays. Delightedly, chemically modified proteins with a 1-pyrenyl fluorophore through 254 nm UV irradiation and the sequential azodation and click reaction of protein prenyl functionality, which enable the incorporation of naphthene, indeed increase the fluorescence energy transferred since we observed significantly enhanced absorption located at 218 nm in lysed HEK293T cells and a clearly strengthened greenish fluorescence in living HEK293T cells.

Hyperglycemic Conditions Promote Rac1-Mediated Serine536 Phosphorylation of p65 Subunit of NFκB (RelA) in Pancreatic Beta Cells.

We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions. INS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry. HG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions. Activation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.

Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells.

Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.

Non-Nitrogen-Containing Bisphosphonates Prevent Pyrophosphorylation of Exocytosis Proteins.

Clodronate, a non-nitrogen-containing bisphosphonate (non-NBP), is intracellularly converted into non-hydrolyzable ATP analogs. Clodronate and its analogs impair normal cell functions, including the exocytosis process. However, how this occurs in mast cells is still not well characterized. To summarize the possible mechanisms of clodronate-mediated exocytosis inhibition in mast cells. Non-NBPs display several possible mechanisms of exocytosis inhibition in various cell types, including vesicular nucleotide transporter (VNUT) and purinergic receptor inhibition. Inhibition of purinergic receptors has been shown in mast cells, but VNUT inhibition remains to be confirmed. Inhibition of protein prenylation by non-NBPs has also been shown; however, direct evidence of non-NBPs in prenylated exocytosis proteins is still contradictory. Finally, non-NBPs may inhibit mast cell exocytosis via impairment of protein pyrophosphorylation. This mechanism is less studied, and direct evidence of the involvement of pyrophosphorylated proteins in exocytosis is still lacking. Non-NBPs may affect mast cell exocytosis by interacting with purinergic receptors or VNUT or by preventing post-translational modifications of exocytosis protein(s), i.e., prenylation and pyrophosphorylation. The latter needs further investigation to provide direct evidence of a role for non- NBPs.

Pleiotropic properties of statins via angiogenesis modulation in cardiovascular disease

Inhibition of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase by statins is affected by inhibiting the active site of the enzyme in a competitive manner. Statins reduce plasma cholesterol by inhibiting its de novo synthesis. In addition, statins impart 'pleiotropic' activities that do not directly relate to their ability to decrease cholesterol. The proangiogenic and antiangiogenic characteristics of statins are among these pleiotropic effects. These angiogenic-modifying properties could offer new therapeutic applications. Statins stimulate or suppress angiogenesis in a biphasic manner. Whereas low doses of statin stimulate angiogenesis, high doses reduce protein prenylation and limit cell development and angiogenesis. In this review, we discuss how statins impact angiogenesis, with a particular focus on angiogenesis in stroke and cardiovascular disease (CVD).

Functional classification and validation of yeast prenylation motifs using machine learning and genetic reporters.

Protein prenylation by farnesyltransferase (FTase) is often described as the targeting of a cysteine-containing motif (CaaX) that is enriched for aliphatic amino acids at the a1 and a2 positions, while quite flexible at the X position. Prenylation prediction methods often rely on these features despite emerging evidence that FTase has broader target specificity than previously considered. Using a machine learning approach and training sets based on canonical (prenylated, proteolyzed, and carboxymethylated) and recently identified shunted motifs (prenylation only), this study aims to improve prenylation predictions with the goal of determining the full scope of prenylation potential among the 8000 possible Cxxx sequence combinations. Further, this study aims to subdivide the prenylated sequences as either shunted (i.e., uncleaved) or cleaved (i.e., canonical). Predictions were determined for Saccharomyces cerevisiae FTase and compared to results derived using currently available prenylation prediction methods. In silico predictions were further evaluated using in vivo methods coupled to two yeast reporters, the yeast mating pheromone a-factor and Hsp40 Ydj1p, that represent proteins with canonical and shunted CaaX motifs, respectively. Our machine learning-based approach expands the repertoire of predicted FTase targets and provides a framework for functional classification.

The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens.

A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.

A Mathematical Model of In Vitro Cellular Uptake of Zoledronic Acid and Isopentenyl Pyrophosphate Accumulation.

The mevalonate pathway is an attractive target for many areas of research, such as autoimmune disorders, atherosclerosis, Alzheimer’s disease and cancer. Indeed, manipulating this pathway results in the alteration of malignant cell growth with promising therapeutic potential. There are several pharmacological options to block the mevalonate pathway in cancer cells, one of which is zoledronic acid (ZA) (an N-bisphosphonate (N-BP)), which inhibits the farnesyl pyrophosphate (FPP) synthase enzyme, inducing cell cycle arrest, apoptosis, inhibition of protein prenylation, and cholesterol reduction, as well as leading to the accumulation of isopentenyl pyrophosphate (IPP). We extrapolated the data based on two independently published papers that provide numerical data on the uptake of zoledronic acid (ZA) and the accumulation of IPP (Ag) and its isomer over time by using in vitro human cell line models. Two different mathematical models for IPP kinetics are proposed. The first model (Model 1) is a simpler ordinary differential equation (ODE) compartmental system composed of 3 equations with 10 parameters; the second model (Model 2) is a differential algebraic equation (DAE) system with 4 differential equations, 1 algebraic equation and 13 parameters incorporating the formation of the ZA+enzyme+Ag complex. Each of the two models aims to describe two different experimental situations (continuous and pulse experiments) with the same ZA kinetics. Both models fit the collected data very well. With Model 1, we obtained a prevision accumulation of IPP after 24 h of 169.6 pmol/mgprot/h with an IPP decreasing rate per (pmol/mgprot) of ZA () equal to 13.24/h. With Model 2, we have comprehensive kinetics of IPP upon ZA treatment. We calculate that the IPP concentration was equal to 141.6 pmol/mgprot/h with a decreasing rate/percentage of 0.051 (). The present study is the first to quantify the influence of ZA on the pharmacodynamics of IPP. While still incorporating a small number of parameters, Model 2 better represents the complexity of the biological behaviour for calculating the IPP produced in different situations, such as studies on T cell-based immunotherapy. In the future, additional clinical studies are warranted to further evaluate and fine-tune dosing approaches.

Tumor-Suppressive Effect of Metformin via the Regulation of M2 Macrophages and Myeloid-Derived Suppressor Cells in the Tumor Microenvironment of Colorectal Cancer.

Simple SummaryThe tumor suppressing effect of metformin has been reported, and tumor microenvironment including immune cells contribute to tumor progression in colorectal cancer. However, the effects of metformin on tumor-promoting MDSCs and M2 macrophages and its mechanisms remain unclarified. Here, we demonstrated that metformin-induced activation of AMPK and subsequent mTOR inhibition decreased the MDSC and M2 macrophage fractions by downregulating the mevalonate pathway. Metformin may be a valuable drug for potential CRC prevention and treatment strategies by regulating the immune cells of the tumor microenvironment and tumor cells.Myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the tumor microenvironment contribute to tumor progression by inducing immune tolerance to tumor antigens and cancer cells. Metformin, one of the most common diabetes drugs, has shown anti-inflammatory and anti-tumor effects. However, the effects of metformin on inflammatory cells of the tumor microenvironment and its underlying mechanisms remain unclarified. In this study, we investigated the effect of metformin on M2 macrophages and MDSCs using monocyte THP-1 cells and a dextran sodium sulfate (DSS)-treated ApcMin/+ mouse model of colon cancer. Metformin decreased the fractions of MDSCs expressing CD33 and arginase, as well as M2 macrophages expressing CD206 and CD163. The inhibitory effect of metformin and rapamycin on MDSCs and M2 macrophages was reversed by the co-treatment of Compound C (an AMP-activated protein kinase (AMPK) inhibitor) or mevalonate. To examine the effect of protein prenylation and cholesterol synthesis (the final steps of the mevalonate pathway) on the MDSC and M2 macrophage populations, we used respective inhibitors (YM53601; SQLE inhibitor, FTI-277; farnesyl transferase inhibitor, GGTI-298; geranylgeranyl transferase inhibitor) and found that the MDSC and M2 populations were suppressed by the protein prenylation inhibitors. In the DSS-treated ApcMin/+ mouse colon cancer model, metformin reduced the number and volume of colorectal tumors with decreased populations of MDSCs and M2 macrophages in the tumor microenvironment. In conclusion, the inhibitory effect of metformin on MDSCs and M2 macrophages in the tumor microenvironment of colon cancers is mediated by AMPK activation and subsequent mTOR inhibition, leading to the downregulation of the mevalonate pathway.

Statins reduce mortality of patients with myelodysplastic syndromes.

e19063 Background: Statins can inhibit cholesterol synthesis through inhibition of mevalonic acid synthesis, and reduce prenylation of proteins involved in tumor growth. Their anti-neoplastic potential has been explored for many malignancies (e.g., multiple myeloma) with improvement in progression free- and overall- survival. Preclinical data support anti-neoplastic activity of statins for myeloid malignancies. In this study, we examined the effect of statins on mortality of patients with myelodysplastic syndromes (MDS) in this nationwide retrospective cohort study. Methods: We identified patients diagnosed with MDS through ICD-O-03 codes in the SEER-Medicare database between 2007 and 2017. Statins users were required to have at least one claim for a statin prescription within 6 months prior to MDS diagnosis. Propensity score matching was done to identify non-statin users with a similar cardiovascular risk profile. We analyzed the effect of statins on mortality (from any cause) in a Cox proportional hazards model that adjusted for demographics, SEER-Medicare MDS Risk Score ( SMMRS comprises of age, comorbidities, MDS histological subtype, cytopenias, transfusion-dependence and hospital admissions), treatments for lower risk MDS (growth factors or lenalidomide), and higher risk MDS (hypomethylating agents/stem cell transplant). Results: Our cohort of 6878 MDS patients (3439 statin users and 3439 non-statin users) had a median age of 80 years at diagnosis, and most were Caucasians. Non-statin users were more likely to have higher risk MDS (by SMMRS) at diagnosis. Statins users had a 33% {95% Confidence Interval (CI) 25%-40%; p <.001} lower risk of death than non-statin users. The beneficial effect of statins on mortality diminished over time (Table). Sub-group analysis (data not shown) showed a significant reduction in mortality with statin use in both patients with lower risk {low or intermediate-1 risk: Adjusted hazards ratio (aHR) 0.72; 95% CI 0.58-0.90; p = 0.003)} and higher risk {intermediate-2 or high-risk: aHR 0.67; 95% CI 0.59-0.76; p < 0.001)} MDS (categorized by SMMRS). Conclusions: Use of statins at time of MDS diagnosis is associated with reduced mortality among these patients. The beneficial effect of statins on death from cardiovascular events and myeloid malignancy should be further explored in clinical trials enrolling MDS patients.[Table: see text]

Targeting of Mevalonate-Isoprenoid Pathway in Acute Myeloid Leukemia Cells by Bisphosphonate Drugs

Metabolic reprogramming represents a hallmark of tumorigenesis to sustain survival in harsh conditions, rapid growth and metastasis in order to resist to cancer therapies. These metabolic alterations involve glucose metabolism, known as the Warburg effect, increased glutaminolysis and enhanced amino acid and lipid metabolism, especially the cholesterol biosynthesis pathway known as the mevalonate pathway and these are upregulated in several cancer types, including acute myeloid leukemia (AML). In particular, it was demonstrated that the mevalonate pathway has a pivotal role in cellular transformation. Therefore, targeting this biochemical process with drugs such as statins represents a promising therapeutic strategy to be combined with other anticancer treatments. In the last decade, several studies have revealed that amino-bisphosphonates (BP), primarily used for bone fragility disorders, also exhibit potential anti-cancer activity in leukemic cells, as well as in patients with symptomatic multiple myeloma. Indeed, these compounds inhibit the farnesyl pyrophosphate synthase, a key enzyme in the mevalonate pathway, reducing isoprenoid formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This, in turn, inhibits the prenylation of small Guanosine Triphosphate-binding proteins, such as Ras, Rho, Rac, Rab, which are essential for regulating cell survival membrane ruffling and trafficking, interfering with cancer key signaling events involved in clonal expansion and maturation block of progenitor cells in myeloid hematological malignancies. Thus, in this review, we discuss the recent advancements about bisphosphonates’ effects, especially zoledronate, analyzing the biochemical mechanisms and anti-tumor effects on AML model systems. Future studies will be oriented to investigate the clinical relevance and significance of BP treatment in AML, representing an attractive therapeutic strategy that could be integrated into chemotherapy.

Protein Prenyltransferases and Their Inhibitors: Structural and Functional Characterization.

Protein prenylation is a post-translational modification controlling the localization, activity, and protein–protein interactions of small GTPases, including the Ras superfamily. This covalent attachment of either a farnesyl (15 carbon) or a geranylgeranyl (20 carbon) isoprenoid group is catalyzed by four prenyltransferases, namely farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase-I), Rab geranylgeranyltransferase (GGTase-II), and recently discovered geranylgeranyltransferase type III (GGTase-III). Blocking small GTPase activity, namely inhibiting prenyltransferases, has been proposed as a potential disease treatment method. Inhibitors of prenyltransferase have resulted in substantial therapeutic benefits in various diseases, such as cancer, neurological disorders, and viral and parasitic infections. In this review, we overview the structure of FTase, GGTase-I, GGTase-II, and GGTase-III and summarize the current status of research on their inhibitors.

Choroideremia: molecular mechanisms and therapies.

Choroideremia (CHM) is a monogenic X-linked chorioretinal dystrophy affecting the photoreceptors, retinal pigment epithelium (RPE), and choroid; it is caused by mutations involving the CHM gene. CHM is characterized by night blindness in early childhood, progressing to peripheral visual field loss and eventually to complete blindness from middle age. CHM encodes the ubiquitously expressed Rab escort protein 1 (REP1), which is responsible for prenylation of Rab proteins and is essential for intracellular trafficking of vesicles. In this review we explore the role of REP1 in the retina and its newly discovered systemic manifestations, and discuss the therapeutic strategies for tackling this disease, including the outcomes from recent clinical trials.

Molecular basis of sequence‐dependent differential prenylation inhibition and statin sensitivity of G proteins

Prenylation is a universal and irreversible post‐translational modification occurring on proteins involved in various cellular processes, including survival, proliferation, and migration. The prenylation process consists of enzymatically catalyzed covalent attachment of an isoprenyl lipid, either a 15‐C farnesyl or 20‐C geranylgeranyl moiety, to the Cys residue located in the four residues from the CT in the target protein via a thioether linkage. Proteins undergoing prenylation share a C‐terminally conserved consensus recognition CaaX motif, of which X determines the type of prenylation on the protein. The mevalonate (HMG‐CoA reductase) pathway synthesizes lipids for protein prenylation. The rate‐limiting enzyme; HMG‐CoA reductase of the mevalonate pathway is the primary target for cholesterol‐lowering medications and anticancer drug development. Our previous study shows that clinically used mevalonate pathway inhibitors, statins not only significantly disrupt PM localization of Gβγ but also perturb GPCR‐G protein signaling and associated cell behaviors in a Gγ subtype‐dependent manner. However, in the present work, we identified crucial residues on Gγ that regulate the efficacy of its prenylation and during post‐prenylations processing. Therefore, our findings demonstrate the molecular underpinnings of differential prenylation sensitivities of G proteins under cholesterol synthesis inhibition and hypolipidemic conditions.