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Cloning, Purification, and Characterization of the Catalytic C-Terminal Domain of the Human 3-Hydroxy-3-methyl glutaryl-CoA Reductase: An Effective, Fast, and Easy Method for Testing Hypocholesterolemic Compounds

  • Rosita Curcio
  • Donatella Aiello
  • Angelo Vozza
  • Luigina Muto
  • Emanuela Martello
  • Anna Rita Cappello
  • Loredana Capobianco
  • Giuseppe Fiermonte
  • Carlo Siciliano
  • Anna NapoliEmail author
  • Vincenza DolceEmail author
Original Paper
  • 50 Downloads

Abstract

3-hydroxy-3-methyl glutaryl-CoA reductase, also known as HMGR, plays a crucial role in regulating cholesterol biosynthesis and represents the main pharmacological target of statins. In mammals, this enzyme localizes to the endoplasmic reticulum membrane. HMGR includes different regions, an integral N-terminal domain connected by a linker-region to a cytosolic C-terminal domain, the latter being responsible for enzymatic activity. The aim of this work was to design a simple strategy for cloning, expression, and purification of the catalytic C-terminal domain of the human HMGR (cf-HMGR), in order to spectrophotometrically test its enzymatic activity. The recombinant cf-HMGR protein was heterologously expressed in Escherichia coli, purified by Ni+-agarose affinity chromatography and reconstituted in its active form. MALDI mass spectrometry was adopted to monitor purification procedure as a technique orthogonal to the classical Western blot analysis. Protein identity was validated by MS and MS/MS analysis, confirming about 82% of the recombinant sequence. The specific activity of the purified and dialyzed cf-HMGR preparation was enriched about 85-fold with respect to the supernatant obtained from cell lysate. The effective, cheap, and easy method here described could be useful for screening statin-like molecules, so simplifying the search for new drugs with hypocholesterolemic effects.

Keywords

HMGR Bacterial expression Affinity chromatography MALDI MS and MS/MS Enzymatic activity Screening of statin-like molecules 

Notes

Acknowledgements

This work was supported by Associazione Italiana per la Ricerca sul Cancro (FG Grant No. 15404/2014) and by Ministero Italiano della Ricerca e dell’Università (MIUR–PRIN 2015, Prot. 201545245K_002).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12033_2019_230_MOESM1_ESM.docx (52 kb)
Supplementary material 1 (DOCX 51 kb)

References

  1. 1.
    Goldstein, J. L., & Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature,343, 425–430.CrossRefGoogle Scholar
  2. 2.
    Baigent, C., Keech, A., Kearney, P. M., Blackwell, L., Buck, G., Pollicino, C., et al. (2005). Efficacy and safety of cholesterol-lowering treatment: Prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet,366, 1267–1278.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Brown, M. S., & Goldstein, J. L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science,232, 34–47.CrossRefGoogle Scholar
  4. 4.
    Stancu, C., & Sima, A. (2001). Statins: Mechanism of action and effects. Journal of Cellular and Molecular Medicine,5, 378–387.PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Dolce, V., Cappello, A. R., Lappano, R., & Maggiolini, M. (2011). Glycerophospholipid synthesis as a novel drug target against cancer. Current Molecular Pharmacology,4, 167–175.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Wang, X., Sato, R., Brown, M. S., Hua, X., & Goldstein, J. L. (1994). SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell,77, 53–62.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Tsai, N. W., Lee, L. H., Huang, C. R., Chang, W. N., Chang, Y. T., Su, Y. J., et al. (2014). Statin therapy reduces oxidized low density lipoprotein level, a risk factor for stroke outcome. Critical Care,18, R16.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Li, H., Horke, S., & Forstermann, U. (2013). Oxidative stress in vascular disease and its pharmacological prevention. Trends in Pharmacological Sciences,34, 313–319.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Liao, J. K., & Laufs, U. (2005). Pleiotropic effects of statins. Annual Review of Pharmacology and Toxicology,45, 89–118.PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Saeedi Saravi, S. S., Saeedi Saravi, S. S., Arefidoust, A., & Dehpour, A. R. (2017). The beneficial effects of HMG-CoA reductase inhibitors in the processes of neurodegeneration. Metabolic Brain Disease,32, 949–965.PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Hamada, M., Sugimoto, M., Matsui, H., Mizuno, T., Shida, Y., Doi, M., et al. (2011). Antithrombotic properties of pravastatin reducing intra-thrombus fibrin deposition under high shear blood flow conditions. Thrombosis and Haemostasis,105, 313–320.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Iannelli, F., Lombardi, R., Milone, M. R., Pucci, B., De Rienzo, S., Budillon, A., et al. (2018). Targeting mevalonate pathway in cancer treatment: Repurposing of statins. Recent Patents on Anti-cancer Drug Discovery,13, 184–200.PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Fiorillo, M., Peiris-Pages, M., Sanchez-Alvarez, R., Bartella, L., Di Donna, L., Dolce, V., et al. (2018). Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism. Biochimica et Biophysica Acta,1859, 984–996.PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Safwat, S., Ishak, R. A., Hathout, R. M., & Mortada, N. D. (2017). Statins anticancer targeted delivery systems: Re-purposing an old molecule. Journal of Pharmacy and Pharmacology,69, 613–624.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Istvan, E. S., & Deisenhofer, J. (2001). Structural mechanism for statin inhibition of HMG-CoA reductase. Science,292, 1160–1164.PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Istvan, E. S., Palnitkar, M., Buchanan, S. K., & Deisenhofer, J. (2000). Crystal structure of the catalytic portion of human HMG-CoA reductase: Insights into regulation of activity and catalysis. The EMBO Journal,19, 819–830.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Luskey, K. L., & Stevens, B. (1985). Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation. The Journal of Biological Chemistry,260, 10271–10277.PubMedPubMedCentralGoogle Scholar
  18. 18.
    du Souich, P., Roederer, G., & Dufour, R. (2017). Myotoxicity of statins: Mechanism of action. Pharmacology & Therapeutics,175, 1–16.CrossRefGoogle Scholar
  19. 19.
    Malachowski, S. J., Quattlebaum, A. M., & Miladinovic, B. (2017). Adverse effects of statins. JAMA,317, 1079–1080.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Alsheikh-Ali, A. A., & Karas, R. H. (2009). The relationship of statins to rhabdomyolysis, malignancy, and hepatic toxicity: Evidence from clinical trials. Current Atherosclerosis Reports,11, 100–104.PubMedCrossRefGoogle Scholar
  21. 21.
    Karalis, D. G. (2014). Achieving optimal lipid goals in the metabolic syndrome: A global health problem. Atherosclerosis,237, 191–193.PubMedCrossRefGoogle Scholar
  22. 22.
    Joy, T. R., & Hegele, R. A. (2009). Narrative review: Statin-related myopathy. Annals of Internal Medicine,150, 858–868.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Mollace, V., Sacco, I., Janda, E., Malara, C., Ventrice, D., Colica, C., et al. (2011). Hypolipemic and hypoglycaemic activity of bergamot polyphenols: From animal models to human studies. Fitoterapia,82, 309–316.PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Gorinstein, S., Leontowicz, H., Leontowicz, M., Krzeminski, R., Gralak, M., Martin-Belloso, O., et al. (2004). Fresh Israeli Jaffa blond (Shamouti) orange and Israeli Jaffa red Star Ruby (Sunrise) grapefruit juices affect plasma lipid metabolism and antioxidant capacity in rats fed added cholesterol. Journal of Agricultural and Food Chemistry,52, 4853–4859.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Cappello, A. R., Dolce, V., Iacopetta, D., Martello, M., Fiorillo, M., Curcio, R., et al. (2016). Bergamot (Citrus bergamia Risso) flavonoids and their potential benefits in human hyperlipidemia and atherosclerosis: An overview. Mini Reviews in Medicinal Chemistry,16, 619–629.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Di Donna, L., Iacopetta, D., Cappello, A. R., Gallucci, G., Martello, E., Fiorillo, M., et al. (2014). Hypocholesterolaemic activity of 3-hydroxy-3-methyl-glutaryl flavanones enriched fraction from bergamot fruit (Citrus bergamia): ‘‘In vivo’’ studies. Journal of Functional Foods,7, 558–568.CrossRefGoogle Scholar
  27. 27.
    Gopal, G. J., & Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. Protein Journal,32, 419–425.PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Snijder, H. J., & Hakulinen, J. (2016). Membrane protein production in E. coli for applications in drug discovery. Advances in Experimental Medicine and Biology,896, 59–77.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Hartley, J. L. (2006). Cloning technologies for protein expression and purification. Current Opinion in Biotechnology,17, 359–366.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Madeo, M., Carrisi, C., Iacopetta, D., Capobianco, L., Cappello, A. R., Bucci, C., et al. (2009). Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells. Journal of Bioenergetics and Biomembranes,41, 289–297.PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Salplachta, J., Rehulka, P., & Chmelik, J. (2004). Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digests. Journal of Mass Spectrometry,39, 1395–1401.PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Di Donna, L., Taverna, D., Indelicato, S., Napoli, A., Sindona, G., & Mazzotti, F. (2017). Rapid assay of resveratrol in red wine by paper spray tandem mass spectrometry and isotope dilution. Food Chemistry,229, 354–357.PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Persike, M., & Karas, M. (2009). Rapid simultaneous quantitative determination of different small pharmaceutical drugs using a conventional matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system. Rapid Communications in Mass Spectrometry,23, 3555–3562.PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Persike, M., Zimmermann, M., Klein, J., & Karas, M. (2010). Quantitative determination of acetylcholine and choline in microdialysis samples by MALDI-TOF MS. Analytical Chemistry,82, 922–929.PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Di Donna, L., Benabdelkamel, H., Taverna, D., Indelicato, S., Aiello, D., Napoli, A., et al. (2015). Determination of ketosteroid hormones in meat by liquid chromatography tandem mass spectrometry and derivatization chemistry. Analytical and Bioanalytical Chemistry,407, 5835–5842.PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Aiello, D., Cardiano, P., Cigala, R. M., Gans, P., Giacobello, F., Giuffre, O., et al. (2017). Sequestering ability of oligophosphate ligands toward Al3+ in aqueous solution. Journal of Chemical and Engineering Data,62, 3981–3990.CrossRefGoogle Scholar
  37. 37.
    Aiello, D., Furia, E., Siciliano, C., Bongiorno, D., & Napoli, A. (2018). Study of the coordination of ortho-tyrosine and trans-4-hydroxyproline with aluminum(III) and iron(III). Journal of Molecular Liquids,269, 387–397.CrossRefGoogle Scholar
  38. 38.
    Aiello, D., Casadonte, F., Terracciano, R., Damiano, R., Savino, R., Sindona, G., et al. (2016). Targeted proteomic approach in prostatic tissue: A panel of potential biomarkers for cancer detection. Oncoscience,3, 220–241.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Napoli, A., Aiello, D., Aiello, G., Cappello, M. S., Di Donna, L., Mazzotti, F., et al. (2014). Mass spectrometry-based proteomic approach in Oenococcus oeni enological starter. Journal of Proteome Research,13, 2856–2866.PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Aiello, D., Materazzi, S., Risoluti, R., Thangavel, H., Di Donna, L., Mazzotti, F., et al. (2015). A major allergen in rainbow trout (Oncorhynchus mykiss): Complete sequences of parvalbumin by MALDI tandem mass spectrometry. Molecular BioSystems,11, 2373–2382.PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Aiello, D., Siciliano, C., Mazzotti, F., Di Donna, L., Athanassopoulos, C. M., & Napoli, A. (2018). Molecular species fingerprinting and quantitative analysis of saffron (Crocus sativus L.) for quality control by MALDI 13 mass spectrometry. RSC Advance,8, 36104–36113.CrossRefGoogle Scholar
  42. 42.
    Aiello, D., Giambona, A., Leto, F., Passarello, C., Damiani, G., Maggio, A., et al. (2018). Human coelomic fluid investigation: A MS-based analytical approach to prenatal screening. Scientific Reports,8, 10973.PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Li, Y., Cappello, A. R., Muto, L., Martello, E., Madeo, M., Curcio, R., et al. (2018). Functional characterization of the partially purified Sac1p independent adenine nucleotide transport system (ANTS) from yeast endoplasmic reticulum. Journal of Biochemistry,164, 313–322.PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Kussmann, M., & Roepstorff, P. (2000). Sample preparation techniques for peptides and proteins analyzed by MALDI-MS. Methods in Molecular Biology,146, 405–424.PubMedPubMedCentralGoogle Scholar
  45. 45.
    Buchner, J., Pastan, I., & Brinkmann, U. (1992). A method for increasing the yield of properly folded recombinant fusion proteins: Single-chain immunotoxins from renaturation of bacterial inclusion bodies. Analytical Biochemistry,205, 263–270.PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Bonofiglio, D., Santoro, A., Martello, E., Vizza, D., Rovito, D., Cappello, A. R., et al. (2013). Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-gamma on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes. Biochimica et Biophysica Acta,1831, 1027–1036.PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Iacopetta, D., Madeo, M., Tasco, G., Carrisi, C., Curcio, R., Martello, E., et al. (2011). A novel subfamily of mitochondrial dicarboxylate carriers from Drosophila melanogaster: Biochemical and computational studies. Biochimica et Biophysica Acta,1807, 251–261.PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    Curcio, R., Muto, L., Pierri, C. L., Montalto, A., Lauria, G., Onofrio, A., et al. (2016). New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier. Biochimica et Biophysica Acta,1864, 1473–1480.PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Lunetti, P., Cappello, A. R., Marsano, R. M., Pierri, C. L., Carrisi, C., Martello, E., et al. (2013). Mitochondrial glutamate carriers from Drosophila melanogaster: Biochemical, evolutionary and modeling studies. Biochimica et Biophysica Acta,1827, 1245–1255.PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Vozza, A., De Leonardis, F., Paradies, E., De Grassi, A., Pierri, C. L., Parisi, G., et al. (2017). Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster. Biochimica et Biophysica Acta,1858, 137–146.PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Kurauskas, V., Hessel, A., Ma, P., Lunetti, P., Weinhaupl, K., Imbert, L., et al. (2018). How detergent impacts membrane proteins: Atomic-level views of mitochondrial carriers in dodecylphosphocholine. The Journal of Physical Chemistry Letters,9, 933–938.PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Santoro, A., Cappello, A. R., Madeo, M., Martello, E., Iacopetta, D., & Dolce, V. (2011). Interaction of fosfomycin with the glycerol 3-phosphate transporter of Escherichia coli. Biochimica et Biophysica Acta,1810, 1323–1329.PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Bolanos-Garcia, V. M., & Davies, O. R. (2006). Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochimica et Biophysica Acta,1760, 1304–1313.PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Kleinsek, D. A., & Porter, J. W. (1979). An alternate method of purification and properties of rat liver beta-hydroxy-beta-methylglutaryl coenzyme A reductase. The Journal of Biological Chemistry,254, 7591–7599.PubMedPubMedCentralGoogle Scholar
  55. 55.
    Rodwell, V. W., Beach, M. J., Bischoff, K. M., Bochar, D. A., Darnay, B. G., Friesen, J. A., et al. (2000). 3-Hydroxy-3-methylglutaryl-CoA reductase. Methods in Enzymology,324, 259–280.PubMedCrossRefPubMedCentralGoogle Scholar
  56. 56.
    Zara, V., Dolce, V., Capobianco, L., Ferramosca, A., Papatheodorou, P., Rassow, J., et al. (2007). Biogenesis of eel liver citrate carrier (CIC): Negative charges can substitute for positive charges in the presequence. Journal of Molecular Biology,365, 958–967.PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Bonesi, M., Brindisi, M., Armentano, B., Curcio, R., Sicari, V., Loizzo, M. R., et al. (2018). Exploring the anti-proliferative, pro-apoptotic, and antioxidant properties of Santolina corsica Jord. & Fourr. (Asteraceae). Biomedicine & Pharmacotherapy,107, 967–978.CrossRefGoogle Scholar
  58. 58.
    Frattaruolo, L., Carullo, G., Brindisi, M., Mazzotta, S., Bellissimo, L., Rago, V., et al. (2019). Antioxidant and anti-inflammatory activities of flavanones from Glycyrrhiza glabra L. (licorice) leaf phytocomplexes: Identification of licoflavanone as a modulator of NF-kB/MAPK pathway. Antioxidants (Basel),8, 186.CrossRefGoogle Scholar
  59. 59.
    Frimpong, K., Darnay, B. G., & Rodwell, V. W. (1993). Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase expressed in Escherichia coli: Production of homogeneous protein. Protein Expression and Purification,4, 337–344.PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology,49, 66–71.PubMedCrossRefGoogle Scholar
  61. 61.
    Ohto, C., Muramatsu, M., Obata, S., Sakuradani, E., & Shimizu, S. (2009). Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols. Applied Microbiology and Biotechnology,82, 837–845.PubMedCrossRefGoogle Scholar
  62. 62.
    Mayer, R. J., Debouck, C., & Metcalf, B. W. (1988). Purification and properties of the catalytic domain of human 3-hydroxy-3-methylglutaryl-CoA reductase expressed in Escherichia coli. Archives of Biochemistry and Biophysics,267, 110–118.PubMedCrossRefGoogle Scholar
  63. 63.
    Song, A. A., Abdullah, J. O., Abdullah, M. P., Shafee, N., Othman, R., Tan, E. F., et al. (2012). Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production. PLoS ONE,7, e52444.PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Lukacs, G., Papp, T., Somogyvari, F., Csernetics, A., Nyilasi, I., & Vagvolgyi, C. (2009). Cloning of the Rhizomucor miehei 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in Mucor circinelloides. Antonie van Leeuwenhoek,95, 55–64.PubMedCrossRefGoogle Scholar
  65. 65.
    Takahashi, S., Kuzuyama, T., & Seto, H. (1999). Purification, characterization, and cloning of a eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme involved in biosynthesis of terpenoids. Journal of Bacteriology,181, 1256–1263.PubMedPubMedCentralGoogle Scholar
  66. 66.
    Li, J., Xie, Z., Shi, L., Zhao, Z., Hou, J., Chen, X., et al. (2012). Purification, identification and profiling of serum amyloid A proteins from sera of advanced-stage cancer patients. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences,889–890, 3–9.PubMedCrossRefGoogle Scholar
  67. 67.
    Easterling, M. L., Colangelo, C. M., Scott, R. A., & Amster, I. J. (1998). Monitoring protein expression in whole bacterial cells with MALDI time-of-flight mass spectrometry. Analytical Chemistry,70, 2704–2709.PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Amado, F. M. L., Santana-Marques, M. G., Ferrer-Correia, A. J., & Tomer, K. B. (1997). Analysis of peptide and protein samples containing surfactants by MALDI-MS. Analytical Chemistry,69, 1102–1106.CrossRefGoogle Scholar
  69. 69.
    Cohen, S. L., & Chait, B. T. (1996). Influence of matrix solution conditions on the MALDI-MS. Analysis of peptides and proteins. Analytical Chemistry,68, 31–37.PubMedCrossRefPubMedCentralGoogle Scholar
  70. 70.
    Zhou, J., & Lee, T. D. (1995). Charge state distribution shifting of protein ions observed in matrix-assisted laser desorption ionization mass spectrometry. Journal of the American Society for Mass Spectrometry,6, 1183–1189.PubMedCrossRefPubMedCentralGoogle Scholar
  71. 71.
    Frankevich, V., Zhang, J., Dashtiev, M., & Zenobi, R. (2003). Production and fragmentation of multiply charged ions in ‘electron-free’ matrix-assisted laser desorption/ionization. Rapid Communications in Mass Spectrometry,17, 2343–2348.PubMedCrossRefPubMedCentralGoogle Scholar
  72. 72.
    Lehoux, J. G., Kandalaft, N., Belisle, S., & Bellabarba, D. (1985). Characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase in human adrenal cortex. Endocrinology,117, 1462–1468.PubMedCrossRefPubMedCentralGoogle Scholar
  73. 73.
    Bischoff, K. M., & Rodwell, V. W. (1996). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: Purification, characterization, and expression in Escherichia coli. Journal of Bacteriology,178, 19–23.PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Koh, K. K., Sakuma, I., & Quon, M. J. (2011). Differential metabolic effects of distinct statins. Atherosclerosis,215, 1–8.PubMedCrossRefPubMedCentralGoogle Scholar
  75. 75.
    Hosomi, N., Kitagawa, K., Nagai, Y., Nakagawa, Y., Aoki, S., Nezu, T., et al. (2018). Desirable low-density lipoprotein cholesterol levels for preventing stroke recurrence: A post hoc analysis of the J-STARS Study (Japan Statin Treatment Against Recurrent Stroke). Stroke,49, 865–871.PubMedCrossRefPubMedCentralGoogle Scholar
  76. 76.
    Arinze, N., Farber, A., Sachs, T., Patts, G., Kalish, J., Kuhnen, A., et al. (2018). The effect of statin use and intensity on stroke and myocardial infarction after carotid endarterectomy. Journal of Vascular Surgery,68, 1398–1405.PubMedCrossRefPubMedCentralGoogle Scholar
  77. 77.
    Bolego, C., Poli, A., Cignarella, A., Catapano, A. L., & Paoletti, R. (2002). Novel statins: Pharmacological and clinical results. Cardiovascular Drugs and Therapy,16, 251–257.PubMedCrossRefPubMedCentralGoogle Scholar
  78. 78.
    McTaggart, F., Buckett, L., Davidson, R., Holdgate, G., McCormick, A., Schneck, D., et al. (2001). Preclinical and clinical pharmacology of Rosuvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. The American Journal of Cardiology,87, 28B–32B.PubMedCrossRefPubMedCentralGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Pharmacy, Health, and Nutritional SciencesUniversity of CalabriaArcavacata di RendeItaly
  2. 2.Department of Chemistry and Chemical TechnologiesUniversity of CalabriaArcavacata di RendeItaly
  3. 3.Laboratory of Biochemistry and Molecular Biology, Department of Biosciences, Biotechnologies and BiopharmaceuticsUniversity of BariBariItaly
  4. 4.Department of Biological and Environmental Sciences and TechnologiesUniversity of SalentoLecceItaly

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