CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function.

A three-nucleotide deletion in cystic fibrosis transmembrane conductance regulator/ATP-binding cassette transporter C7 (CFTR/ABCC7) resulting in the absence of phenylalanine at 508 leads to mis-fold of the mutated protein and causes cystic fibrosis. We have used a comparable three-nucleotide deletion mutant in another ABCC family member, multidrug resistance-associated protein (MRP1)/ABCC1, to determine whether CRISPR-Cas9-mediated recombination can safely and efficiently knock in three-nucleotide to correct the mutation. We have found that the rate of homology-directed recombination mediated by guideRNA (gRNA) complementary to the deletion mutant is significantly higher than the one mediated by gRNA complementary to the wild-type (WT) donor.

Expression of the cereblon binding protein argonaute 2 plays an important role for multiple myeloma cell growth and survival.

Background: Immunomodulatory drugs (IMiDs), such as lenalidomide, are therapeutically active compounds that bind and modulate the E3 ubiquitin ligase substrate recruiter cereblon, thereby affect steady-state levels of cereblon and cereblon binding partners, such as ikaros and aiolos, and induce many cellular responses, including cytotoxicity to multiple myeloma (MM) cells. Nevertheless, it takes many days for MM cells to die after IMiD induced depletion of ikaros and aiolos and thus we searched for other cereblon binding partners that participate in IMiD cytotoxicity.

Methods: Cereblon binding partners were identified from a MM cell line expressing histidine-tagged cereblon by pulling down cereblon and its binding partners and verified by co-immunoprecipitation.

Potential sites of CFTR activation by tyrosine kinases.

The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences.

Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma.

Cereblon (CRBN) mediates immunomodulatory drug (IMiD) action in multiple myeloma (MM). Using 2 different methodologies, we identified 244 CRBN binding proteins and established relevance to MM biology by changes in their abundance after exposure to lenalidomide. Proteins most reproducibly binding CRBN (>fourfold vs controls) included DDB1, CUL4A, IKZF1, KPNA2, LTF, PFKL, PRKAR2A, RANGAP1, and SHMT2.

Human Breast Cancer Stem Cells Have Significantly Higher Rate of Clathrin-Independent and Caveolin-Independent Endocytosis than the Differentiated Breast Cancer Cells.

Breast Cancer Stem (BCS) cells play critical roles in self-renewal, Multi Drug Resistance (MDR), differentiation and generation of secondary tumors. Conventional chemotherapy may efficiently kill the bulk of differentiated drug sensitive breast cancer cells, but not the MDR self-renewable BCS cells, leading to enrichment of the MDR BCS cells. In order to target the MDR BCS cells, we have isolated: 1) BCS cells from either breast cancer cell lines or fresh breast cancer specimens; 2) ATP binding cassette (ABC) transporter group G number 2 (ABCG2)-specific aptamers; and 3) BCS cell-binding aptamers.

What is the functional role of the thalidomide binding protein cereblon?

It has been found that nonsense mutation R419X of cereblon (CRBN) is associated with autosomal recessive non-syndromic mental retardation. Further experiments showed that CRBN binds to the cytosolic C-terminus of large-conductance Ca(++) activated potassium channel (BK(Ca)) α-subunit and the cytosolic C-terminus of a voltage-gated chloride channel-2 (ClC-2), suggesting that CRBN may play a role in memory and learning via regulating the assembly and surface expression of BK(Ca) and ClC-2 channels. In addition, it has also been found that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK) and prevents formation of a functional holoenzyme with regulatory subunits β and γ.

Infection of H69AR cells with retroviral particles harboring interfering RNAi significantly reduced the multidrug resistance of these small cell lung cancer cells.

Incubation of the drug-sensitive H69, a small cell lung cancer cell line, with increased concentrations of adriamycin yielded multidrug resistant (MDR) H69AR cells that over-express multidrug resistance-associated protein (MRP1). MRP1 co-transports its substrate with glutathione (GSH), leading to lower intracellular GSH. In this report we tested whether depleting intracellular GSH in MRP1-expressing cells could hyper-sensitize them to anticancer drugs or not.

Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide.

The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan.

Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor.

Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotriene-C4 transport.

Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.

Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers.

Effects of putative catalytic base mutation E211Q on ABCG2-mediated methotrexate transport.

ABCG2 is a half-ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a transmembrane domain. This half-ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. Substitution of the putative catalytic residue E211 with a nonacidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport.

Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, DeltaF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and DeltaF508 CFTR.

Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport.

Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport.

The hydroxyl group of S685 in Walker A motif and the carboxyl group of D792 in Walker B motif of NBD1 play a crucial role for multidrug resistance protein folding and function.

Structural analysis of MRP1-NBD1 revealed that the Walker A S685 forms hydrogen-bond with the Walker B D792 and interacts with magnesium and the beta-phosphate of the bound ATP. We have found that substitution of the D792 with leucine resulted in misfolding of the protein. In this report we tested whether substitution of the S685 with residues that prevent formation of this hydrogen-bond would also cause misfolding.

(R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1.

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells.

Multidrug resistance protein 1 is not associated to detergent-resistant membranes.

Multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette superfamily. Using the energy provided by ATP hydrolysis, it transports a broad spectrum of substrates across the plasma membrane, including hormones, leukotriene C(4), bile salts, and anti-cancer drugs. Recent works have suggested that P-glycoprotein is associated to cholesterol and sphingolipid-rich membrane microdomains and that cholesterol upregulates its ATPase and drug transport activities.

A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.

Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers.

Misassembled mutant DeltaF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.

Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells.

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport.

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated.

Domain interdependence in the biosynthetic assembly of CFTR.

The dimerization of their two nucleotide binding domains (NBDs) in a so-called "nucleotide-sandwich" is the hallmark of ATP cassette binding (ABC) proteins and the basis of their catalytic activities. The major disease-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7), deletion of Phe508 in NBD1, does not grossly alter the structure of that domain but prevents conformational maturation of the whole CFTR protein, possibly by disrupting the native interaction between NBD1 and NBD2. However, the role of inter-domain interactions in CFTR folding has been brought into question by a recent report that all CFTR domains fold independently.

F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive.

Most cystic fibrosis (CF) patients carry the F508del mutation in the CFTR chloride channel protein resulting in its misassembly, retention in the endoplasmic reticulum (ER), and proteasomal degradation. Therefore, characterization of the retention and attempts to rescue the mutant CFTR are a major focus of CF research. Earlier, we had shown that four arginine-framed tripeptide (AFT) signals in CFTR participate in the quality control.

Nucleotide dissociation from NBD1 promotes solute transport by MRP1.

MRP1 transports glutathione-S-conjugated solutes in an ATP-dependent manner by utilizing its two NBDs to bind and hydrolyze ATP. We have found that ATP binding to NBD1 plays a regulatory role whereas ATP hydrolysis at NBD2 plays a dominant role in ATP-dependent LTC4 transport. However, whether ATP hydrolysis at NBD1 is required for the transport was not clear.

Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1.

Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein.

Verapamil and its derivative trigger apoptosis through glutathione extrusion by multidrug resistance protein MRP1.

This study demonstrates that verapamil and a newly synthesized verapamil derivative, NMeOHI(2), behave as apoptogens in multidrug resistance protein 1 (MRP1)-expressing cells. When treated with either verapamil or NMeOHI(2), surprisingly, baby hamster kidney-21 (BHK) cells transfected with human MRP1 were killed. Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter.