Chemistry and Biochemistry

Aileen F. Knowles

Adjunct Professor
Biochemistry

Office: CSL-336
Office Phone: 619-594-5811
e-mail: aknowles_at_chemistry.sdsu.edu

Research Interests

My laboratory studies several cell surface ATPases or ecto-ATPases. Many of the well-characterized membrane ATPases (F-, P-, V-type ATPases) are transport proteins and use intracellular ATP to drive active ion transport. In contrast, the cell-surface ATPases, which are also ubiquitous, hydrolyze extracellular ATP (and UTP, ADP). Their functions are related to the regulation of the concentrations of extracellular nucleotides that are ligands of the P2 purinergic receptors, which mediate numerous physiological responses, including vasoconstriction, neurotransmission, secretion, pain etc. The ecto-ATPases belong to the nucleoside triphosphate diphosphohydrolase (NTPDase) family because of their ability to hydrolyze nucleoside triphosphates and/or nucleoside diphosphates. Members of the NTPDase family are distinguished by the extent of their sequence homology, substrate specificity, and tissue and subcellular distribution. NTPDases 1, 2, 3, and 8 are ectoenzymes that have variable glycosylation. All possess a large extracellular domain (ECD), two transmembrane domains (TMD) at their N- and C- termini, and short cytoplasmic domains. The conserved regions of NTPDases belong to the sugar kinase-actin-hsp70 superfamily. The 3-D structures of the membrane bound NTPDases have not been solved. However, the structure of the ECD of NTPDase1 was reported recently.

Our recent research focuses on the structure-function relationship of three NTPDases that were cloned in our lab: the human NTPDase2 and the chicken and human liver NTPDase8. Characterization of the expressed proteins revealed markedly different regulatory mechanisms of these enzymes, aside from their different substrate specificity. The human NTPDase2 is particularly interesting in that it is inactivated by parameters that disrupt interaction of the two TMDs. These results indicate that catalysis at the active site, which resides in the ECD, is affected by interaction of the TMD which are situated a distance away. Another unusual property of the human NTPDase2 is its inactivation by substrates. In contrast to the human NTPDase2, the chicken NTPDase8 is stable under most conditions.

To understand the different properties of the two NTPDases, we generated 1) chimeras in which the TMD of the unstable human NTPDase2 and that of the stable chicken NTPDase8 are exchanged, and 2) the soluble ECD of both NTPDases. The results obtained with these engineered proteins showed that 1) the two TMD of the chicken ecto-ATPDase can impart resistance to detergent to the human NTPDase2; 2) the two TMD of the human ecto-ATPase can impart lability to detergents to the chicken ecto-ATPDase8; 3) the presence of TMD is required to achieve maximal activity of the two NTPDases; 4) the presence and absence of TMDs affect substrate and divalent ion preferences, pH and temperature depenence of the NTPDases; 5) after removal of the TMDs, the human NTPDase2 becomes insensitive to substrate inactivation; 6) in contrast, the chicken NTPDase8 becomes susceptible to inactivation by both substrate and reaction products after removal of the TMDs; and 7) anchorage of the chicken NTPDase8 ECD by a single TMD at either the N-or C-terminus is insufficient to restore the stability of the enzyme; 8) protein expression (biogenesis) of the engineered NTPDases varies depending on the ECD and the membrane anchors. All our results highlight how much we have yet to learn about membrane protein biogenesis, assembly, the influence of lipid environment on membrane protein functions, communication between the TMDs and communications between the TMDs with the soluble domains of most membrane proteins.

Selected Publications

1. Li, C. S., Lee, Y. and Knowles, A. F. (submitted, 2009) "The stability of chicken NTPDase8 requires both of its transmembrane domains."
2. Knowles, A. F. (2009) "The single NTPase gene of Drosophila melanogaster encodes an intracellular nucleoside triphosphate diphosphohydrolase 6 (NTPDase6)," Arch. Biochem. Biophys. 484, 70-79.
3. Chiang, W.-C. and Knowles, A. F. (2008) "nhibition of the human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains ," Biochemistry 47, 8775-8785.
4. Brunschweiger, A., Iqbal, J., Umbach, F., Scheiff, A. B., Munkonda, M. N., Sevigny, J., Knowles, A. F. and Muller, C. A. (2008) "Selective nucleoside triphosphate diphopshohydrolase 2 (NTPDase2) inhibitors: nucleotide mimetics derived from uridine-5'-carboxamide," J. Med. Chem. 51, 4518-4528.
5. Chiang, W.-C. and Knowles, A. F. (2008) "Transmembrane domain interactions affect the stability of the extracellular domain of the human NTPDase2," Arch. Biochem. Biophys. 472, 89-99.
6. Javed, R., Yarimizu, K., Pelletier, N., Li, C. and Knowles, A. F. (2007) "Mutagenesis of lysing 62, asparagines 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2)," Biochemistry 46, 6617-6627.
7. Knowles, A. F. and Li, C. (2006) "Molecular Cloning and Characterization of Expressed Human Ecto-Nucleoside Triphosphate Diphosphohydrolase 8 (E-NTPDase 8) and Its Soluble Extracellular Domain," Biochemistry 45, 7323-7333.
8. Mukasa, T., Lee, Y. and Knowles, A. F. (2005) "Either carboxyl- or amino-terminal  region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8)," Biochemistry 44, 11160-70.
9. Kukulski, F., Levesque, S. A., Lavoie, E. G., Lecka, J., Bigonnessee, F., Knowles, A. F., Robson, S. C., Kirley, T. L., Sevigny, J. (2005) "Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3, and 8," Pur. Sig. 1, 193-204.
10. Dranoff J. A., Kruglov, E. A., Toure, J., Braun, N., Zimmermann, H., Jain, D., Knowles, A. F., Sevigny J. (2004) "Ectonucleotidase NTPDase2 is selectively down-regulated in biliary cirrhosis," J. Investig. Med. 52, 475-82.
11. Wen L. T. and Knowles A. F. (2003) "Extracellular ATP and adenosine induce cell apoptosis of human hepatoma Li-7A cells via the A3 adenosine receptor," British Journal of Pharmacology 140, 1009-1018.
12. Knowles A. F., Chiang W. C. (2003) "Enzymatic and transcriptional regulation of human ecto-ATPase/E-NTPDase 2," Archives of Biochemistry and Biophysics 418, 217-227.
13. Swank, D. M., Knowles, A. F., Kronert, W. A., Suggs, J. A., Morrill, G. E., Nikkhoy, M., Manipon, G. G., Bernstein, S. I. (2003) "Variable N-terminal regions of muscle myosin heavy chain modulate ATPase rate and actin sliding velocity," J. Biol. Chem. 278, 17475-17482.
14. Wen, L. T., Caldwell, C. C., and Knowles, A. F. (2003) "Poly(ADP-ribose)polymerase activation and changes in Bax protein expression associated with extracellular ATP-mediated apoptosis in human embryonic kidney 293-P2X₇ cells" Mol. Pharmacol. 63, 706-713.
15. Knowles, A. F., Nagy, A. K., Strobel, R. S., and Wu-Weis, M. (2002) "Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase" Eur. J. Biochem. 269, 2373-2382.
16. Swank, D. M., Bartoo, M. L., Knowles, A. F, et al. (2001) "Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity" J. Biol. Chem. 276, 15117-15124.
17. Caldwell, C. C., Hornyak, S. C., Pendleton, E., Campbell, D. and Knowles, A. F. (2001) "Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status" Arch. Biochem. Biophys. 387, 107-116.

Theses & Dissertations

• Cheryl Li (2009) "The effect of membrane anchorage on the enzymatic properties of the chicken NTPDase8."
• Wei-chie Chiang (2008) Ph.D. dissertation, "Regulation of the human NTPDase2 activity by its transmembrane domains."
• Kyoko Shindo (2004) "Mutagenesis of N-glycosylation sites of human ecto-ATPase (E-NTPDase 2)"
• Takashi Mukasa (2004) "Carboxyl-terminus of the human ecto-ATPase confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase"
• Yong hee Lee (2006) "Enzymatic properties of the chicken ecto-ATP-diphosphohydrolase are determined by its transmembranous domains"
• Reem Javed (2006) "Mutagenesis of conserved lysine and arginine residues in the human ecto-ATPase (NTPDase 2)"

Previous Knowles Students at SDSU