Aileen F. Knowles
Adjunct Professor
Biochemistry
Office: GMCS-203
Office Phone: 619-594-2065
e-mail: aknowles_at_chemistry.sdsu.edu
Research Interests
My laboratory is studying several cell surface E-type 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. P2 receptors mediate numerous physiological responses, including vasoconstriction, neurotransmission, secretion, pain etc. Because the ecto-ATPases have the ability to hydrolyze nucleoside triphosphates other than ATP, and some also hydrolyze nucleoside diphosphates, they are more appropriately called ecto-nucleoside triphosphate diphosphohydrolases or E-NTPDases.
Currently, the E-NTPDases and related proteins constitute the NTPDase family. Members of the 8 different subfamilies are distinguished by the extent of their sequence homology, substrate specificity, and tissue and subcellular distribution. NTPDases 1, 2, 3, and 8 are cell surface ectonucleotidases that possess a large extracellular domain (ECD), two transmembranous domains (TMD) at their N- and C- termini, and short cytoplasmic domains. The conserved regions of the ECD of the cell-surface ATPases indicate that they belong to the sugar kinase-actin-hsp70 superfamily. At present, the 3-dimensional architecture of E-ATPase has not been solved, although structures of the ECD based on computer modeling have been proposed.
Our recent research focuses on the structure-function relationship of three NTPDases that were cloned in our lab: the human ecto-ATPase (NTPDase 2) and the chicken and human liver ecto-ATP-diphosphohydrolase (ecto-ATPDase, NTPDase 8). Characterization of the expressed enzymes revealed markedly different regulation mechanisms of these enzymes, aside from their different substrate specificity. The human ecto-ATPase is particularly interesting in that it is inactivated by amphiphilic molecules and is less active at higher assay temperatures, conditions that increase membrane protein dissociation and disrupt TMD interaction. The ecto-ATPase is also one of the few enzymes that undergo substrate inactivation. However, decrease of ecto-ATPase activity by high temperature, detergents, and substrates is diminished when the membranes are treated with cross-linking agents, suggesting that an oligomeric protein is more stable. It has become apparent that catalysis at the active site, which resides in the ECD, is affected by interaction of the TMD which are situated a distance away. In contrast to the human ecto-ATPase, the chicken ecto-ATPDase is stable under most conditions.
To understand this regulation, we generated 1) chimeras in which the TMD of the unstable human ecto-ATPase and that of the stable chicken ecto-ATPDase are exchanged, and 2) the soluble ECD of the NTPDases. The results obtained with these engineered proteins showed that 1) the two TMD of the chicken ecto-ATPDase can impart resistance to detergent and high temperature to the human ectco-ATPase, 2) the two TMD of the human ecto-ATPase can impart lability to detergents and high temperature to the chicken ecto-ATPDase, 3) the presence and absence of TMD influences the substrate and divalent ion preferences of the enzymes, 4) removal of TMD causes the chicken ecto-ATPDase to become susceptible to substrate and reaction products whereas it is the opposite for the human ecto-ATPase, and 5) protein expression of the chicken ecto-ATPDase is more tolerant of "foreign" TMD whereas that of the human ecto-ATPase is less so. Above all, 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 TM segments and communications between TM segments with the soluble loops of most membrane proteins.
Selected Publications
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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
| Kristel Weaver, M.A. (2002) | DVM, UC Davis (2006) |
| Sam Sihapong, B.S. (2003) | Nanogen |
| Kyoko Yarimizu, M.S. (2004) | Amylin |
| Takashi Mukasa, M.S. (2004) | BD Sciences |
| Justin Brazil, B.S. (2004) | Biosite |
| Yonghee Lee, M.S. (2006) | Amylin |
| Nicole Pelletier, B.S. (2006) | Nexbio |
| Cheryl Li, B.S. (2006) | SDSU Chem & Biochem grad program |
| Reem Javed, M.S. (2006) | Norvatis |