TTUHSC School of Medicine
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Ina Urbatsch, Ph. D.

Assistant Professor ina.urbatsch@ttuhsc.edu

Research Interests

ABC Transporters in Atherosclerosis and Cancer

ABC transporter Superfamily :

The ABC transporter superfamily comprises a large number of membrane spanning transport proteins. We distinguish full-size transporters with two TMDs and two NBDs encoded by a single gene and half-size transporters with one NBD and one TMD, that either form homodimers or heterodimers. The TMDs provide the transport pathway for a particular substrate, and the NBDs power the transport by ATP hydrolysis. While high-resolution X-ray structures are now available for many NBDs from both prokaryotic and eukaryotic proteins, the information for TMDs is limited to a few structures from bacteria. The X-ray structure of Sav1866 showed a homodimer with closely interacting intertwined TMDs and NBDs (Fig. 2B). This structure is indeed similar to our low-resolution map of membrane-bound P-glycoprotein (Pgp) by electron microscopy (Fig. 2A and C), and a cavity is clearly visible although the transport pathway is still unclear (Fig. 2D-F).

Figure 2: Comparison of the 3-D Map of ATPγS-bound Pgp resolved by electron microscopy with the X-ray crystal structure of MgADP-bound Sav1866. Form Lee, Urbatsch and Wilkins, J. Biol. Chem. 283(9), 5769-5779, 2008.

Role of P-glycoprotein (Pgp) in multidrug resistance of cancers:

Breast cancer is a major health problem with one million new cases annually and roughly 370,000 deaths worldwide. Often chemotherapy fails because the patient’s tumors either were resistant at the outset of treatment or eventually develop resistance after exposure to the cancer drugs. Several molecular pumps similar to Pgp such as the Breast Cancer Resistance Protein (BCRP) and the Multidrug Resistance Associated Proteins (MRP), as well as a number of unrelated mechanisms have been implicated in drug resistance. Yet, Pgp is the most prevalent and best-studied cause of multidrug resistance. This protein is an ATP-driven pump that transports anti-cancer drugs out of tumor cells and thus, the cells are not killed and appear resistant. Inhibition of Pgp offers an opportunity to improve the concentration in tumor cells of cytotoxic agents that are substrates for Pgp and potentially improve anti-tumor activity. However, as inhibitors used so far in clinical trials have revealed unacceptable toxicity towards normal cells and side effects on the liver and other organs, developing novel more specific inhibitors may be required to effectively target this well-established mechanism of multidrug resistance. Another alternative is to identify novel cytotoxics that are preferentially cytotoxic for Pgp-expressing malignant cells relative to normal cells or Pgp-low tumor cells.  We believe such compounds will open up new treatment strategies for cancer patients by preferentially killing the Pgp-expressing tumor cell lines and excluding normal cells.

Role of ABCG5 and ABCG8 in Sterol and Cholesterol Transport:

Hypercholesterolemia is a major risk factor for cardiovascular disease. It develops from an imbalance in cholesterol homeostasis, a finely tuned process involving many steps including de novo synthesis, absorption from the diet, trafficking through the body via lipoproteins and excretion through the liver and intestines. Sitosterolemia is a hypercholesterolemia-like disease characterized by significantly elevated plasma levels of plant and shellfish sterols. This genetic disorder also causes hyperabsorption and insufficient excretion of cholesterol, although plasma levels appear normal in half of the cases because de novo biosynthesis of cholesterol is downregulated. The clinical syndromes include the development of tendon and tuberous xanthomas and premature coronary arteriosclerosis due to significantly increased absorption from the diet and decreased excretion of sterols into bile. Sitosterolemia is caused by mutations in the ABCG5 and ABCG8 genes, suggesting that these gene products play a role in the efficient removal of plant sterols and cholesterol (Fig. 1) and thus, are directly involved in the regulation of cholesterol homeostasis.

Figure 1: Putative topology of ABCG5 and ABCG8 with mutations known to cause sitosterolemia.ABCG5 and ABCG8 are half-size ABC transporter each containing six transmembrane α-helices (green and blue cylinders) and a cytoplasmic nucleotide binding domain (NBD). Mutations that cause sitosterolemia are indicated by a red asterix (stop, premature termination; ins, insertion; Δ, deletion). The conserved Walker A, Walker B, and C motifs are shown in cyan circles, and N-glycosylation sites in brown diamonds. Based on Swiss-Prot entries Q9H222 and Q9H221.

The biomechanics of the sterol transport mechanism by ABCG5/G8 is still largely unknown, but can be elucidated from studies of their biochemical properties and their molecular structures. Such information is critical to understand how ABCG5 and ABCG8 regulate cholesterol in the body, and will provide insights into strategies for modulating sterol uptake and excretion in cases of sitosterolemia and more importantly, arteriosclerosis.

Recent Publications

  1. Lee JY, Urbatsch IL, Senior AE, and Wilkens S. Nucleotide-induced Structural Changes in P-glycoprotein observed by Electron Microscopy. J. Biol. Chem., 2008, 283(9), 5769–5779.
  2. Chloupkova M, Pickert A, Lee, JY, Souza S, Trinh Y, Connelly SM, Dumont ME, Dean M, and Urbatsch IL. Expression of 25 human ABC transporters in the yeast Pichia pastoris and Characterization of the Purified ABCC3 ATPase Activity.Biochemistry 2007;46(27):7992-8003.
  3. Urbatsch IL, Chloupkova M. Localization and processing of the human mitochondrial ABC transporter ABCB7 in Pichia pastoris. J. Prot. Express. Purif., 2007 submitted.
  4. Harvey BH, Lee JY and Urbatsch IL. Sitosterolemia, book chapter in Encyclopedia of Molecular Mechanisms of Disease, Springer 2008, accepted for publication.
  5. Carrier I, Urbatsch IL, Senior AE, Gros P. Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3). FEBS Lett. 2007; 581(2): 301-8.
  6. Wang Z, Stalcup LD, Harvey BJ, Weber J, Chloupkova M, Dumont ME, Dean M, Urbatsch IL. Purification and ATP Hydrolysis of the Putative Cholesterol Transporters ABCG5 and ABCG8. Biochemistry 2006; 45(32), 9929-9939.
  7. Tombline G, Urbatsch IL, Virk N, Muharemagic A, White LB, Senior AE. Expression, purification, and characterization of cysteine-free mouse P-glycoprotein. Arch Biochem Biophys. 2006;445(1):124-8.
  8. Delannoy S, Urbatsch IL, Tombline G, Senior AE, Vogel PD. Nucleotide Binding to the Multidrug Resistance P-Glycoprotein as Studied by ESR Spectroscopy. Biochemistry 2005;44(42):14010-14019.
  9. Tombline, G., Bartholomew, L.A., Tyndall G.A., Gimi, K., Urbatsch, I.L., and Senior, A.E. (2004) Properties of P-glycoprotein with mutations in the “catalytic carboxylate” glutamate residues. J. Biol. Chem., 2004, 279: 46518-46526.
  10. Tombline G, Bartholomew LA, Urbatsch IL, Senior AE. Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly. J Biol Chem. 2004 Jul 23;279(30):31212-20.
  11. Urbatsch, I.L., Tyndall G.A., Tombline, G., and Senior, A.E. (2003) P-glycoprotein catalytic mechanism. Studies of the ADP-vanadate inhibited state. J. Biol. Chem.278, 23171 - 23179.
  12. Lee, J.-Y., Urbatsch, I.L., Senior, A.E., and Wilkens, S. (2002) Projection Structure of P-glycoprotein by electron microscopy: evidence for a “closed” conformation of the nucleotide binding domains. J. Biol. Chem.277, 40125-31.
  13. Urbatsch, I.L., Gimi, K., Wilke-Mounts, S., Lerner-Marmarosh, N., Rousseau, M.-E., Gros, P., and Senior, A.E. (2001) Cysteine-431 and Cysteine-1074 are responsible for inhibitory disulfide crosslinking between the two nucleotide binding sites in human P-glycoprotein. J. Biol. Chem. 276, 26980-26987.
  14. Urbatsch, I.L., Wilke-Mounts, S., Gimi, K., and Senior, A.E. (2001) Purification and characterization of N-glycosylation mutant mouse and human P-glycoproteins expressed in Pichia pastoris cells. Arch. Biochem. Biophys. 388, 171-177.
  15. Urbatsch, I.L., Gimi, K., Wilke-Mounts, S., and Senior, A.E. (2000) Investigation of the role of Glutamine-471 and Glutamine-114 in the two catalytic sites of P-glycoprotein. Biochemistry 39, 11921-11927.
  16. Urbatsch, I.L., Julien, M., Carrier, I., Rousseau, M.-E., Cayrol, R., and Gros, P. (2000) Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein. Biochemistry 39, 14138-14149.
  17. Urbatsch, I.L., Gimi, K., Wilke-Mounts, S., and Senior, A.E. (2000) Conserved Walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step. J. Biol. Chem.275, 25031-25038.
  18. Kwan, T., Loughrey, H., Brault, M., Gruenheid, S., Urbatsch, I.L., Senior, A.E., and Gros, P. (2000) Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy. Mol. Pharm. 58, 37-47.
  19. Lerner-Marmarosh, N., Gimi, K., Urbatsch, I.L., Gros, P., and Senior, A.E. (1999) Large-scale purification of detergent-soluble P-glycoprotein from Pichia Pastoris and characterization of nucleotide-binding properties of wild-type, Walker A, and Walker B mutant proteins. J. Biol. Chem. 274, 34711-34718.
  20. Senior, A.E., Al-Shawi, M.K., and Urbatsch, I.L. (1998) ATPase activity of Chinese hamster P-glycoprotein. Methods Enzymol. 292, 414-523.
  21. Senior, A.E., Gros, P., and Urbatsch, I.L. (1998) Residues in P-glycoprotein catalytic sites that react with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Arch.Biochem. Biophys. 357, 121-125.
  22. Gros, P., Beaudet, L., and Urbatsch, I.L. (1998) Yeast as an expression system for the study of P-glycoprotein and other ABC transporters. Acta Physiol. Scand. Suppl. 643, 219-225.
  23. Beaudet, L., Urbatsch, I.L., and Gros, P. (1998) High level expression of mouse MDR3 P-glycoprotein in yeast Pichia Pastoris and characterization of ATPase activity. Methods Enzymol. 292, 397-413.
  24. Beaudet, L., Urbatsch, I.L., and Gros, P. (1998) Mutations in the nucleotide-binding sites of P-glycoprotein that modulate substrate induced ATPase activity. Biochemistry 37, 9073-9082.
  25. Urbatsch, I.L., Beaudet, L., Carrier, I., and Gros, P. (1998) Mutations in either nucleotide binding site prevent vanadate trapping of nucleotide at both sites. Biochemistry 37, 4592-4602.
  26. Senior, A.E., Al-Shawi, M.K., and Urbatsch, I.L. (1995) The catalytic cycle of P-glycoprotein.FEBS Lett. 377, 285-289.
  27. Urbatsch, I.L., Sankaran, B., Baghat, S., and Senior, A.E. (1995) Both P-glycoprotein nucleotide binding sites are cata¬lytically active. J. Biol. Chem. 270, 26956-26961.
  28. Urbatsch, I.L., Sankaran, B., Weber, A., and Senior, A.E. (1995) P-plycoprotein is stably inhibited by vanadate-in¬duced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270, 19383-19390.
  29. Senior, A.E., Al-Shawi, M.K., and Urbatsch, I.L. (1995) ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells. J. Bioenerg. Biomembr. 27, 31-16.
  30. Urbatsch, I.L., and Senior, A.E. (1994) Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein. Arch. Biochem. Biophys. 316, 135-140.
  31. Urbatsch, I.L., Al-Shawi, M.K., and Senior, A.E. (1994) Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. Biochemistry 33, 7069-7076.
  32. Al-Shawi, M.K., Urbatsch, I.L., and Senior, A.E. (1994) Covalent inhibitors of P-glycoprotein ATPase activity. J. Biol. Chem. 269, 8986-8992.
  33. Urbatsch, I.L., Sterz, R.K., Peper, K., and Trommer, W.E. (1993) Antigen-specific therapy of experimental myasthenia gravis with acetylcholine receptor-gelonin conjugates in vivo. Eur. J. Immunol. 23, 776-779.