1. Regulation of pyruvate dehydrogenase kinase 4 (PDK4) through protein- protein interaction and its role in apoptosis By Patricia Coliwe Ntlabati CSIR Biosciences

2. © CSIR 2006 www.csir.co.za Outline: ● Background on diabetes, PDK4 and apoptosis ● Objectives of the study ● Experimental Procedures ● Results

3. © CSIR 2006 www.csir.co.za Diabetes ● is a metabolic disorder characterized by: ➢ impaired insulin secretion ➢ insulin resistance ● due to environmental and genetic factors ● It affects 10 % of the world population ● chronic complications are leading cause of death ● There are two main types: ➢ Type 1 (T1D) ➢ Type 2 (T2D)

4. © CSIR 2006 www.csir.co.za Type 2 diabetes (T2D) ● Approximately 85 % of diabetic patients have T2D ● It is associated with: ➢ obesity ➢ hypertension ➢ stress ● T2D results from inability of tissues to respond to insulin due to insulin resistance ➢ results in continuous secretion of insulin by pancreatic β cells ➢ leads to pancreatic β cell failure

5. © CSIR 2006 www.csir.co.za Role of PDK4 in diabetes ● PDK4 is a member of mitochondrial serine kinases ➢ other isoforms ➢ PDK1,2,3,4 ● PDK4 is believed to be the isoform involved in diabetes ● Based on : ➢ high levels in obesity and diabetes ➢ expression of PDK4 in tissues involved in metabolic clearance of glucose ➢ expression of PDK4 is regulated by insulin

6. Regulation of PDK4 activity NADH Acetyl- CoA NAD + Pyruvat e ADP CoA PDK4 PDC

7. Regulation of PDK4 gene expression PDK4 gene expression Diabet es High fat diet Starvati on Stress Insulin

8. © CSIR 2006 www.csir.co.za Hypothesis: ● PDK4 activity is also regulated through protein- protein interaction

9. © CSIR 2006 www.csir.co.za Objectives: ● To uncover whether there are proteins that can possibly interact with PDK4 ● To understand the physiological implications of such interaction

10. © CSIR 2006 www.csir.co.za Experimental procedure Bioinformatics Analysis Cell Culture Studies Two components

11. Bioinformatics Identification of domains that potentially interact with hPDK4 (InterWeaver database) Examination of sequence similarity between hPDK4 and other proteins that may interact with the identified domains (e.g NFκB) (Gene Explorer/ClustalW) Identification of the possible interacting region on hPDK4 structure (Protein structure visualiser)

12. © CSIR 2006 www.csir.co.za InterWeaver results

13. © CSIR 2006 www.csir.co.za Physiological role of p100/p49 (I κ B-like) ● p100/p49 is an IκB like subunit of nuclear kappa B (NFκB) ● IκB is an inhibitor protein involved in regulation of apoptosis ● Apoptosis is the clearance of unwanted or damaged cells ● Apoptosis is partly induced by activation of NfκB ● IκBs inhibit apoptosis by interacting with NFκB

14. Physiological role of p100/p49 (Cont'd)

15. © CSIR 2006 www.csir.co.za Does hPDK4 contain a potential I  B binding sequence like NFκB IBIB NF  B IBIB hPDK4 ?

16. Bioinformatics (Cont'd) Identification of domains that potentially interact with hPDK4 (InterWeaver database) Examination of sequence similarity between hPDK4 and other proteins that may interact with the identified domains (e.g NFκB) (Gene Explorer/ClustalW) Identification of the possible interacting region on hPDK4 structure (Protein structure visualiser)

17. Potential interacting sequence of hPDK4 with consensus binding sequence of NFκB 341 360 PDK4_hs AKYFQGDLNL YSLSGYGTDA PDK4_rn AKYFQGDLNL YSMSGYGTDA PDK2_hs AKYFQGDLQL FSMEGFGTDA PDK2_rn AKYFQGDLQL FSMEGFGTDA PDK3_hs ARYFQGDLKL YSMEGVGTDA PDK3_rn ARYFQGDLKL YSMEGVGTDA PDK1_hs AQYFQGDLKL YSLEGYGTDA PDK1_rn AQYFQGDLKL YSLEGYGTDA *:******:* :*:.* **** NFKB GGRNNY YCC

18. Bioinformatics (Cont'd) Identification of domains that potentially interact with hPDK4 (InterWeaver database) Examination of sequence similarity between hPDK4 and other proteins that may interact with the identified domains (e.g NFκB) (Gene Explorer/ClustalW) Identification of the possible interacting region on hPDK4 structure (Protein structure visualiser)

19. Locating the conserved amino acids on rat PDK2 Rat PDK4 structures showing conserved amino acids to that of NF κB. Glycine (346), Glutamine (349) and phenylalanine (351) represented by Gly346, Gln349 and Phe351 respectively A- dimer interface B - homodimer

20. © CSIR 2006 www.csir.co.za Bioinformatics Summary ● InterWeaver tools suggested potential interaction between hPDK4 and I  B. ● Sequence alignment between hPDK4 and NFκB showed potential conservation of the I  B binding region. ● The conserved amino acids are located in a region of PDK4 that is accessible to nucleotide and protein substrates of PDK4. ● These findings support the hypothesis that PDK4 may be interacting with proteins other than PDC, specifically IκB. This protein-protein interaction may contribute to regulation of apoptosis.

21. © CSIR 2006 www.csir.co.za Relationship between PDK4 & Apoptosis Examine by: ● Investigate whether inducers of apoptosis such as TNF  ceramide and linoleic acid increase PDK4 expression levels

22. Cell culture work TNF , ceramide, linoleic acid (inducers of apoptosis) HeLa/HepG2 Cell viability Apoptosis Assay (e.g DNA laddering) Real time PCR (Levels of PDK4 mRNA)

23. © CSIR 2006 www.csir.co.za Effects of TNFα on cell viability HeLa and HepG2 cells treated with 50ng/ml TNFα over a 48 hr time period. Cell viability was performed using a trypan blue exclusion method.

24. © CSIR 2006 www.csir.co.za Effects of ceramide on cell viability HeLa and HepG2 cells treated with 40 uM ceramide over a 48 hr time period. Cell viability was performed using a trypan blue exclusion method.

25. © CSIR 2006 www.csir.co.za Effects of linoleic acid (LA) on cell viability HeLa and HepG2 cells treated with 1 mM linoleic acid over a 48 hr time period. Cell viability was performed using a trypan blue exclusion method.

26. Confirmation of apoptosis by DNA laddering assay M 1 2 3 4 5 6 7 8 9 10 11 12 DNA ladder bands DNA laddering assay results after treating HeLa cells with TNFα, linoleic acid and ceramide. Lane M - DNA marker Lanes 1, 5 and 9 are controls (untreated) Lanes 2, 3 and 4: cells treated with 50 ng/ml TNFα, Lanes 6, 7 and 8: cells treated with 40 µM C2-ceramide, Lanes 10, 11 and 12: cells treated with 300 µM linoleic acid

27. Effects of TNFα, ceramide and linoleic acid on PDK4 mRNA TNFα Ceramide Linoleic acid

28. © CSIR 2006 www.csir.co.za Summary ● TNFα, ceramide and linoleic acid which are known to induce insulin resistance and diabetes also induce apoptosis ● DNA laddering assay confirmed cell death was due to apoptosis ● RT-PCR demonstrated down regulation of PDK4 by the three factors ● These findings indicates that anti-apoptotic agents supress the levels of PDK4 expression ● This suggests that PDK4 may be anti-apoptotic

29. © CSIR 2006 www.csir.co.za Future Work ● Overexpression of PDK4 ● Inhibition of PDK4 – transfection studies

30. Acknowledgements Dr L. Shoba-Zikhali at CSIR Biosciences (Supervisor) Dr Z. Dlamini at Wits Anatomical Sciences (Supervisor) The CSIR Parlimentary Grant The NRF Thuthuka fund

31. Thank you