Exam 2: Biochemistry

Passage The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis.  Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors.  Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane.  The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation.  Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1). Figure 1  Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site.  The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing.  This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed.  Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively.  They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2). Figure 2  Percent cleavage of wild-type and mutant Tcd by autoprocessing Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002. -Based on information given in the passage, stabilizing interactions are most likely to occur between phosphate groups on IP6 and which of the following amino acids lining the Tcd binding pocket?

Passage Porphyrin biosynthesis is a multistep process that is required for production of porphyrin derivatives such as heme, which is a crucial component of hemoglobin, myoglobin, and the cytochrome cofactors of the electron transport chain.  In the first step of this pathway, the enzyme porphobilinogen synthase catalyzes the production of porphobilinogen (PBGN) from two δ-aminolevulinate molecules, as shown in Reaction 1. Reaction 1In the next step, the monomeric enzyme hydroxymethylbilane synthase (HMBS) combines four PBGN molecules to form hydroxymethylbilane (Reaction 2).  Several additional steps are required to convert hydroxymethylbilane to heme. Reaction 2Defects in HMBS activity can lead to accumulation of δ-aminolevulinate and porphobilinogen in the liver and blood plasma.  The accumulation of these molecules can cause acute intermittent porphyria, a disorder characterized by episodes of severe abdominal pain.  Researchers expressed, purified, and assessed several variants of HMBS for enzymatic activity (Table 1) and thermal stability (Figure 1).Table 1  Michaelis-Menten equations generated from several HMBS variants, with V₀ and V_max measured in nmol/hr and [PBGN] and K_m measured in μM.  (Note: All reactions were carried out with 1 μM HMBS.) Figure 1  Thermal stability of several HMBS variants -What type of reaction does porphobilinogen synthase catalyze?

Passage Spinocerebellar ataxia 3 (SCA3) is a neurodegenerative disease that manifests as a progressive decrease in coordination of limbs and frequently results in impaired speech and eye coordination.  It is associated with extension of the polyglutamine (polyQ) region of the ataxin-3 protein.  The polyQ region is encoded by a series of consecutive CAG codons known as poly-CAG repeats.  These regions vary in length from one person to another, ranging from as few as 10 to as many as 80 repeats.  SCA3 has been reported in patients with polyQ regions exceeding ~55 repeats, and increased polyQ length correlates with earlier onset of the disease and greater clinical severity.Circular dichroism (CD) is a method of assessing protein secondary structure.  It measures a molecule's ability to absorb left- and right-handed circularly polarized light of varying wavelengths.  The absorption difference is known as "ellipticity," denoted by Δε.  Alpha-helices have a maximum Δε at 190 nm, and beta-sheets have a maximum Δε at 200 nm.CD results show that as the length of the ataxin-3 polyQ region increases, alpha-helical nature is lost and the protein aggregates, ultimately precipitating out of solution and depositing in brain and other tissues.  Furthermore, as proteins aggregate, they form beta-sheets.  These beta-sheets are in the parallel orientation, and each sheet can interact with sheets in other proteins to form long, insoluble structures known as amyloid fibers (Figure 1). Figure 1  Depiction of the ataxin-3 polyQ region shifting from alpha-helical (cylinders) to beta-sheet (arrows) structure and aggregatingGlutamine can be encoded by both CAG and CAA codons.  Researchers investigated survival rates in fruit flies expressing ataxin-3 with polyQ regions of equal length encoded by either CAG-CAG or CAA-CAG repeats (Table 1).  The protein products had identical amino acid sequences.Table 1  Fruit Fly Survival Rates Relative to Ataxin-3 Expression Adapted from Bevivino AE, Loll PJ. An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel beta -fibrils. Proc Natl Acad Sci USA. 2001;98(21):11955-60. -Researchers hypothesized that overexpression of the chaperone protein HSP70 could reduce toxicity associated with ataxin-3 containing long polyQ regions.  Which line of reasoning might have led them to this hypothesis?

Passage The innate immune system relies heavily on the phagocytic action of neutrophils, mobile white blood cells that engulf and degrade bacteria and viruses as the first line of host defense against invading pathogens.  One mechanism for eliminating viruses engulfed in the phagosome involves the degradation of viral proteins by neutrophil serine proteases (NSPs).NSPs, which form part of the chymotrypsin family of serine proteases, cleave viral proteins using a catalytic triad relay system.  Originally discovered in granules released by neutrophils, NSP4 is a novel monomeric serine protease that has been studied to elucidate the catalytic mechanism of NSPs.  Prior experimental data suggest that NSP4 alters its tertiary structure upon substrate binding and cleaves viral proteins using the acylation reaction shown in Figure 1. Figure 1  Degradation of viral proteins by acylation of NSP4Computational methods were used to identify the energetic parameters of reactions between NSP4 and synthetic viral proteins.  To ensure that simulated reactions resembled physiological conditions, algorithms were adjusted to account for the enzyme being in an aqueous environment.  The rate-determining step in the acylation reaction was determined by calculating the changes in free energy in both the enzyme and substrate (Figure 2). Figure 2  Free energy profiles of synthetic viral protein cleavageKnowledge of amino acid frequency distribution at an enzymatic active site may facilitate the design of novel active sites and enzyme-specific inhibitors.  Researchers analyzed the composition of amino acids that form the NSP4 active site during protein folding to study the characteristics of residues involved in active site formation (Figure 3). Figure 3  Frequency distribution of amino acid residues at the NSP4 active site Adapted from Stapels DA, Geisbrecht BV, Rooijakkers SH. Neutrophil serine proteases in antibacterial defense. Curr Opin Microbiol. 2015;23:42-8. -Which of the following is the most accurate statement regarding NSP4 binding of viral proteins?

Thermolysin is a protease that can cleave peptides at the N-terminal side of branched alkyl residues.  Which of the following peptides has the most thermolysin cleavage sites?

A certain enzyme uses Mn²⁺ as a cofactor under physiological conditions, and scientists hypothesize that Mn²⁺ is required for stabilization of tertiary structure but not for enzyme activity.  Which of the following observations is consistent with this hypothesis?

Passage Nerve cells must maintain the composition of their myelin sheaths to function properly.  Galactocerebroside (GC), shown in Figure 1, is a hydrolyzable lipid and an important component of the myelin sheath. Figure 1  Structure of the myelin sheath component galactocerebrosideHomeostasis of GC is maintained by its dynamic synthesis and degradation, as shown in Figure 2.  GC is synthesized by the enzyme ceramide galactosyltransferase (CGT) using UDP-galactose (UDP-Gal) and ceramide (Cer) as substrates.  It is degraded by the enzyme galactocerebrosidase (GalC), which hydrolyzes the glycosidic bond to release galactose (Gal) and ceramide.  A deficiency in GalC activity leads to a buildup of GC, and prior research has connected this buildup with neurodegeneration. Figure 2  Schematic of GCT and GalC activity in healthy and GalC deficient cellsOver 100 inactivating GalC mutations have been identified.  Five of the most common mutations are listed in Table 1.  The changes to both DNA and amino acid sequence are shown.Table 1  Five Common Galactocerebrosidase Mutations -Based on Table 1, the D187V form of GalC resulted from what type of nucleotide change?

Passage Although the structure of the cell membrane has been investigated repeatedly, few studies examine the contribution of its lipid components.  Lipid composition influences the fluidity and permeability of cell membranes in the human body.  Chronic diseases such as atherosclerosis and psoriasis arise from alterations to the lipid composition.Atherosclerosis is caused by the accumulation of cholesterol in the endothelial cells lining the arterial wall.  Cholesterol carried by low-density lipoprotein (LDL) is deposited into cell membranes, where it forms cholesterol crystalline domains (CCDs).  In damaged cells, these CCDs increase membrane permeability and contribute to the formation of cholesterol plaques that can potentially block the artery itself.Omega-3 fatty acids (O3FAs) derived from marine animals may be used to slow the development of atherosclerosis.  Formulations of O3FA contain varying concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1).  Present research suggests that formulations of O3FA should contain EPA exclusively because it prevents cholesterol from organizing into CCDs.  Nonetheless, atherosclerosis is generally treated by administering drugs referred to as statins, which decrease the free cholesterol in blood plasma by inhibiting the production of a cholesterol precursor.  Statin therapy combined with O3FAs may be used to reduce atherosclerosis in patients with persistent hypertriglyceridemia (high triglyceride plasma levels). Figure 1  Organization of membranes in endothelial and epidermal cellsWater loss from the uppermost layer of the epidermis, the stratum corneum (SC), is prevented by specialized lipids called ceramides (cer).  Psoriasis is a skin disease in which the permeability of the SC is compromised due to a significant decrease in the level of ceramides.  The scarcity of ceramides impedes the formation of a lipid matrix called the long periodicity phase (LPP) in the cell membrane.  Increasing the concentration of ceramides such as esterified omega-hydroxyacyl-sphingosine (cer-EOS) has been shown to reduce the permeability of SC cell membranes and may be used to treat psoriasis scabs (Figure 1). Adapted from Mason RP, Jacob RF, Shrivastava S, Sherratt SC, Chattopadhyay A. Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes. Biochim Biophys Acta. 2016;1858(12):3131-3140. -Structural lipids in the cell membrane can be derived directly from dietary sources or from lipase-mediated catabolism.  Which statement correctly describes lipid processing during digestion?

Which of the following most accurately describes the ribose component of a nucleotide triphosphate?

Passage Whole grains have been shown to reduce the risk of cardiovascular disorders.  A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism.  Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell.  Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria.  Based on the structures of 5-AVAB and L-carnitine (Figure 1), scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine. Figure 1  Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours.  Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources.  The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2). Figure 2  Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB. Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1):13036. -The information in the passage and the data in Figure 2 best support which statement about cardiomyocytes when palmitate is their only energy source?

Passage The K-ras gene codes for a small guanine nucleotide-binding protein, which acts as a molecular activator in numerous signaling pathways that promote DNA replication along with cell growth and survival.  K-ras mutations at codons 12 and 13 function as biological markers for diagnosis of tumors.  The DNA of SW-480 cells, a line originating from a colon adenocarcinoma, is the standard positive control used to identify codon 12 mutations in K-ras testing. Figure 1  Structural comparison of K-ras codon 12 in wild-type cells and SW-480 cellsPCR sequence analysis has been the preferred method for studying genetic mutations such as those in K-ras.  However, challenges in analyzing PCR amplicons have prompted the development of DNA melting curve analysis (MCA).  In the MCA technique, DNA is extracted from clinical samples and amplified by PCR.  The amplified DNA is then melted (30-70°C) and analyzed by assessing the fluorescence (at 640 nm) of LC Green Plus, an intercalating dye that fluoresces when bound to double-stranded DNA (Figure 2). Figure 2  Melting curves of wild-type (WT) and SW-480 K-ras DNA.To study K-ras activity in colorectal cancer cells, short tandem repeats (STRs) were introduced into the K-ras gene.  STRs are short stretches of DNA with repeating nucleotides, and four different STR sequences were inserted into the K-ras gene and analyzed via MCA under different experimental conditions (Table 1).Table 1  Sequences Studied in K-ras DNA MCA Analysis. Liu YP, Wu HY, Yang X, et al. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis. Sci Rep. 2014;4:7521. -Assuming that additional mutations similar to the one shown in Figure 1 occur throughout the mutant K-ras gene, what would be the best explanation for the difference in denaturation between wild-type (WT) and mutant K-ras DNA?

Thermophiles are microorganisms that thrive at temperatures above 40 °C.  Which of the following characteristics LEAST accurately describes an enzyme isolated from a thermophile?

Passage Synthesis of coenzyme A (CoA) requires vitamin B₅ as a substrate.  In bacteria, vitamin B₅ is synthesized by condensation of β-alanine and pantoic acid.  β-alanine is produced by decarboxylation of L-aspartate, mediated by the enzyme aspartate α-decarboxylase (ADC).  Active ADC is formed when its inactive precursor, PanD, binds to a regulatory complex composed of acetyl coenzyme A (AcCoA) and a protein known as PanZ.  Formation of the PanD-PanZ-AcCoA complex causes the activation loop of PanD to fold into a β-pleated sheet, resulting in PanD autocleavage.  Researchers hypothesize that this conformational change allows the Thr57 residue to cleave the N-terminal end of PanD to form ADC.The limited information on the role of PanZ in the synthesis of CoA has stimulated an investigation of its effect on ADC activity.  Escherichia coli cells were provided with varying concentrations of PanZ, and the rate of β-alanine production was measured in three different conditions (Figure 1).  Reaction vessels were maintained at pH 6 to ensure optimal decarboxylation activity. Figure 1  Catalytic activity of ADC in E. coli with increasing concentrations of L-aspartate Adapted from Monteiro DC, Patel V, Bartlett CP, et al. The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chem Biol. 2015;22(4):492-503. -Which experiment could best provide data to support the proposed mechanism of autocleavage described in the passage?

Passage Mitochondria are double membrane-bound cellular organelles that house several connected metabolic reactions.  The citric acid cycle takes place inside the mitochondrial matrix and produces two reduced electron carriers, NADH and FADH₂.  These two carriers then pass their electrons to ubiquinone (UQ), which is reduced to ubiquinol (UQH₂).  UQH₂ passes its electrons to oxidized cytochrome C (cyt-C_ox), regenerating UQ and forming reduced cytochrome C (cyt-C_red).  Finally, cyt-C_red transfers electrons to oxygen, regenerating cyt-C_ox and forming water.  Each reaction is facilitated by one of four protein complexes that are collectively known as the electron transport chain (ETC).Researchers isolated mitochondria and treated them with a novel ETC inhibitor, "drug X."  After an overnight incubation, they measured levels of several ETC-reduced electron carriers in the presence of drug X relative to untreated mitochondria.  Their results are shown in Figure 1. Figure 1  Relative levels of reduced electron carriers in the presence and absence of drug XNext, the researchers exposed mitochondria to FCCP, a chemical that can transport protons across membranes.  They observed that in the presence of FCCP, the citric acid cycle was fully active, and all reduced cofactors were present at the same levels as in untreated mitochondria.  However, the rate of ATP synthesis was significantly reduced.  ATP synthesis was immediately restored when FCCP was washed away. -Based on Figure 1, which electron transport chain complex is inhibited by drug X?

Passage ATP synthase, also known as Complex V, produces the majority of the ATP in eukaryotic cells.  It is a transmembrane protein encoded by both nuclear and mitochondrial genes in humans, and is found in the inner mitochondrial membrane.  Complex V consists of over 20 subunits, including three sets of heterodimers arranged in a circle in the mitochondrial matrix.  Each dimer contains an α subunit and a β subunit, and each β subunit exists in one of three conformations: one that binds ATP, one that binds ADP and inorganic phosphate, and one that binds neither.As protons flow from the intermembrane space through Complex V and into the matrix, they cause the transmembrane γ subunit to rotate and interact with each β subunit in turn.  Four protons provide enough energy for rotation from one β subunit to the next, and each rotation causes a β subunit to release a new ATP molecule as it changes from the ATP-binding to the nonbinding conformation (Figure 1).  Thus 12 protons produce 3 ATP molecules each time the γ subunit makes a complete revolution.  Every NADH molecule that enters the electron transport chain (ETC) pumps 10 protons into the intermembrane space for use by Complex V whereas only 6 protons are pumped per FADH₂ molecule. Figure 1  Depictions of ATP synthase viewed from the side (left) and the bottom (right)Researchers visualized the rotation of the γ subunit by attaching it to a fluorescently labeled actin filament.  The observed rotation over time is shown in Figure 2. Figure 2  Counterclockwise rotation of the γ subunit of ATP synthase (bottom view) over time in seconds as visualized by a fluorescent labelAn L156P mutation in subunit A of Complex V is linked to neuropathy, ataxia, and retinal degeneration.  Although the exact molecular bases of these pathological states are unknown, the following hypotheses are currently under study:Hypothesis 1L156P Complex V functions normally but is less abundant than wild-type Complex V.Hypothesis 2The mutation partially decouples proton flow from ATP synthesis.Hypothesis 3The mutation leads to increased oxidative stress from the ETC, resulting in apoptosis. Adapted from Kucharczyk R, Zick M, Bietenhader M, et al. Mitochondrial ATP synthase disorders: molecular mechanisms and the quest for curative therapeutic approaches. Biochim Biophys Acta. 2009;1793(1):186-99. -Based on the information in the passage and Figures 1 and 2, approximately how many protons flow through wild-type Complex V per second under the experimental conditions?

Passage Whole grains have been shown to reduce the risk of cardiovascular disorders.  A team of scientists discovered that the compound 5-aminovaleric acid betaine (5-AVAB) was present at high levels in cardiomyocytes of mice given a diet rich in whole grains and decided to investigate its role in metabolism.  Preliminary evidence indicated that 5-AVAB did not alter the citric acid cycle in these cardiomyocytes, but that it did alter fatty acid metabolism.The plasma membrane transport protein organic cation/carnitine transporter 2 (OCTN2) transports extracellular L-carnitine into the cell against its concentration gradient using the energy provided by the simultaneous transport of Na⁺ into the cell.  Cellular L-carnitine is required for the transport of fatty acids from the cytosol into the mitochondria.  Based on the structures of 5-AVAB and L-carnitine (Figure 1), scientists hypothesized that 5-AVAB may interfere with OCTN2-mediated transport of L-carnitine. Figure 1  Structures of L-carnitine and 5-AVABTo test their hypothesis, scientists incubated mouse cardiomyocytes with various concentrations of 5-AVAB for 24 hours.  Cells were then provided with the fatty acid palmitate for energy and were deprived of all other energy sources.  The cardiomyocytes were also given 50 μM L-carnitine (endogenous extracellular concentration) or 2 mM L-carnitine, and the rate of oxygen consumption was monitored (Figure 2). Figure 2  Oxygen consumption rate of cardiomyocytes at various concentrations of 5-AVAB and L-carnitineIn a follow-up experiment, the scientists used siRNA to silence expression of OCTN2 and found that intracellular L-carnitine levels were similarly depleted in siRNA-treated cells as in cells treated with 100 μM 5-AVAB. Adapted from Kärkkäinen O, Tuomainen T, Koistinen V, et al. Whole grain intake associated molecule 5-aminovaleric acid betaine decreases ß-oxidation of fatty acids in mouse cardiomyocytes. Sci Rep. 2018;8(1):13036. -A colleague suggested that 5-AVAB may interfere with the electron transport chain directly.  Which experiment could provide evidence for the effect (if any) of 5-AVAB on the electron transport chain?

Which of the following steps in the citric acid cycle does NOT produce reduced electron carriers?

Passage The bacterium Clostridium difficile secretes protein toxins TcdA and TcdB, two homologues associated with C. difficile-related colitis.  Tcd proteins enter host intestinal cells via endocytosis induced by interactions between the Tcd C-terminal domain and cell surface receptors.  Endocytosis is followed by translocation of the N-terminal glucosyltransferase domain (GTD) across the endosomal membrane.  The GTD remains anchored to endosomes by the central region of the protein, a transmembrane domain believed to form membrane pores and facilitate translocation.  Inositol hexakisphosphate (IP6) binds Tcd proteins, an interaction that is stabilized by salt bridges in the binding pocket (Figure 1). Figure 1  Salt bridges formed between IP6 and the amino acids in the binding pocketThis interaction induces a conformational change that causes a molecular flap to move away from the catalytic site.  The catalytic site then mediates intramolecular cleavage in a reaction known as autoprocessing.  This reaction results in the release of GTD into the cytosol, where it becomes active.To better understand the relationship between IP6 binding and catalytic activity, autoprocessing assays were performed.  Researchers mutated histidine to alanine at positions 759 and 757 in the flap regions of TcdA and TcdB, respectively.  They then compared the autoprocessing activities of H759A and H757A mutants to those of wild-type TcdA and TcdB at varying concentrations of IP6 (Figure 2). Figure 2  Percent cleavage of wild-type and mutant Tcd by autoprocessing Adapted from Chumbler NM, Rutherford SA, Zhang Z, et al. Crystal structure of Clostridium difficile toxin A. Nat Microbiol. 2016;1:15002. -Based on Figure 2, residues H757 and H759 of wild-type TcdA and TcdB, respectively, may function to:

Passage The sirtuins are a class of enzymes that regulate various metabolic processes by removing post-translational modifications from enzymes.  SIRT4 is an autosomally encoded sirtuin protein that colocalizes with the pyruvate dehydrogenase complex.  It deacylates lysine residues on certain metabolic proteins by removing methylglutaryl and hydroxymethylglutaryl units.  Methylglutaryl-CoA and hydroxymethylglutaryl-CoA are intermediates in leucine catabolism, and scientists hypothesized that SIRT4 regulates this pathway.  To test their hypothesis, livers from wild-type (WT) and SIRT4 knockout (KO) mice were treated with α-keto acid derivatives of several amino acids, including the derivative of leucine, α-ketoisocaproate (αKIC).  Figures 1 and 2 show the rate of oxidation of α-keto acids and oxygen consumption in both WT and KO livers, respectively. Figure 1  Box plot showing the rate of oxidation of α-ketoisocaproate (αKIC), α-ketoglutarate (αKG), and pyruvate in SIRT4 knockout livers (Note: Asterisk indicates p < 0.05 relative to WT level) Figure 2  Oxygen consumption in WT and KO livers in the presence and absence of succinate and reduced cytochrome C (Cyt-C)Because leucine is known to regulate insulin secretion, scientists injected WT and KO mice with leucine and measured plasma insulin levels over time.  They then injected mice with insulin and monitored blood glucose levels (Figure 3). Figure 3  (A) Plasma insulin levels after injection with leucine and (B) blood glucose levels after injection with insulin in both WT and KO mice Adapted from Anderson KA, Huynh FK, Fisher-Wellman K, et al. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion. Cell Metab. 2017;25(4):838-855.e15. -Which conclusion about the effect of SIRT4 on blood glucose levels can be drawn from the data in Figure 3?

Passage The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride ion channel involved in the production of mucus, sweat, and digestive fluids.  CFTR is composed of five domains (Figure 1):  Two transmembrane domains (TMD1 and TMD2), two nucleotide binding domains (NBD1 and NBD2), and one regulatory domain (R domain). Figure 1  Schematic diagram of CFTR domainsThe NBD domains regulate CFTR activity by binding ATP, which causes a conformational change in the TMD domains.  This change allows chloride ions to leave cells by passively crossing the membrane down their concentration gradient.  The R domain regulates activity further through dynamic phosphorylation at several positions.  The movement of chloride ions facilitates the movement of water out of the cell by osmotic pressure and helps keep secretions thin.Cystic fibrosis (CF) arises from recessively inherited mutations in the CFTR gene.  The most common mutation is a three-base-pair deletion that removes a phenylalanine residue at position 508 in NBD1 (ΔF508).  This mutation causes the protein to fold incorrectly, making it more susceptible to degradation by proteases.  The resulting decrease in CFTR abundance inhibits chloride ion transport, leading to thickening of normally thin secretions and increased risk of life-threatening pulmonary infections. -A scientist proposed that phosphorylation of the R domain increases CFTR activity.  Which of the following functional groups in the R domain could be removed by mutation to test this hypothesis in vivo?