Exam 2: Biochemistry

Passage Interactions between positively charged histones and negatively charged nucleic acids are altered by acetylation and methylation of histone subunits.  These different post-translational modifications are recognized by histone-binding proteins such as BROMO domain Adjacent to Zinc finger 2B (BAZ2B).  BAZ2B has a net negative charge but contains a lysine-rich regulatory region between its two histone-binding domains.NMR data revealed potential interactions between BAZ2B and histone subunit H3 (Figure 1).  Binding was confirmed by isothermal titration calorimetry (ITC), which measures enthalpy of binding when a protein solution is titrated with ligand injections of equal volume and concentration.  Each injection results in a negative peak as heat is released.  Figure 2 shows ITC results when wild-type BAZ2B (WT) or BAZ2B with the regulatory region removed (10M+) were titrated with unmodified histone 3 (H3) or H3 that was acetylated at lysine-14 (H3K14ac).  In each case, the Hill coefficient was approximately 1.To test binding in a biological setting (in vivo), WT and 10M+ BAZ2B were extracted from cell lysates on affinity columns containing H3 or H3K14ac covalently attached to beads.  BAZ2B was then eluted and binding was quantified by Western blot (Figure 3). Figure 1  Electrostatic interactions between H3 and BAZ2B amino acid residues Figure 2  ITC traces (A) and K_d values (B) of WT BAZ2B and 10M+ BAZ2B titrated with either unmodified H3 or H3Ka4ac Figure 3  Western blot of BAZ2B recovered from unmodified or acetylated H3 columns Adapted from Kostrhon S, Kontaxis G, Kaufmann T, et al. A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding. J Biol Chem. 2017;292(43):17643-17657. -Based on the information in the passage, why does the size of the ITC peaks decrease over the course of each experiment in Figure 2?

Passage The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis.  Mtb secretes over 70 serine proteases that may be used as viable drug targets.  Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response.  Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme.  Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo.  A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1). Figure 1  Schematic design of Hip1 fluorogenic substrates.  P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide.  Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1  Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1 Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11):807-815. -In another experiment, researchers assessed peptides of various compositions for enzymatic cleavage to survey the substrate preferences of Hip1.  The results are shown in the table below. Going from P4 to P1, which sequence would be most suitable for the design of a selective Hip1 substrate?

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. -Which of the following could researchers add to restore oxygen consumption in the presence of drug X?

Passage The recent advent of multidrug-resistant Mycobacterium tuberculosis (Mtb) has led to the search for novel agents to combat tuberculosis.  Mtb secretes over 70 serine proteases that may be used as viable drug targets.  Hydrolase important for pathogenesis 1 (Hip1) is a serine protease found in Mtb with proteolytic activity that is required for dampening the host inflammatory response.  Given the role of Hip1 in Mtb virulence, analyzing Hip1 substrate specificity is a critical step in designing selective inhibitors for effective tuberculosis treatment.Fluorogenic substrates are nonfluorescent compounds that release a fluorescent reporter group when acted upon by an enzyme.  Such substrates are useful biomolecular imaging tools that allow monitoring of enzymatic activity in vitro and in vivo.  A large library of peptides was screened against Hip1, and enzymatic cleavage was analyzed using mass spectrometry to facilitate the design of fluorogenic substrates (Figure 1). Figure 1  Schematic design of Hip1 fluorogenic substrates.  P1, P2, P3, and P4 in Group 1 represent the positions of variable amino acids in the peptide.  Group 2 depicts the fluorescent compound released on Hip1 cleavage.Researchers reported Michaelis-Menten kinetic parameters for substrates from two peptide families: the WKLL substrates and the CSL substrates.Table 1  Kinetic Parameters of Fluorogenic Substrates for Mtb Hip1 Adapted from Lentz CS, Ordonez AA, Kasperkiewicz P, et al. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis. ACS Infect Dis. 2016;2(11):807-815. -Assuming that product formation is the rate-limiting step for all substrates, which of the following statements regarding the formation of the Hip1 enzyme-substrate (ES) complex is correct?

Each of the following molecules can be converted to oxaloacetate by a single enzymatic reaction EXCEPT:

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. -Which of the following statements correctly characterizes the bond that is broken during the reaction catalyzed by the IP6-dependent Tcd protease?

An infant who experiences seizures and has lactic acid buildup is shown to have deficient pyruvate dehydrogenase complex (PDHC) activity.  Genetic analysis shows that both the maternal and paternal alleles for all PDHC subunits are normal.  These results could indicate:

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. -The first step of the autoprocessing reaction mechanism described in the passage involves modification of a thiol in the active site by an amino acid functioning as a general base.  This action is likely to enhance protease activity by:

Passage Interactions between positively charged histones and negatively charged nucleic acids are altered by acetylation and methylation of histone subunits.  These different post-translational modifications are recognized by histone-binding proteins such as BROMO domain Adjacent to Zinc finger 2B (BAZ2B).  BAZ2B has a net negative charge but contains a lysine-rich regulatory region between its two histone-binding domains.NMR data revealed potential interactions between BAZ2B and histone subunit H3 (Figure 1).  Binding was confirmed by isothermal titration calorimetry (ITC), which measures enthalpy of binding when a protein solution is titrated with ligand injections of equal volume and concentration.  Each injection results in a negative peak as heat is released.  Figure 2 shows ITC results when wild-type BAZ2B (WT) or BAZ2B with the regulatory region removed (10M+) were titrated with unmodified histone 3 (H3) or H3 that was acetylated at lysine-14 (H3K14ac).  In each case, the Hill coefficient was approximately 1.To test binding in a biological setting (in vivo), WT and 10M+ BAZ2B were extracted from cell lysates on affinity columns containing H3 or H3K14ac covalently attached to beads.  BAZ2B was then eluted and binding was quantified by Western blot (Figure 3). Figure 1  Electrostatic interactions between H3 and BAZ2B amino acid residues Figure 2  ITC traces (A) and K_d values (B) of WT BAZ2B and 10M+ BAZ2B titrated with either unmodified H3 or H3Ka4ac Figure 3  Western blot of BAZ2B recovered from unmodified or acetylated H3 columns Adapted from Kostrhon S, Kontaxis G, Kaufmann T, et al. A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding. J Biol Chem. 2017;292(43):17643-17657. -Which statement best explains why the 10M+ form of BAZ2B binds H3 with greater affinity than its wild-type counterpart?

Passage The HIV-derived transactivator of transcription (Tat) is a short peptide with the sequence RKKRRQRRRPPQ.  When paired with liposomes (ie, synthetic vesicles) composed of cationic lipids, Tat can transport large molecules across cell membranes and has been studied as a vector for gene therapy.  However, Tat delivers cargo to cells with relatively low efficiency.  Researchers hypothesized that a Tat homodimer could yield stronger DNA binding than the monomer, which may improve the efficiency of DNA delivery.To study this hypothesis, Tat and two variant peptides (Variants 1 and 2) were produced by solid-phase peptide synthesis, in which the growing peptide chain is immobilized on polystyrene beads.  Unlike biological systems, solid-phase synthesis assembles peptides from the C-terminus to the N-terminus, as shown in Figure 1. Figure 1  Simplified schematic of one round of solid-phase peptide synthesis.  The protecting group is removed during deprotection.  The process is repeated until the peptide is complete.Variants 1 and 2 contained a C-terminal and an N-terminal cysteine residue, respectively.  Each variant was allowed to dimerize by disulfide bond formation.  Peptide-DNA binding interactions were then studied by mixing varying amounts of each peptide with plasmid DNA encoding luciferase protein (pLuc).  The resulting peptide-DNA complexes were run on agarose gels, with results displayed in Figure 2. Figure 2  Agarose gel results for pLuc plasmid exposed to varying amounts of each peptideEqual concentrations of each type of peptide-DNA complex were then incubated with cationic liposomes and exposed to cultured cells for 24 hours.  The cells were then assessed for luciferase activity.  Greater luciferase activity indicated more efficient delivery of the pLuc plasmid.  The results are shown in Figure 3. Figure 3  Plasmid delivery efficiency of three peptides, as measured by induced luciferase activity Adapted from Lee S-J, Yoon S-H, Doh K-O. Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond. J Microbiol Biotechnol. 2011;21(8):802-807. -Do the data in Figure 2 and Figure 3 support the hypothesis that stronger peptide-DNA interactions yield more efficient plasmid delivery?

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 -After galactose is released from GC and linearized, it contains all of the following functional groups EXCEPT:

Passage The Ankyrin repeat and SOCS box containing protein 4 (Asb-4) is highly expressed in the cytosol of hypothalamus cells during the well-fed state but is downregulated during fasting.  In addition, the protein insulin receptor substrate 4 (IRS4), known to be involved in appetite regulation, is expressed in the cytosol of the same cells as Asb-4.  Accordingly, it was hypothesized that Asb-4 suppresses appetite through interactions with IRS4.Small peptides such as myc (EQKLISEEDL) and flag (DYKDDDDK) are often attached to recombinant proteins to facilitate their purification and detection.  HEK293 cells were transfected with plasmids encoding Asb-4 and IRS4 tagged with myc or flag at their N-termini (myc-Asb4 and flag-IRS4, respectively).  Cell lysates were incubated with beads linked to α-flag, an antibody that binds flag, and proteins that did not bind were washed away.  The bound proteins were then visualized by Western blot using α-myc antibodies.  The α-myc antibody detected the presence of myc, indicating that myc-Asb-4 binds to flag-IRS4.To better understand interactions between Asb-4 and IRS4, cells were transfected with 1 μg of flag-IRS4 plasmid and varying amounts of either myc-Asb-4 plasmid or a plasmid with the sb domain deleted (myc-Asb-4Δsb).  Flag-IRS4 levels from each set of cells were then quantified, as shown in Figure 1. Figure 1  Flag-IRS4 levels in cells with varying amounts of myc-Asb-4 or myc-Asb-4Δsb plasmid addedIn a follow-up experiment, lysates from transfected cells were again incubated with the α-flag beads, and a Western blot was performed on the precipitated proteins using α-flag, α-myc, and an antibody against ubiquitin (α-ub).  The results are shown in Figure 2. Figure 2  Ubiquitin, flag-IRS4, and myc-Asb-4 levels in cells transfected with flag-IRS4 and either an empty vector (control), myc-Asb-4 plasmid, or myc-Asb-4Δsb plasmid Adapted from Li JY, Chai B, Zhang W, et al. Ankyrin repeat and SOCS box containing protein 4 (Asb-4) colocalizes with insulin receptor substrate 4 (IRS4) in the hypothalamic neurons and mediates IRS4 degradation. BMC Neurosci. 2011;12:95. -Which of the following is the most likely mechanism by which the sb region of Asb-4 decreases IRS4 levels?

Passage The ABO blood groups are determined by the polysaccharide antigens linked to sphingolipids on red blood cell membranes.  The polysaccharide known as the H-antigen (O-type blood) consists of a polysaccharide (R-group) glycosidically linked to D-galactose, which is connected to L-fucose in an α-1,2 linkage.  Type A and type B blood express the A- and B-antigens, respectively (Figure 1).  These antigens are modified forms of the H-antigen, in which D-galactose (B-antigen) or a derivative of D-galactose called GalNAc (A-antigen) is added in an α-1,3 linkage to the D-galactose moiety of the H-antigen. Figure 1  Structures of the H-, A-, and B-antigens.  The R-group represents additional carbohydrates.The addition of GalNAc or galactose to the H-antigen is catalyzed by an enzyme encoded by the ABO gene.  One allele of the gene encodes GalNAcyl transferase and produces the A-antigen, while another allele encodes galactosyl-transferase and yields the B-antigen (Figure 1).  The two alleles differ at seven nucleotide positions, summarized in Table 1.Table 1  Nucleotide Positions Where the Alleles Encoding GalNAcyl Transferase and Galactosyl Transferase Differ Individuals with both alleles express both antigens and have type AB blood.  Those with type O blood have a third allele that encodes an inactive form of the enzyme.  A study of blood type abundance was conducted, and the number of participants with each type is shown in Table 2.  Table 3 shows several fatty-acyl chains found in the sphingolipids to which the antigens were attached.Table 2  Number of Participants That Express Each Blood Type in a Study of 10,000 Individuals Table 3  Types of Sphingolipids to Which Polysaccharide Antigens Were Bound, as Determined by Mass Spectrometry (Note:  For fatty acid chains, the numbers on the left and right of the colon indicate number of carbons and number of C=C double bonds, respectively) -The D-galactose moiety of the A-antigen participates in how many glycosidic bonds?

Passage The ABO blood groups are determined by the polysaccharide antigens linked to sphingolipids on red blood cell membranes.  The polysaccharide known as the H-antigen (O-type blood) consists of a polysaccharide (R-group) glycosidically linked to D-galactose, which is connected to L-fucose in an α-1,2 linkage.  Type A and type B blood express the A- and B-antigens, respectively (Figure 1).  These antigens are modified forms of the H-antigen, in which D-galactose (B-antigen) or a derivative of D-galactose called GalNAc (A-antigen) is added in an α-1,3 linkage to the D-galactose moiety of the H-antigen. Figure 1  Structures of the H-, A-, and B-antigens.  The R-group represents additional carbohydrates.The addition of GalNAc or galactose to the H-antigen is catalyzed by an enzyme encoded by the ABO gene.  One allele of the gene encodes GalNAcyl transferase and produces the A-antigen, while another allele encodes galactosyl-transferase and yields the B-antigen (Figure 1).  The two alleles differ at seven nucleotide positions, summarized in Table 1.Table 1  Nucleotide Positions Where the Alleles Encoding GalNAcyl Transferase and Galactosyl Transferase Differ Individuals with both alleles express both antigens and have type AB blood.  Those with type O blood have a third allele that encodes an inactive form of the enzyme.  A study of blood type abundance was conducted, and the number of participants with each type is shown in Table 2.  Table 3 shows several fatty-acyl chains found in the sphingolipids to which the antigens were attached.Table 2  Number of Participants That Express Each Blood Type in a Study of 10,000 Individuals Table 3  Types of Sphingolipids to Which Polysaccharide Antigens Were Bound, as Determined by Mass Spectrometry (Note:  For fatty acid chains, the numbers on the left and right of the colon indicate number of carbons and number of C=C double bonds, respectively) -Ignoring the anomeric carbon, the fucose shown in Figure 1 is the dehydroxylated enantiomer of:

Passage The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD), a proline-rich domain, a DNA-binding domain (DBD), a tetramerization domain (TD), and a C-terminal regulatory domain (REG) on each subunit (Figure 1). Figure 1  Organization of the p53 domains (1-393 amino acids)Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage.  Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue.  However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2.  The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers.  Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex. Figure 2  ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1), which enhances signaling for the degradation of Nedd8 proteins.  Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1.  To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3). Figure 3  Half-lives of p53, phosphorylated p53 (p53-PO₄), and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16):6591-6. -Which statement best explains the relationship between Hdm2 and Nedd8 protein concentration?

Certain RNA molecules can fold into structures known as hairpins, as depicted below. Which of the following RNA hairpins is the most stable?

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 -Based on the data in Figure 1, a mutation of which amino acid residue has the greatest impact on HMBS stability?

Passage The liver plays a central role in maintaining blood glucose homoeostasis.  It contains the highest concentration of glycogen of any organ in the body and is one of few organs that can regenerate glucose from metabolic byproducts.  The liver can replenish glucose in muscle and brain tissue during times of fasting, and it can help prevent lactate accumulation during intense physical activity.Von Gierke disease is a rare autosomal recessive disorder that arises from inactivating mutations in the liver enzyme glucose 6-phosphatase (G6Pase).  It affects roughly 1 in 100,000 individuals, and the resulting loss of both hormonal sensitivity and enzymatic activity leads to hypoglycemia (low blood sugar).  Von Gierke can also cause an increase in blood acidity concurrent with lactate build-up.  Patients typically become symptomatic shortly after birth with convulsions, hyperventilation, and tremors.  Left untreated, patients may develop gout, osteoporosis, and life-threatening complications such as kidney failure and liver tumors.In an effort to examine the biochemical consequences of this disorder, scientists generated a genetically engineered mouse model with liver G6Pase expression under the control of the Tet-Off system at both alleles.  Under this system, addition of the small molecule tetracycline in the water source of the mouse rapidly shuts off G6Pase gene expression and therefore can recapitulate von Gierke disease.  Blood glucose and pH levels were measured daily in two groups of genetically engineered mice over 20 days: group A mice received no tetracycline and group B mice were given tetracycline starting at day 10.  All mice were fed identical low-sugar diets throughout the experiment. Adapted from Froissart R, Piraud M, Boudjemline AM, et al. Glucose-6-phosphatase deficiency. Orphanet J Rare Dis. 2011;6:27. -Treatment with which of the following could help counteract hypoglycemia in patients with von Gierke disease?

An increase in which of the following would lead to decreased ethanol production in yeast?

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. -In Figure 2, the most likely reason that researchers added succinate was to stimulate: