To answer the following questions read the paper by Iram and

To answer the following questions, read the paper by Iram and Cole --> http://www.jbc.org/content/286/9/7202.full.pdf+html

What hypothesis do the authors propose? (2 marks)

Describe the structure and outline the function of MRP1. (3 marks)

What is the CL5 region? For the purposes of this study how did the authors define the CL5 region? What amino acids comprise this region? (4 marks)

Briefly, how did the authors determine the role of the CL5 region in the expression and activity of MRP1? (6 marks)

What were the overall results of their findings? (2 marks)

What is the implication of this study? (2 marks)

Solution

1. The hypothesis is simple. MRP1 a multidrug resistant protein which when expressed in cells confers resistance to them by effluxing the drugs. This MRP has a cytoplasmic domain and an extracellular domain. in the intracellular cytoplasmic domain there are cytoplasmic loops which are basically polypeptides and therefore made of amino acids which are structural and functional units of the polypeptide. Any change in the sequence of the aminoacids or replacement of one aminoacid by another will lead to a non functional protein. With the same concept the authors substituted particular amino acid in the CL5 loop and some on the interface of CL5 loop and nucleotide binding domain which reduced the protein MRP1 expression in the cells. This confirms that certain amino acids substituted for or utated in these domains must play an important role in function and in its presence in the membrane.

2. this protein belongs to the ABC family of proteins. These proteins are multi domain transporters and have a domain with the ATPase activity hence the name ATP-Binding Casette (ABC). The MDR1 has 3 membrane spannind domains (MSD), 2 nucleotide binding domains (NBDs) on the cytoplasmic site. Two of the MSDs are composed of 6 transmembrane alpha helices between which lays a channel through which the substrate translocates. On the other hand NBDs are arranged in such a way to form a sit where the nucleotide binds, basically ATP to provide the energy for the translocation of the substrate. the third part of the structure the Cytoplasmic loops CLs, they are responsible for cpupling the activities of the MSDs and NBDs. Therefore, the function of the MRP1 is active (ATP coupled) translocation of the substrate against a concentration gradient.

3. CL are the cytoplasmic loops that connect the two domains the extracellular domain MSDs and the intracellular domain NBDs. the CL5 links the MSD1 and NBD2. It is composed of Lys513, Lys516, Glu521 and Glu535.

4. they mutated the amino acids in the CL5 region and at its interface with NBD2. The engineered/mutated sequences was harvested and ligased with a plasmid, an expression vector. The expression vector was transfected into the cell lines. The expression i.e. transcription-translation-expression of a functional protein was assayed by Bradford assay. Low levels of MRP1 for a particular known mutation indicates the role of the amino acid in structural expression and functions of the MRP1.

5. Lys513, Lys516, Glu521, His1364, and Arg1367 these residues were predicted to present at the interface between the CL5 and NBD2. they play a critical role in the function as their mutants showed absence of substrate selectivity. With the previous results from the CL7 mutants which were transport deficient, studies of CL5 mutants has lead to the clearance of their role in the normal funcitoning of MRP1.

6. elucidating the function of various structural features of MRP1 is necessary in understanding the functioning of the protein transporter as a whole. Even the analysis of MRP1\'s expression patterns, distribution in various cells, overexpression and underexpression in various pathophysiology is of utmost importance. For example an overexpression of these cells in tumors confers a resistance to these cells against the anticancer drugs; in such situations, targetting of these overexpressed MRPs is of therapeutic potenial. Only the complete understanding of the nomral physiology of teh transporter can lead to such a therapeutic approach and such mutant studies help in better comprehension of MRP1.