Molecular biology of gene functions involved in pathological conditions
Group Leader: Reinhold Hofbauer
Postdocs:
PhD Student: Elke Litzlbauer
Diploma students:
Technician: Thomas Sauer (part time)
| Lectures |
* our own results
Cardio myopathy and genetical factors involved in the pathogenesis of the disease One of my basic aims has been the identification of novel cDNA clones, being differentially expressed between two time points in the cell cycle (center of G1 versus early S-phase). I established collection of differentially regulated clones which were the outcome of a comparative screen. Upon closer analysis one of these differentially expressed genes turned out to be the murine carnitine acetyltransferase (CARAT). Our study showed that the CARAT mRNA, which is coding for a key function of the energy metabolism in mitochondria and peroxisomes, is not only strictly regulated by growth but also during the real cell cycle. We have very carefully analyzed the consequences of sense and antisense treatment of cells with oligo nucleotides and single stranded phagemids constructed from the cDNA clone. The inhibition experiments clearly argued for an indispensable cellular function. Which fits to the fact that no deletion of defective mutants are known in the literature regarding this gene function.
Together with Prof.Dr. Alfred Lohninger (Inst. f. Medical Chemistry, University of Vienna), who has accomplished the analytical (GC and MS of L-carnitine and esters in patients and relevant experimental model systems), we concentrated on several questions: the consequences of L-carnitine deficiency as well as supplementation on the gene expression of the carnitine acyl transferases, establishment of cell culture model systems mimicking real physiological conditions (dialysis, hyper-triglyceridemia, erythrocyte differentiation), aging and carnitine metabolism and finally together with clinicians patient pilot studies of carnitine supplementation. In cellular samples we analyzed the key enzyme functions of the carnitine interplay (CARAT, CPT I (liver and muscle form) and CPT 2), by northern blot analysis and developed semi- and quantitative RT-PCR tools. For our in vitro cell culture studies we cultivated human and mouse fibroblast cell lines (MRC-5, NIH-3T3) under specific conditions (increasing carnitine concentrations, different media and sera including standard and dialyzed fetal and calf sera, human umbilical vein sera as well as sera from hemodialysis (HD) patients). We have generated very convincing data that carnitine itself is closely regulating the expression behavior of its metabolizing genes and that there is an almost immediate effect of low carnitine levels in the medium (carnitine deficiency, see fig 1).
Fig. 1: effect of carnitine supplementation on expression levels of CPT I mRNA in mrc-5 cells, cultivated in serum from short and long term HD patients versus control serum
Based on the recombinant CARAT/GST and CPT I/HIS-tag-fusion proteins we currently produce specific antibodies and will establish further immunological tools (ELISA) to complement our studies on mRNA level. To bring more insight into the molecular regulation we have isolated genomic clones of the CARAT and liver CPT I to clarify the promoter of these genes. A whole set of different "normal" physiological conditions, where carnitine deprivation is the normal situation was reconstructed by us in cell culture. In the closer future I plan to complement the analytical tools to put us in a situation to study the carnitine metabolizing genes from mRNA production (promoter activity) to protein synthesis and activity. In parallel to these investigations I want to reveal the genetic link between the carnitine metabolizing genes and those responsible for lipid metabolism and anabolism (e.g. ß-oxidation, phospholipid biosynthesis).
Carnitine deficiency and genetical consequences of its supplementation
In another collaborative research project together with Prof.Dr. S. Aharinejad from the Inst. f. Anatomy of the University of Vienna we investigated endothelin-1 (ET-1), angiopoetin, VEGF, CPT1, CPT2, CARAT, and GAPDH (ß-actin) as internal standard myocardial biopsy samples obtained from both explanted hearts of CMP patients and from the donated transplanted hearts. Cardiomyopathy (CMP) defines a heterogeneous group of diseases, where predominately the heart muscle is damaged. With the exception of the ischemic heart disease, the toxic and post infectious CMP, the primary cause still remains unknown (see table 1). The incidence of the disease is relatively high and a causal therapeutic regimen is still missing. Knowledge on mRNA steady state levels of certain cardiovascular genes would give us a better insight into the ongoing pathophysiological processes involved in CMP. Therefore, we decided to evaluate these levels by semi-quantitative RT-PCR. We sought to find differences in expression levels of certain genes in CMP and donated
Table 1: different forms of CMP and causes of the disease
conditions causing CMP classification of CMP hypertension
ET1*
catecholamines
NOS*
ischemic CMP
dilatative CMP ?toxic factors toxic CMP hypoxia ( O2
VEGF
NOS
ischemic CMP infections infectious CMP
genetic predisposition (?) infectious CMP
congestive CMP
unclear genesis
ET1
ECE1
NOS
TGFß
IGF1
VEGF ??
dilatative CMP
idiopathic CMP
(dilatative or congestive)cardiomyocytes. The selection criterion for gene functions was based on the etiology of CMP. Dependent on the CMP type, levels of the studied gene expressions were increased. In general, all gene functions were induced between 2-5 fold higher than the normal heart myocytes. VEGF and CPT1 exhibited a constant induction pattern, whereas angiopoetin and ET-1 showed the highest steady state levels but also the highest variations among the CMP types studied. A follow-up study after heart transplantation indicated primarily a decrease of all the analyzed gene functions and a time-dependent in/deduction of CPT1 and VEGF. Subsequently, different CMP mRNAs were used to screen an ATLAS array (CLONTECH), specific for cardiovascular gene functions in order to generate an overview on the complexity of all involved factors.
In a separate study we have started to establish an antisense gene therapy based on VEGF and CSF1 specific oligo nucleotides or recombinant constructs to treat human testis carcinoma in a mouse model organism.Structure and function of the human thymidine kinase 1
Enzymes, which are part of the purine and pyrimidine pathway, are mostly regulated due to the demands of the cell cycle, because their substrates and products are highly dependent on enhanced DNA-metabolism. The faster the cell grows, the higher the enzymatic activities of those enzymes, based on changes at different levels: induced expression of the genes, increased mRNA stability, different splicing, expression of different isoenzymes, or diminuation of protein degradation and enhanced protein stability can be the answer(s) to this basic challenge.
The thymidine kinase 1 (TK1), a key enzyme of the salvage pathway, is a central function for antiviral and/or cytostatic treatment and therefore of high pharmacological interest. The expression of the human TK1 gene is strictly cell cycle regulated and the enzyme itself is active only as a di- or tetramer, which consists in two or four 24 kD polypeptides. Beside the human TK1 or also named cytosolic TK, due to its cellular location, there also exists a human TK2 which is located in the mitochondria. With 95% of the whole activity the TK1 is considered to be main form.
At the beginning of G1 during cell cycle the activity is very low but at the starting point of S-phase there is a rapid and strong enhancement and therefore this enzyme is so suitable to be used as a marker for cell proliferation as well as serological marker for tumors. Our current hypothesis describes the active center as a cavity built up by two palm-like structures of each monomer (fig. 3: structural model of the human TK1 monomer, Prof. G. Folkers ETH Zürich) anchored by antiparallel pairing of predominately a-helical regions. These sub-domains we called the "thumb region" (aa 1- 100 of
Fig. 3: human tk1-structural model
the amino-terminus of TK1) of the TK1 monomer. The latter being a hypothetical structure we have postulated based on theoretical predictions and observations we have made with site-specific mutants. In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships, identifying intramolecular regions or amino acids, which play key roles in function. In collaboration with Prof.Dr. Birgitte Munch-Petersen (Dept. of Life Sciences, Roskilde University, DK), we set out to verify our hypothesis by biochemical and immunological means based on newly developed recombinant mutant TK1 constructs. By only mutating very specific amino acids we were able to generate a set of "super" versus "feeble" thymidine kinases.
Publications
- Biochem. J. 322, 403-410 (1997), Cloning and characterisation of murine carnitine acetyltransferase: Evidence for a requirement during cell cycle progression. S. Brunner, K. Kramar, D.T. Denhardt, and R. Hofbauer.
- Biochem. Soc. Trans. 26 (1), S63 (1998), An investigation of thymidine kinase 1 from normal and transformed mammary cell lines. F. Britton, B. Munch-Petersen, R. Hofbauer, and Y.A. Barnett.
- Biotechniques 26(5), 846-850 (1999), Reverse strand priming: A versatile cDNA radiolabelling method for differential hybridization on nucleic acid arrays, Mikulits W., Dolznig H., Hofbauer R., and E.W. Müllner.
- Prot. Express. Purification 18 (3), 338-345 (2000), Highly Purified Recombinant Varicella Zoster Virus Thymidine Kinase Id a Homodimer. Amrhein I., Wurth C., Bohner T., Hofbauer R., Folkers G., and L. Scapozza.
- Effects of carnitine deficiency on plasma lipids in hemodialysis patients and on carnitine acyltransferases in cultured fibroblasts. Lohninger A., Godarova A., Lohninger S., Kletzmayr J., Agu A.C., and Hofbauer R., submitted to Kidney International (2000).
- Valine, not Methionine, is Amino Acid 106 in Human Cytosolic Thymidine Kinase (TK1) - Impact on Oligomerisation, Stability and Kinetic Properties Berenstein D., Christensen J. F.,Kristensen T, HofbauerR. and Munch-Petersen B., submitted to J. Biol. Chem. (2000)
Collaborations:
Prof.Dr. Alffred Lohninger, Inst.f. Med.Chemistry, University of Vienna
Prof.Dr. Seyedhossein Aharinejad, Inst.f. Antomy. University of Vienna
Prof. Dr. David T. Denhardt, Nelson Biological Labs., Rutgers University, NJ, USA
Prof. Dr. Gerd Folkers, Dept. of Pharmacy, ETH Zürich, Zürich, Switzerland
Prof.Dr. Birgitte Munch-Petersen, Dept. of Life Sciences, Roskilde University, DKProjects:
Herzfelder´sche Familienstiftung, OeNB Jubiläumsfonds (6998, 7111), Onkologiefonds, Anton Dreher Stiftung, Hochschuljubiläumsstiftung City of Vienna, collaborative projects with Boehringer Mannheim GermanyDiploma thesises:
In vitro and in vivo analysis of mRNA levels of carnitine acyl transferases and their recombinant expression in bacteria (Agu Allison Chukwuma)
Molecular study of the human cardio myopathy (CMP) heart (Romana Schäfer)
Recombinant expression of a super versus feeble human thymidine kinase 1 (TK1) and amino/carboxy-terminal subfragments (Sven Budik)
Comparative secondary structure calculations of eukaryotic cDNAs and their experimental verification (Andreas Bauer)
Ph.d. thesis:
Molecular analysis of mitochondrial enzyme functions involved in energy and nucleotide metabolism (Alzbeta Godarova)