David M. Clarke
|Address||Medical Sciences Building, Room 7342
Toronto, ON M5S 1A8
Structure and Function of Membrane Transport Proteins
Our research is focused on the study of two medically important membrane proteins.
2. To understand how mutations in the CFTR protein of patients with cystic fibrosis (CF) cause protein misfolding and to develop a method that corrects the folding defects.
Both P-gp and CFTR are members of the very important and large family (48 members in humans) of the A TP- B inding C assette (ABC) family of transporters. These proteins are generally characterized as having a transmembrane domain (TMD) containing six transmembrane segments and a hydrophilic domain containing a nucleotide-binding site (NBD). CFTR has an additional regulatory (R) domain (Fig. 1).
1. The Human Multidrug Resistance P-glycoprotein: Mechanism and Inhibition.
P-glycoprotein (P-gp) (product of the MDR1 or ABCB1 gene) is present in the plasma membrane and can transport many structurally diverse compounds out of the cell. P-gp is often highly expressed in cancer cells and is a major cause of multidrug resistance during cancer and AIDS chemotherapy. Chemotherapy would be much more effective if P-gp was shut off during treatment. Most efforts to inhibit P-gp have focused on the identification of modulator compounds that inhibit P-gp activity during chemotherapy. Potential drawbacks of modulators are that they can induce/increase P-gp synthesis and maturation to the cell surface. Many different methods are used in our laboratory to study the structure and mechanism of how ATP hydrolysis is coupled to drug efflux. Studies involving the folding of P-gp, determining the location of critical residues involved in drug binding, determining the number of drug-binding sites , how substrates bind to P-gp and conformational changes (Fig. 2) that occur during drug binding and drug efflux provide important clues that could be used to develop strategies to shut down the pump during chemotherapy.
2. Cystic Fibrosis, Pharmacological/Chemical Chaperones, Small Molecules and Protein Misfolding
Cystic Fibrosis (CF) is a lethal genetic disease. The disease is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is a chloride channel on the apical surface of epithelial cells. Defects in CFTR result in impaired epithelial salt and water transport. Most patients with severe CF have mutations in their CFTR proteins. The mutations cause the protein to be misfolded such that it does not reach the cell surface. The misfolded protein is retained in the endoplasmic reticulum (ER) (processing mutants) and is rapidly degraded (Fig. 3).
Knowledge about how mutations interfere in the proper folding of CFTR would aid in the development of strategies to correct the folding defects in CFTR mutants. We hypothesize that processing mutations disrupt domain-domain interactions. This is based on our observation that cystic fibrosis-type processing mutations disrupt domain-domain interactions in P-glycoprotein and CFTR . An exciting discovery was that modulators and substrates acted as specific chemical chaperones to rescue misfolded P-gp mutants. These specific chemical chaperones (substrates/agonists, modulators/antagonists) are often small molecules that are now called pharmacological chaperones .
The exact mechanism of how these pharmacological chaperones rescue misfolded proteins is unknown. We hypothesized that the substrate or modulator binding site(s) in the mutant protein are formed early during biosynthesis and exist very transiently during synthesis of the misfolded protein. Occupation of the drug-binding site(s) by substrates during the folding process must stabilize the folding intermediate and allow proper domain-domain interactions to occur that results in proper folding of the mutant protein. The rescued protein then escapes the cell’s ER quality control mechanism and is trafficked to the cell surface (Fig. 4).
Therefore, the drug rescue approach could be used to correct the folding defect in any protein. It has been quite difficult however, to identify compounds that can correct the folding defect caused by the most common CF mutation (deletion of residue Phe508 in CFTR, Delta F508). This is because many known modulators bind to the CFTR protein with relatively low affinity. With the recent development of high through-put screening of chemical compound libraries however, it may be possible to identify ligands that may bind with relatively high affinity to CFTR and correct the folding defects in Delta F508-CFTR (Fig. 5). These compounds could then lead to the synthesis of more potent correctors of mutant CFTR proteins.
View all publications on PubMed
Cysteines introduced into extracellular loops 1 and 4 of human P-glycoprotein that are close only in the open conformation spontaneously form a disulfide bond that inhibits drug efflux and ATPase activity.
Loo TW, Clarke DM.
J Biol Chem. 2014 Sep 5;289(36):24749-58. Read
Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.
Loo TW, Clarke DM.
J Biol Chem. 2014 Mar 21;289(12):8484-92. Read
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Loo TW, Clarke DM.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. Read