P2Y Receptor trafficking in polarized epithelial cells
Signaling mechanisms of P2Y receptors
Antibiotic Action in Neisseria gonorrhoeae
Mechanisms of chromosomally mediated antibiotic resistance
Structure/function of PilQ and other pilus-related proteins
Research Synopsis:
P2Y Receptor Trafficking. My lab is focused on defining the mechanisms by which the eight subtypes of P2Y receptors are targeted to distinct membrane domains in polarized epithelial cells. Epithelial cells, which form a tight monolayer between two compartments, have two distinct membrane surfaces: the apical membrane and the basolateral membrane. Membrane proteins can either be targeted to one of the two membrane surfaces, or they can be non-sorted. The proper targeting of proteins to either the apical or basolateral surface is critical for normal epithelial cell function. Multiple P2Y receptor subtypes are expressed in epithelial cells and are intimately involved in regulating epithelial cell physiology. We have recently begun to define the targeting sequences within each member of the P2Y receptors (Fig. 1). These experiments have utilized construction and expression of chimeric receptors in MDCK (Manin-Darby Canine Kidney) cells, a useful polarized epithelial cell line, and subsequent localization of these receptors by confocal microscopy. Our data indicate that each of the P2Y receptors achieves steady-state localization at a particular membrane surface by a different mechanism.
Antibiotic Resistance Mechanisms. My laboratory
is also interested in the mechanisms of antibiotic resistance in the pathogenic
bacterium Neisseria gonorrhoeae, which is the etiologic agent
for the sexually transmitted infection gonorrhoea. Although penicillin
used to be the antibiotic of choice in treating a gonococcal infection,
increased resistance to this antibiotic necessitated its replacement by
3rd generation cephalosporins or fluorinated quinolones for the treatment
of infected individuals (resistance to fluoroquinolones has recently emerged).
Resistance to penicillin and other antibiotics occurs through chromosomally-mediated
alterations in endogenous genes. At least five genes are involved in mediating
high level resistance to penicillin. These include penA and ponA,
which encode alterations in the two essential PBPs of this organism (PBPs
1 and 2) that decrease their affinity for penicillin; mtrR, which
increases expression of an efflux pump; penC, which is a spontaneously
arising mutation in the PilQ secretin; and penB, which encodes
mutations in the major outer membrane porin, PIB (Fig.
2). Our research utilizes biochemistry, structural biology, and genetics
to define how these resistance genes synergize with one another to increase
resistance to antibiotics.
Recent Publications:
P2Y Receptors
Qi, A.D., Wolff, S.C., and Nicholas, R.A. (2005) The apical targeting signal of the P2Y2 receptor is located in its first extracellular loop. J Biol Chem 280: 29169-75. Abstract
Wolff, S.C., Qi, A.D., Harden, T.K., Nicholas, R.A. (2005) Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon. Amer J Physiol (Cell Physiol) 288(3): C624-32. Abstract
Herold, C.L., Qi, A.-D., Harden, T.K., Nicholas, R.A. (2004) Agonist versus antagonist action of ATP at the P2Y4 receptor is determined by the second extracellular loop. J Biol Chem 279: 11456-64. Abstract
Lee, S.Y., Wolff, S.C., Nicholas, R.A., and O'Grady, S.M. (2003) P2Y receptors modulate ion channel function through interactions involving the C-terminal domain. Mol Pharmacol 63: 878-885. Abstract
Antibiotic Resistance
Zhao, S., Tobiason, D., Seifert, H.S., and Nicholas, R.A. (2005) The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer assembly and prevents antibiotic influx. Mol Microbiol 57: 1238-1251. Abstract
Nicola, G., Peddi, S., Stefanova, M., Nicholas, R.A., Gutheil, W.G., and Davies, C. (2005) Crystal structure of Escherichia coli PBP 5 bound to a tripeptide boronic acid inhibitor: a role for Ser110 in deacylation. Biochemistry 44: 8207-17. Abstract
Stefanova, M., Tomberg, J., Olesky, M., Höltje, J.V., Gutheil, W.G., and Nicholas, R.A. (2003) Neisseria gonorrhoeae Penicillin-Binding Protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities. Biochemistry 42: 4614-25. Abstract
Nicholas, R.A., Krings, S., Tomberg, J., Nicola, G., and Davies, C. (2003) Crystal Structure of Wild-type Penicillin-Binding Protein 5 at 1.85 Å Resolution: Implications for Deacylation. J Biol Chem 278: 52826-33. Abstract
