Exam 2: Biochemistry

Passage Antimicrobial peptides are an important component of innate immunity.  Since their discovery in 1939, over 5,000 peptides with antimicrobial properties have been isolated from organisms in every domain of life.  They have demonstrated activity against enveloped viruses, bacteria, fungi, and even tumor cells and have recently gained interest for their potential in fighting antibiotic-resistant bacteria.  Most known antimicrobial peptides are positively charged, but some with negative charges have also been discovered.Frogs are known to secrete a variety of antimicrobial peptides as a major source of immunity.  Secretions from a new species of frog were collected and the peptides were separated by cation exchange chromatography.  Four of the isolated peptides demonstrated strong antibacterial activity and were characterized by isoelectric focusing and sequencing: Figure 1  Results of isoelectric focusing of four antimicrobial peptidesTable 1  Amino Acid Compositions of Four Antimicrobial Peptides Adapted from Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res. 2004;32(Database issue):D590-2. -In which order will the four peptides elute from the cation exchange column as pH is increased from 7 to 12?

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 step is NOT required to generate the western blot shown in Figure 3?

Passage Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is one of the most abundant enzymes on Earth.  In carbon-fixing organisms, it consumes CO₂ to carboxylate the sugar ribulose-1,5-bisphosphate (Ru1,5BP), and forms two molecules of 3-phosphoglycerate (3PG) as a product.  The functional form of rubisco from the bacterium Rhodospirillum rubrum is a noncovalent homodimer composed of two 51 kDa monomers.  Each subunit requires a magnesium ion as a prosthetic group in the active site.Rubisco is often used as a model for protein folding studies.  On their own, rubisco monomers typically cannot fold to completion and instead are trapped in a form known as a "kinetic intermediate."  These kinetically trapped monomers cannot dimerize correctly and are prone to aggregation.Scientists interested in studying protein folding dynamics labeled the rubisco kinetic intermediate with a set of fluorophores known as a FRET (fluorescence resonance energy transfer) pair, with one fluorophore at the N-terminus and the other at the C-terminus.  A FRET signal occurs when the emission spectrum of one fluorophore (the donor) overlaps with the excitation spectrum of the other (the acceptor).  When the two fluorophores are sufficiently close to each other, the donor can transfer energy from the light it absorbs directly to the acceptor, causing the acceptor to emit light when the donor absorbs light.The GroEL/ES complex is a chaperone protein that binds and releases misfolded proteins, hydrolyzing ATP each time the protein is released.  A misfolded protein may undergo multiple rounds of binding and release before adopting its correct conformation, or it may never fold correctly and instead be targeted for destruction (Figure 1). Figure 1  Representation of GroEL/ES-mediated protein foldingResearchers diluted the labeled kinetic intermediate into a refolding buffer in the presence or absence of 200 nM GroEL/ES and then monitored the FRET signal over time.  The results are shown in Figure 2. Figure 2  FRET signal of labeled rubisco with and without GroEL Lin Z, Rye HS. Expansion and compression of a protein folding intermediate by GroEL. Mol Cell. 2004;16(1):23-34. -Which of the following is true concerning magnesium in rubisco?The rubisco-Mg²⁺ interaction has a small K_d.Mg²⁺ is a cofactor of rubisco.Mg²⁺ activates rubisco by binding at an allosteric site.

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. -Prolonged starvation in patients diagnosed with hypertriglyceridemia would most likely promote:

Passage Myalgic encephalopathy (ME), or chronic fatigue syndrome (CFS), is a physiological disease that affects 0.15% of the human population.  Compared with healthy individuals, persons with ME experience reduced phospholipid levels, muscular degeneration, urinary urgency, and other symptoms that contribute to a lower quality of life.  Studies in resting subjects have yielded inconsistent data and failed to identify a unique metabolic defect in patients with ME.Analyzing the blood plasma of male and female patients with ME, physicians have observed that male patients display elevated levels of 3-methylhistidine, a marker of protein catabolism.  Researchers expanded this finding by measuring the serum concentration of amino acids in blood samples obtained from nonfasting ME patients and healthy controls.  Amino acids were classified into separate categories according to their metabolic intermediates.Table 1  Classification of Amino Acids in Blood Plasma Figure 1  Serum concentrations of amino acids in patients with ME and healthy controls (HC)Next, researchers hypothesized that the inhibition of pyruvate dehydrogenase (PDH) and impairment of fatty acid oxidation were responsible for the fluctuations in amino acid levels.  PDH function and lipid metabolism were studied in peripheral blood mononuclear cells (PBMCs) by measuring the gene expression of proteins within regulatory pathways.  Researchers monitored the mRNA levels of the inhibitor pyruvate dehydrogenase kinase 1 (PDK1) and of sirtuin 4 (SIRT4), which removes lipoic acid groups from the PDH complex.  They also examined lipid metabolism by measuring the mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), a transcription factor that upregulates the uptake of fatty acids.  The mRNA level of each protein in PBMCs of patients with ME was evaluated against healthy controls, as shown in Figure 2. Figure 2  Gene expression of PDK1, SIRT4, and PPARα in PBMCs Adapted from Fluge Ø, Mella O, Bruland O, et al.. JCI Insight. 2016;1(21):e89376. -Lipids serve as energy stores for healthy patients and patients with ME.  If both groups were fed the same diet, would the rate of lipid synthesis after a meal be faster in healthy individuals or in patients with ME?

Passage Dystroglycan (Dg) is a transmembrane protein that mediates interactions between the cytoskeleton and the extracellular matrix (ECM).  It binds the ECM protein laminin in an interaction that is required for intercellular communication and muscle membrane structural stability.  The Dg-laminin interaction is facilitated by a long carbohydrate chain called an O-mannosyl glycan, which is covalently linked to Dg (Figure 1). Figure 1  Schematic of interaction between Dg and laminin, mediated by the Dg-linked O-mannosyl glycanThe O-mannosyl glycan is added to Dg by a series of enzymes called glycosyltransferases.  The first enzyme in this series, protein O-mannosyltransferase (POMT), attaches D-mannose to the hydroxyl groups of serine and threonine residues in an α-linkage through the anomeric carbon.  The next sugar in the chain then forms a β-1,4 linkage to the mannose residue, followed by several more steps to produce the full glycan.  Genetic inactivation of any of the enzymes in the pathway results in an incomplete glycan and failure to bind laminin, and is the underlying cause of several severe forms of muscular dystrophy known as dystroglycanopathies.Researchers investigating dystroglycanopathies recently discovered the functions of two enzymes in the pathway:  protein O-mannose kinase (POMK) and fukutin.  POMK phosphorylates Dg-linked mannose at carbon 6, and fukutin adds the sugar ribitol 5-phosphate to the glycan chain.  To assess the roles of POMK and fukutin relative to each other, scientists conducted experiments in which lysates from muscle cells deficient in either POMK or fukutin were incubated with ³²P-labeled ATP or ³H-labeled activated ribitol 5-phosphate.  Combined lysates containing both fukutin and POMK were also tested.  Dg was then purified and detected by autoradiography and western blot using an antibody against Dg, as shown in Figure 2. Figure 2  Autoradiograms and western blots of Dg purified from lysates incubated with ³²P-labeled ATP or ³H-labeled ribitol 5-phosphate Adapted from Kanagawa M, Kobayashi K, Tajiri M, et al. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy. Cell Rep. 2016;14(9):2209-23. -O-linked D-mannose can form a glycosidic bond with any sugar that has a free anomeric carbon. Therefore it could theoretically form a glycosidic bond with all of the following sugars EXCEPT:

Passage Glycolysis and gluconeogenesis are tightly regulated, opposing metabolic pathways that help control blood glucose levels.  Glycolysis converts glucose to two pyruvate molecules, whereas gluconeogenesis consumes 6 ATP equivalents to convert two pyruvate molecules back to glucose.  When glycolysis is upregulated, gluconeogenesis is downregulated, and vice versa.As shown in Figure 1, glycolysis and gluconeogenesis in the liver are largely regulated by the allosteric action of the small molecule fructose-2,6-bisphosphate (F2,6BP) on the enzymes phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F1,6BPase).  PFK-1 is a kinase that uses ATP to phosphorylate fructose-6-phosphate (F6P) in an irreversible step of glycolysis, forming fructose-1,6-bisphosphate (F1,6BP) and ADP.  During gluconeogenesis, F1,6BPase removes the phosphate group by hydrolysis. Figure 1  Activities of (A) PFK-1 and (B) F1,6BPase in the presence (solid lines) and absence (dashed lines) of F2,6BPA bifunctional enzyme that contains a phosphofructokinase-2 (PFK-2) domain and a fructose-2,6-bisphosphatase (F2,6BPase) domain controls F2,6BP levels in the liver.  The PFK-2 domain converts F6P to F2,6BP, and the F2,6BPase domain converts F2,6BP back to F6P.  When blood glucose levels are low, the enzyme becomes phosphorylated.  This phosphorylation event simultaneously activates the F2,6BPase domain and inactivates the PFK-2 domain.  Under high blood glucose conditions, the enzyme becomes dephosphorylated, activating the PFK-2 domain and inactivating the F2,6BPase domain. -All of the following statements about PFK-1 and F1,6BP are correct EXCEPT: