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Department of Lipid Biochemistry

Head: Professor Ewa Swieżewska

The research conducted in our department is focused on the evaluation of the role of lipid components in the cell. There are two research groups at the department. Group supervised by dr Swiezewska is working on various aspects of biochemistry and structure of isoprenoid lipids in plants and mammals. Group supervised by dr Szkopinska investigates the effects of statins on specific classes of lipids in correlation with the mutations in human HMGR gene using as a model a yeast strain with deletions of both HMG1 and HMG2 genes with introduced wild-type or mutant form of human HMGR gene. Other subject are lipid droplets and their proteins. Third field of interest is the methylotrophic yeast Pichia pastoris an expression system for production of proteins of interest.
The main results obtained by the first group are as follows: compilation of a spatial model of dolichol biosynthesis in the root cells, discovery of a new type of α-trans polyprenols in plant leaves, molecular characterization of Rab Escort Protein - an accessory subunit of Rab geranylgaranyltransferase of Arabidopsis, and indication of the protective role exerted by polyisoprenoid alcohols in plant upon biotic stress. The main results obtained by second group are as follows: discovery that the level of egosterol synthesis after statint treatment depends on the type of the mutation in the hHMGR gene, localization to lipid droplet membrane Nus1p protein encoded by NUS1 gene deletion of which is lethal, modification of culturing protocol of Pichia pastoris to increase the amount of secreted model proteins.

 




The occurrence and role of prenyl lipids and prenylated proteins in plant tissues. Studies on the structure and biosynthesis. 

Group leader: Prof. Ewa Świeżewska

Staff:Professor em.: Tadeusz Chojnacki, Ph.D.,

Post-doctoral fellows: Dr. Karolina Skorupińska-Tudek, Dr. Liliana Surmacz, Dr. Agnieszka Witek

Researcher: Dr. Wiesław A. Jankowski

Ph.D. students: M.Sc. Ewa Ciepichał, M.Sc. Katarzyna Gawarecka, M.Sc. Adam Jóźwiak, M.Sc. Magdalena Wojtas

Undergraduate student: Łukasz Woźniak

Lab manager: Józefina Hertel

Former group members: Vo Si Hung, Małgorzata Wanke, Małgorzata Gutkowska, Agnieszka Bajda, Monika Zelman-Femiak, Izabela Szostkiewicz, Julita Terebińska, Julita Sternik


Summary of the recent results:
Complete assignment of 1H and 13C signals in spectra of Prenol-10 with 3D NMR spectroscopy was described [2].

Contribution of the MVA and MEP pathways to the dolichol biosynthesis in plant roots was estimated. ω-terminal isoprenoid units were synthesized with the involvement of both pathways while α-terminal ones were derived exclusively from the MVA pathway. A spatial model of dolichol biosynthesis was compiled indicating plastid-cytoplasm transport of intermediates [6, 8].

Polyisoprenoid alcohols together with plastoquinone were shown to be involved in the resistance response of tobacco plants against Tobacco Mosaic Virus infection, both locally and systemically [3, 11].

Increased content of dolichol was detected in the human lens with different types of cataracts [1].

The occurrence of α-trans polyprenols was detected in the leaves of several plants [14, 16]. Different effects of α-cis and -trans isomers on physical properties of model membranes was noted.

Lipofecting activity of heptaprenyltrimetylammonium iodide, a new cationic polyprenyl derivative, was described [17 and patent application].

Accumulation of polyisoprenoid alcohols in plant tissues cultivated in vitro was detected [19].

Structure of oxidation products of polyisoprenoid alcohols with singlet oxygen were analyzed, and formation of epoxides and epoxialdehydes was detected.

Miss-localization of non-geranylgeranylated Rab proteins in Arabidopsis insertion mutant devoid of functional RabGGTase was established together with the decreased resistance of this plant to salinity. Upon in vitro assay significantly decreased activity of RabGGTase was also shown in this mutant.

A stimulating effect of polyisoprenoid epoxides on biosynthesis of ubiquinone in mammalian cells was described [7 and patent application]. Investigation of ubiquinone biosynthetic system in mammals was performed [9].

‘Collection of Polyprenols’ supplied numerous laboratories with polyisoprenoids and their derivatives.


Current projects:

• Studies on lipofectional properties of polyisoprenoid derivatives
• Mechanisms of early steps of isoprenoid biosynthesis in plants
• Occurrence, structure and biological role of polyisoprenoid lipids in plants and animals
• Chemical oxidation of polyisoprenoids and role of oxidative products
• Plant Rab GGTase - role of REP subunit
• Factors regulation metabolism of ubiquinone in mammalian cells

 

Research grants:
2006-2009 "The role of protein prenylation and prenylated lipids in the vesicular transport in plants",
                 (Ministry of Science and Higher Education)

2004-2008 "The involvement of prenylated lipids in the defence mechanism against biotic and abiotic stress in plant cells",
                 (Ministry of Science and Higher Education)

2006-2007 "Genetics of coenzyme Q deficiency in humans", (18/PRUE/2006/7, Ministry of Science and Higher Education)

2005-2007 "Genetics of coenzyme Q deficiency in humans", (LSHB-CT-2004-00-5151, EU FP6)

 

Patents and patent aplications:
Patent: Chojnacki T., Świeżewska E.
"A method of preparation of tritium labeled ubiquinones and their structural analogues" P364116, Urząd Patentowy RP, 29.09.2008

Patent application: "Polyisoprenoid epoxides useful for decreasing cholesterol and/or increasing Coenzyme Q biosynthesis" No EP08380055.7, (2007)

Patent application: Chojnacki T., Ciepichał E., Jankowski W., Świeżewska E., Chmielewski M., Łysek R.,  Masnyk M., Madeja Z. (2007) w Polskim Urzędzie Patentowym
"Trimetyloaminowe pochodne poli-cis i poli-trans liniowych oligomerów izoprenowych, sposób ich wytwarzania oraz ich zastosowanie" nr PL 382488


Selected publications:

  1. Gajjar  D,  Jozwiak A, Swiezewska E, Alapure B, Parmar T, Johar K, Vasavada AR. (2009) Quantification of dolichol in the human lens with different types of cataracts. Molecular Vision 15: 1573-1579.
  2. Misiak M, Koźmiński, W, Kwasiborska M, Wójcik J, Ciepichał E, Swiezewska E. (2009) Complete 1H and 13C spectral assignment of prenol-10 with 3D NMR spectroscopy Magn. Resonance Chem 28: 167-175.
  3. Bajda A, Konopka-Postupolska D, Krzymowska M, Hennig J, Skorupinska-Tudek K, Surmacz L, Wojcik J, Matysiak Z, Chojnacki T, Skorzynska-Polit E, Drazkiewicz M, Patrzylas P, Tomaszewska M,  Kania M,  Swist M, Danikiewicz W, Piotrowska W, Swiezewska E. (2009)  Role of polyisoprenoids in tobacco resistance against biotic stresses. Physiol. Plantarum 135: 351 – 364.
  4. Liav A, Ciepichal E, Swiezewska E, Bobovská A, Dianišková P, Blaško J, Mikušová KJ, Brennan PJ. (2009) Stereoselective syntheses of heptaprenylphosphoryl beta-D-arabino-and beta-D-ribo-furanoses. Tetrahedron Lett. 50: 2242-2244.
  5. Lewandowska M, Bajda A, Swiezewska E, Sirko A. Influence of short term sulfur starvation on photosynthesis-related compounds and processes in tobacco. In: Sulfur metabolism in plants. Regulatory aspects, significance of sulfur in the food chain, agriculture and the environment. eds. A. Sirko, L.J. De Kok, S. Haneklaus, M.J. Hawkesford, H. Rennenberg, K. Saito, E. Schnug and I. Stulen. Backhuys Publishers, Leiden, Margraf Publishers GmbH, Weikersheim 2009, pp. 79-83.
  6. Skorupinska-Tudek K, Poznanski J, Wojcik J, Bienkowski T, Szostkiewicz I, Zelman-Femiak M, Bajda A, Chojnacki T, Olszowska O, Grunler J, Meyer O, Rohmer M, Danikiewicz W, Swiezewska E (2008) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants. J. Biol. Chem. 283: 21024-21035.
  7. Bentinger M, Tekle M, Brismar K, Chojnacki T, Swiezewska E, Dallner G (2008) Polyisoprenoid epoxides stimulate biosynthesis of CoQ and inhibit cholesterol synthesis. J. Biol. Chem. 283: 14645-14653
  8. Skorupinska-Tudek K, Wojcik J, Swiezewska E. (2008) Polyisoprenoid alcohols – recent results of structural studies. Chemical Records 8: 33 – 45.
  9. Tekle M, Turunen M, Dallner G, Chojnacki T, Swiezewska E. (2008) Investigation of coenzyme Q biosynthetic system in human fibroblast and hepatoblastoma cells. J Biochem Biophys Methods, 70: 909-917.
  10. Maltsev S, Sizova O, Utkina N, Shibaev V, Chojnacki T, Jankowski W, Swiezewska E Prenyl sulfates as alkylating reagents for mercapto amino acids. (2008) Acta Biochim. Pol. 55: 807-815.
  11. Kruk J, Szymanska R, Strzalka K, Bajda A, Swiezewska E, Chojnacki T. (2008) Terpenoids. In Research Methods in Plant Sciences: Allelopathy. Ed R. Bogatek-Leszczynska, in press
  12. Chen MM, Weerapana E, Ciepichal E, Stupak J, Reid CW, Swiezewska E, Imperiali B. (2007) Polyisoprenol specificity in the Campylobacter jejuni N-Linked g lycosylation pathway. Biochemistry 46: 14342-14348.
  13. Chojnacki T, Bajda A, Ciepichal E, Hertel J, Swiezewska E, Behl HM, Sidhu OP. (2007) The search for plant polyprenols in the arboretum of the National Botanical Garden Research Institute in Lucknow (U.P. India) in Monographs of Botanical Gardens. Eds. J. Rybczynski and J. Puchalski. vol. 1, pp.147-153
  14. Ciepichal, E., Wojcik J., Bienkowski T., Swist M., Danikiewicz W., Marczewski A., Hertel J., Matysiak Z., Swiezewska E., Chojnacki T. (2007) Alloprenols: Novel α-trans-polyprenols of Allophylus caudatus. Chem.Phys.Lipids, 147: 103-112.
  15. Grinco M, Kulcicki V, Jankowski W, Chojnacki T, Vlad PF. (2007) Superacid-catalyzed cyclization of methyl (6Z)-geranylfarnesoates. Helvetica Chimica Acta 90 1223-1229.
  16. Liav A, Swiezewska E, Ciepichal E, Brennan PJ. (2006) Stereoselectivity in the synthesis of polyprenylphosphoryl β-D-ribofuranoses. Tetrahedron Lett. 47: 8781-8783.
  17. Madeja Z, Rak M, Wybieralska E, Różański I, Masnyk M, Chmielewski M, Łysek R, Chojnacki T, Jankowski W, Ciepichal E, Swiezewska E, Tekle M, Dallner G. (2007) New cationic polyprenyl derivative proposed as lipofecting agent. Acta Biochim. Pol. 54: 873-876.
  18. Marczewski A, Ciepichal E, Le Xuan Canh , Tran The Bach, Swiezewska E, Chojnacki T. (2007) The search for polyprenols in dendroflora of Vietnam. Acta Biochim. Pol. 54: 727 – 732.
  19. Skorupinska-Tudek K, Pytelewska A, Zelman-Femiak M, Mikoszewski J, Olszowska O, Gajdzis-Kuls D, Urbanska N, Syklowska-Baranek K, Hertel J, Chojnacki T, Swiezewska E. (2007) In vitro plant tissue cultures accumulate polyisoprenoid alcohols. Acta Biochim. Pol. 54: 847 – 852.
  20. van Daum V, Sijbrandi R, Kol M, Swiezewska E, de Kruijff B, Breukink E. (2007) Transmembrane transport of peptidoglycan precursors across model and bacterial membranes. Molecular Microbiology 64: 1105–1114.



Regulation of the biosynthesis of mevalonate-pathway derivatives in yeast Saccharomyces cerevisiae

Group leader: Assoc. Prof. Anna Szkopińska

Staff: Dr. Barbara Kłudkiewicz

Yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways thus conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Our model system is a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene. We investigate the effects of statins  on specific classes of lipids in correlation with the type of mutation in human HMGR gene. This could allow individual treatment of a patient with type and quantity of administered statins. The research is carried out in collaboration with Dr Beata Burzyńska, head of  Project “Genetic analysis of some hereditary human diseases”.

The methylotrophic yeast Pichia pastoris is an expression system for production of proteins of interest. The modified culturing protocol may lead to much higher amounts of secreted glycoproteins, which is of importance for economical reasons. The optimization of model proteins: SPI (silk proteins inhibitor) and USP (ultraspiracle, nuclear receptor) expression and secretion is worked on.

Two yeast cis-prenyltransferases Rer2p and Srt1p synthesize polyprenols which after saturation of α-terminal isoprene residue participate as dolichols in the synthesis of N- and O-glycosidically linked oligosaccharide chains of proteins and the formation of glycosyl-phosphoinosytol membrane (GPI) anchors, and are the constituents of biological membranes. Using TAP tagging method the search for proteins interacting with a yeast cis-prenyltransferase Rer2p has been done. MS analysis of proteins  indicated Nus1p (nuclear undecaprenyl pyrophosphate synthase, putative prenyltransferase) encoded by NUS1 gene, deletion of which is lethal, as a potential candidate. The investigation on the biological role of Nus1p  is in progress. The research is carried out in collaboration with Dr Bożenna Rempoła head of Project “Functional analysis of open reading frames of Saccharomyces cerevisiae”.

 

Selected publications:

  1. Kaliszewki P., Szkopinska A., Ferreira T., Swiezewska E., Berges T., Zoladek T. Rsp5p ubiquitin ligaseand the transcriptional activators Spt23p and Mga2p are involved in co-regulation of biosynthesis of end products of the mevalonate pathway and triacylglycerol in yeast Saccharomyces cerevisiae. BBA - Molecular and Cell Biology of Lipids (2008) 1781: 627-634
  2. Poznański J., Szkopinska A. Precise bacterial polyprenol length control fails in Saccharomyces cerevisiae. Biopolymers (2007) 86: 155-164
  3. Szkopinska A., Swiezewska E., Rytka J. Interplay between the cis-prenyltransferases and polyprenol reductase in the yeast Saccharomyces cerevisiae Biochimie (2006) 88: 271-276
  4. Kaliszewski P., Ferrira T., Gajewska B., Szkopinska A., Berges T., Zoladek T. Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the grpwth of Sachcaromyces cerevisiae cells deficient in Rsp5 ubiquitin ligase. Biochem J. (2006) 395: 173-81
  5. Szkopinska A., Swiezewska E., Karst F. The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy3-methylglutaryl-CoA reductase and Squalene synthase in yeast Saccharomyces cerevisiae. Biochi. Biophys. Res. Commun. (2000) 267: 473-477