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Passage 10

Resistant hypertension is a type of hypertension that remains uncontrolled despite treatment with antihypertensive medicines. It is hypothesized that the signaling of prorenin, an inactive precursor of the renin component of the renin-angiotensin system in the central nervous system, plays a crucial role in the pathophysiology of this vascular disorder.

Researchers have shown that binding of prorenin to its receptor (PRR) in nerve cells of the hypothalamus results in a conformational change in prorenin and activation of its catalytic functions. C17XA, a substrate analog which binds to the active site of activated prorenin has been used to inhibit the catalytic activity of prorenin.

To investigate the roles of prorenin and its hypothalamic receptor in pathways leading to resistant hypertension, researchers cloned the gene encoding PRR (WT-PRR) and constructed several PRR variants. The dissociation constants (Kd) of prorenin for each of the PRR variants were determined (Table 1).

Table 1 Dissociation Constants (Kd) of Prorenin for Various PRR Variants

Variant Kd (µM)
WT 0.034
V109E 0.037
W140L 0.163
D201N 0.441
A269K 0.007

Mouse Neuro-2A cells were transfected with either cloned WT-PRR cDNA or an empty vector as controls for 72 h. Cells were then treated with PBS buffer (control), an angiotensin II receptor antagonist (losartan), prorenin, or a combination losartan and prorenin for 30 min. The levels of reactive oxygen species (ROS), an intermediary molecule responsible for hypertension were measured.

Figure 1 Relative levels of ROS in Neuro-2A cells 3 days after transfection with either an empty vector (black bars) or WT-PRR cDNA (gray bars), treated with various compounds

Questions

74. Based on the data presented in Table 1, which amino acid residues of PRR are involved in binding to prorenin?

  1. Residue 109
  2. Residue 140
  3. Residue 201
  4. Residue 269
  1. I and II only
  2. I, II, and III only
  3. II and III only
  4. II, III, and IV only

75. Lineweaver–Burk plot most likely exhibits the inhibition of prorenin by compound C17XA?

76. Based on the data presented in Figure 1, overexpression of PRR:

  1. increases blood pressure in part through an angiotensin II-dependent pathway.
  2. increases blood pressure in part through an angiotensin II-independent pathway.
  3. decreases blood pressure in part through an angiotensin II-dependent pathway.
  1. I only
  2. II only
  3. III only
  4. I and II only

77. Based on the data presented in Table 1, which amino acid residue of prorenin most likely interacts with the residue at position 201 of PRR?

  1. Alanine
  2. Glutamate
  3. Arginine
  4. Glutamine

78. Which enzyme was LEAST likely used in cloning of WT-PRR cDNA?

  1. DNA polymerase
  2. RNA polymerase
  3. DNA ligase
  4. Reverse transcriptase

79. Compared to WT, what is the most likely effect of the W140L substitution on the stability of the PRR–prorenin complex?

  1. Elimination of a π-stacking interaction decreases stability of the complex.
  2. Elimination of an ionic interaction decreases stability of the complex.
  3. Addition of a hydrophobic interaction increases stability of the complex.
  4. Addition of a hydrophilic interactions increases stability of the complex.

Answers

Question 74 Solution: The correct answer is D. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because amino acid substitutions at positions 140, 201, and 269 result in changes to the Kd, while a non-conservative substitution at position 109 yields no change to the Kd. It is a Data-based and Statistical Reasoning question because you must interpret the data presented in Table 1 and predict the significance of each of the amino acid residues in protein binding.
Question 75 Solution: The correct answer is B. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because the passage states that C17XA is a compound that inhibits the catalytic activity of prorenin by binding to its active site, implying competitive inhibition. The Lineweaver–Burk plot shown in panel B illustrates the pattern of enzyme kinetics in the presence of a competitive inhibitor. It is a Scientific Reasoning and Problem Solving question because you must apply the information from the passage to the scientific model of the double reciprocal plot to reach a conclusion.
Question 76 Solution: The correct answer is D. This is correct. This Biology question falls under the content category “Structure and integrative functions of the main organ system.” The answer to this question is D because compared to control, the PRR overexpression alone increased ROS (an indicator of blood pressure) levels even in presence of losartan (angiotensin II antagonist), indicating that PRR overexpression induces ROS formation in an angiotensin II-independent manner. Moreover, the addition of exogenous prorenin, a ligand for PRR, induced an increase in ROS formation which can be reversed by losartan, indicating its angiotensin II-dependent nature. It is a Data-based and Statistical Reasoning question because you must interpret patterns in data and reach a conclusion about the effect of PRR overexpression and various compounds on blood pressure.
Question 77 Solution: The correct answer is C. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is C because substitution of aspartate (D) at position 201 with asparagine (N) results in a lower PRR affinity (higher Kd) towards prorenin. Since both asparagine and aspartate have equivalent carbon lengths, the decrease in affinity is most likely due to elimination of an ion pair. Among the possible options, only arginine could form an ion pair with aspartate. It is a Data-based and Statistical Reasoning question because you must analyze the change in Kd in D201N substitution and deduce the amino acid residue of prorenin that interacts with aspartate.
Question 78 Solution: The correct answer is B. This is correct. This Biology question falls under the content category “Transmission of genetic information from the gene to the protein.” The answer to this question is B because while DNA polymerase (in DNA amplification), DNA ligase (in ligation of the cDNA to DNA vector) and reverse transcriptase (in reverse transcription of RNA to cDNA) are used during cDNA cloning, RNA polymerase is not used in cDNA cloning. It is a Reasoning about the Design and Execution of Research question because you must identify the appropriate enzymes used in cDNA cloning process.
Question 79 Solution: The correct answer is A. This is correct. This Biochemistry question falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is A because based on results presented in Table 1; in W140L variant, the replacement of tryptophan (a hydrophobic, aromatic amino acid) by leucine (a hydrophobic but non-aromatic amino acid) results in a decrease in stability of the PRR–prorenin complex as indicated by the increase in Kd. This is most likely due to the elimination of a π-stacking interaction with the side chain of tryptophan. It is a Knowledge of Scientific Concepts and Principles question because you must recall potential bonding interactions between the side chains on the amino acids.