Bioc 385 Exam 4

Front 1

HMG-CoA reductase as rate limiting step

Back 1

HMG-CoA to mevalonate by HMG-CoA reductase is the rate limiting step in the biosynthesis of cholesterol. The reaction requires oxidation of 2NADPH + H+ → 2NADP+.

Front 2

Stage 1 of Cholesterol Biosynthesis

Back 2

2 Acetyl-CoA is the input to start the reaction

Acetoacetyl CoA is converted to HMG-CoA via HMG-CoA synthase

HMG-CoA is converted to mevalonate via HMG-CoA reductase (rate limiting)(requires oxidation of 2 NADPH)

Front 3

Stage 2 of Cholesterol Biosynthesis

Back 3

Mevalonate is the input to begin stage 2 and is phosphorylated by mevalonate 5-phosphotransferase and again by phosphomevalonate kinase to ultimately get 5-pyrophosphomevalonate

Pyrophosphomevalonate decarboxylase converts 5-pyrophosphomevalonate to isopentyl pyrophosphate and dimethylallyl pyrophosphate (requires input of ATP → ADP+Pi+CO2)

Isopentyl pyrophosphate and dimethylallyl pyrophosphate are isomers of each other

Result is 5-carbon unit

Front 4

Stage 4 of Cholesterol Biosynthesis

Back 4

Isopentyl pyrophosphate and dimethylallyl pyrophosphate together are converted to geranyl pyrophosphate by prenyltransferase (Pi is lost)

Farnesyl phosphate is converted to squalene via squalene synthase (loss of 2PPi, NADPH + H+ → NADP+)

6 5-carbon units → 30-carbon squalene

Front 5

LCAT

Back 5

lecithin cholesterol acyl transferase

Converts membrane cholesterol to cholesterol esters to be transported back to the liver by HDL

Converts membrane phosphatidylcholine to serum lysolecithin

Catalyzes esterification of cholesterol from peripheral cell membranesHigh LDL levels associated with high risk of atherosclerosis & myocardial infarction bc it becomes oxidized and scavenged by macrophages causing plaques

Front 6

ACAT

Back 6

acetyl-CoA acetyl transferase

Occurs in situations of high cholesterol

Cholesterol is converted to cholesterol esters (fatty acyl-CoA → CoA required as well)

Inside the cellCatalyzes esterification of cholesterols directly synthesized from liver

Front 7

General function to increase LDL receptors and HMG-CoA reductase

Back 7

Pathway is activated when cholesterol levels are low

Low levels of cholesterol initiate proteolytic activation of SREBP

Active SREBP activates the LDL receptor gene, HMG-CoA reductase gene, and HMG-CoA synthase gene

LDL receptor gene increases transcription of LDL receptors; Allows for endocytosis of serum LDL

HMG-CoA synthase and reductase genes; Transcribes HMG-CoA reductase and synthase enzymes; Work in conjunction to increase mevalonate production in stage 1, and increases flux through cholesterol pathway.

All work together to create an overall increase in cholesterol levels

Front 8

SREBP Storage Site

Back 8

stored in the endoplasmic reticulum

Front 9

Three Domains of SREBP

Back 9

1.) an N-terminal domain, encoding the DNA binding and gene regulatory functions

2.) a membrane anchoring domain, consisting of 2 transmembrane alpha helices joined by a 30 amino acid loop protruding into the ER lumen

3.) a C-terminal regulatory domain, which interacts with a cholesterol sensing protein embedded into the ER membrane (SCAP)

Front 10

Three SREBPs characterized in animals

Back 10

1.) SREBP-1cSelective activation of genes involved in lipogenesis

2.) SREBP-1aActivates genes involved in BOTH lipogenesis and cholesterol biosynthesis *** SREBP-1a and 1c are both encoded by the same gene

3.) SREBP-2Activation of genes involved in cholesterol biosynthesis

***all 3 isoforms induce gene expression involved in NADPH production.

Front 11

SCAP and INSIG

Back 11

Both retain precursors of SREBP in the ER

Cholesterol binds to SCAP. 25-hydroxycholesterol binds to INSIG; Results in SCAP-INSIG-SREBP complex; Low cholesterol activates SREBP-2 and 1c, and causing dissociation of SREBP-SCAP from INSIG, and goes to golgi.; SREBP is cleaved by site 1 protease in golgi, binding domain is released by site 2 protease. Binding domain is sent to nucleus.

Front 12

Role of Proteases

Back 12

cut off SCAP and regulatory domain to release it from anchoring domain, allows DNA binding domain to move to the nucleus and bind DNA

Front 13

Benefits of Statin

Back 13

Statins bind and inhibit HMG-CoA reductase, preventing HMG-CoA conversion to mevalonate

Decreases serum cholesterol, and activates SREBP

Increases LDL receptor gene expression

Increases endocytosis of LDL into the cell. This removes excess LDL from blood (incre

Front 14

Chylomicrons

Back 14

contain mainly exogenous lipids (dietary) in the form of triacylglycerides. They are produced in the small intestine and carry lipids from the small intestine to the rest of the body

Front 15

VLDL

Back 15

transport endogenous triglycerides and cholesterol from the liver. They are produced in the liver and converted to IDL then to LDL in the bloodstream

Front 16

IDL

Back 16

Carry cholesterol and a smaller amount of triglycerides from the liver. Produced from breakdown of VLDL

Front 17

LDL

Back 17

transports only cholesterol from the liver and are uptaken into cells via receptor mediated endocytosis

Front 18

HDL

Back 18

removes cholesterol from peripheral tissue and takes it back to liver, making them beneficial.

Front 19

APO CII

Back 19

a component of VLDL and chylomicrons (also IDL and HDL) . Activates lipoprotein lipase in capillaries and hydrolyzes triglycerides to produce free fatty acids.

Front 20

APO E

Back 20

found in chylomicrons, HDL, and IDL. Receptors located on the liver to transport cholesterol into liver.

Front 21

PKU

Back 21

defect in Phenylalanine hydroxylase enzyme

Symptoms bc of phenylalanine metabolite buildup, phenylpyruvate, phenylacetate, and phenyllactate in the brain

Primary treatment: limit amount of phenylalanine in diet (this includes dietary limitation of aspartame-- which is hydrolyzed to phenylalanine and aspartate in the stomach.)

Difficult to limit exogenous phenylalanine because we can’t cut it out completely- we still need some for protein synthesis.

Despite the fact that people with PKU cannot synthesize tyrosine from phenylalanine, they are not albinos b/c they obtain tyrosine in their diets and can make melanin.

Front 22

Albinism

Back 22

mutation in tyrosinase enzyme

Symptoms are due to lack of product (melanin), tyrosine can’t be processed into into eumelanin and pheomelanin

Technically not “treatable” like PKU.

Front 23

Nitrogen Assimilation

Back 23

Key Enzyme: Glutamine synthetase, incorporates NH4+ into glutamate to make glutamine, uses ATP; glutamate + NH4 → Glutamine (ATP → ADP + Pi)

Key Enzyme: Glutamate synthase, works with glutamine synthetase to replenish glutamate so glutamine synthetase isn’t substrate limited; Glutamine + alpha-ketoglutarate → 2 glutamate (requires NADPH+ + H+ → NADP+)

Net reaction is Nitrogen Assimilation: A-ketoglutarate + NH4 + ATP + NADPH + H → Glutamate + NADP + ADP + Pi

Front 24

Nitrogen Fixation

Back 24

Key Enzyme: Bacterial Nitrogenase complex → redox reactions coupled with ATP hydrolysis to convert N2 to 2 NH3; N2 + 8H + 8e- + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi

Front 25

Explain why feeding a single 15N-labeled amino acid to an animal can result in accumulation of many 15N-labeled amino acids in proteins

Back 25

Aminotransferase pairs with many other aminotransferases and keto acids

Front 26

Carbamoyl phosphate synthetase I

Back 26

catalyzes rate limiting rxn, is key regulated step in pathway, happens in mitochondrial matrix (in mito). Bicarbonate is phosphorylated by ATP to form carboxyphosphate which is attacked by NH3 to form carbamate which is then phosphorylated by another ATP to form carbamoyl phosphate

Front 27

Ornithine transcarbamoylase

Back 27

combines carbamoyl phosphate and ornithine to make citrulline which is transported from mitochondrial matrix to cytosol (in mito)

Front 28

Argininosuccinase synthetase

Back 28

converts citrulline to arginosuccinate, in cytosol

Front 29

Arginosuccinase

Back 29

converts arginosuccinate to arginine, in cytosol

Front 30

Arginase

Back 30

converts arginine to ornithine, in cytosol

Front 31

Purines

Back 31

Adenine, Guanine, double ring structure

Front 32

Pyrimidine

Back 32

Cytosine, Thymine, Uracil, single ring structure

Front 33

Nucleoside

Back 33

base+sugar, ends in “ine”

RNA nucleosides: adenosine, guanosine, cytidine, uridine

DNA nucleosides: deoxyadenosine...etc. Plus deoxythymidine

Front 34

Nucleotide

Back 34

(base+sugar+phosphate); phosphorylated nucleoside, ends in “ylate”

RNA nucleotides: adenylate, guanylate, cytidylate, uridylate

DNA nucleotides: deoxyadenylate...etc. Plus deoxythymidylate

Front 35

Severe combined immunodeficiency disease

Back 35

Adenosine deaminase deficiency, accumulate large amounts of adenosine and deoxyadenosine and imbalance of nucleotides so immunodeficiency disease because affect cells involved in immune system

Front 36

Hyperammonemia

Back 36

deficiencies in carbamoyl phosphate synthetase-1, and/or ornithine transcarbamoylase (or any of the enzymes involved in the urea cycle) build up ammonia in mitochondria, diffuse to blood and brain

Front 37

Lesch-Nyhan

Back 37

HGPRT enzyme deficiency -- responsible for recycling purine bases (sticks it onto phosphorylated ribose and regenerates nucleotides)

Front 38

Gout

Back 38

PRPP enzyme over activity causes uric acid build up in synovial fluid of joints causing inflammation

From quiz: Defects in the enzyme hypoxanthine-guanine phosphoribosyltransferase

Front 39

Cancer drug resistance

Back 39

PROTEIN: P-glycoprotein OR “MDR” - multidrug resistance protein

Normal cells: have these P-glycoproteins that normally rid the cell of potentially harmful foreign molecules

CANCER cells: have elevated expression of the P-glycoproteins (selective advantage), that spit out all the cancer-killing drugs (good for cancer, bad for human)

THE DRUG:Verapamil = multidrug resistance protein inhibitor - PREVENTS rapid drug efflux that occurs when cocktail of other drugs used to treat cancer and cell “rebels” by building channels to spit them all out

Now for DHFR (PROTEIN):DHFR (dihydrofolate reductase) is involved in synthesis of dTMP (pathway to DNA synthesis), methotrexate & aminopterin (anti-cancer drugs) inhibit DHFR, thus indirectly inhibiting DNA synthesis.

Cancer cells can resist methotrexate via gene amplification, which increases expression of DHFR, overcoming the inhibitory effects of the drug, allowing for adequate dTMP synthesis for cancer cells to continue to synthesize DNA and grow/divide

Front 40

Thymidylate synthase

Back 40

can be inhibited by uracil based compounds like 5-fluorouracil (5-FU) or 5- fluorodeoxyuridine or Raltitrexed (RTX) which binds to TS active site

Front 41

DHFR

Back 41

targeted by methotrexate (MTX) and aminopterin which prevents DNA synthesis. DHFR is an enzyme involved in dTMP synthesis, so MTX and aminopterin indirectly inhibit DNA synthesis

Front 42

xanthine oxidase

Back 42

Allopurinol inhibits uric acid production here to treat Gout

Front 43

Carbamoyl phosphate synthetase I

Back 43

combines HCO3- and NH4 and uses 2 ATP to make carbamoyl phosphate, catalyzes rate limiting step of urea cycle, occurs in mitochondrial matrix, urea production

Front 44

Carbamoyl phosphate synthetase II

Back 44

uses glutamine not NH4, occurs in cytosol, pyrimidine production

Front 45

Glucagon receptors are only found in liver and adipose cells. Why?

Back 45

Glucagon signaling in liver stimulates glucose export by increasing gluconeogenesis and glycogen degradation.

Activation of glucagon receptors in liver increases levels of cyclic AMP, activating PKA signaling, PKA phosphorylates PFK-2/FBPase-2 leading to increased gluconeogenesis and decreased glycolysis.

No glucagon receptors in skeletal muscle or brain cells because we need these tissues to have an adequate fuel source during starvation for survival.

Muscle and brain cells: generate fuel for that cell only.

Liver and adipose cells: generate fuel for the whole body.

Skeletal Muscle and Brain contain insulin receptors but no glucagon receptors.

Front 46

PPAR Effects in Liver

Back 46

Fasting response, stimulate flux through Pentose Phosphate Pathway, stimulate lipid synthesis, improve insulin sensitivity, fatty acid oxidation.

Front 47

PPAR Effects in Skeletal Muscle

Back 47

Fatty acid oxidation, thermogenesis, energy uncoupling, and improved insulin sensitivity.

Front 48

PPAR Effects in Adipose

Back 48

adipose differentiation, lipid synthesis, insulin sensitivity, fatty acid oxidation, energy uncoupling and thermogenesis

Front 49

Four major changes in Metabolic Flux during starvation

Back 49

1. Release fatty acids from adipose (mobilize fats)

2. Increased gluconeogenesis in liver and kidney

3. Increased ketogenesis in liver (conversion of fatty acids to ketone bodies)(brain needs ketones)

4. Protein degradation in skeletal muscle (provides carbons for glucose synthesis)

**During starvation: body is trying to spare glucose for brain cells and erythrocytes, so it starts utilizing fatty acids (broken down into ketone bodies in liver via ketogenesis) as an energy source.

Front 50

Thrifty Gene Hypothesis

Back 50

Body tries to store fuel in times of plenty to ensure it has enough fuel in times of famine. This was helpful when we were hunters and gatherers and had to work hard to find our food, but is backfiring epicly on us as we are seeing the “diabesity” epidemic get worse and worse because most Americans are always in times of plenty now.

Front 51

Leptin vs. Insulin

Back 51

Leptin: Signaling reduces fat storage. Sends signals to the hypothalamus to release melanocyte signaling hormone (MSH) to decrease fat storage.; α-MSH is released by POMC neurons, signals satiety and stimulates fat metabolism; Controls appetite and energy expenditure; Reduces fat storage through a combination of decreased appetite and increased energy expenditure

Insulin: Associated with fed state--causes glucose uptake, activates glycogen and fatty acid synthesis (sequesters glucose), decreases appetite.; In most tissues, not RBCs. Increases anorexigenic signaling, inhibits orexigenic.

**Leptin and Insulin have same effect on POMC (activate) and NPY/AGRP (inhibit) neurons.

Front 52

Type 1 vs. Type 2

Back 52

Type 2: “Adult onset”: patients usually obese, synthesize insulin but are insulin resistant due to chronic hyperlipidemia and hyperglycemia. Need metformin (drug) which stimulates AMPK activity and reduces glucose and fat; Best treatment: lifestyle modification. Change diet and exercise more.

Type 1- Autoimmune disease, usually not obese. Don’t synthesize insulin but are insulin sensitive, so insulin shots are usually the only necessary treatment

Front 53

Fats

Back 53

Polyunsaturated (omega-3 > omega-6) > saturated > trans.

Saturated and trans fats promote pro-inflammatory diet, polyunsaturated promote anti-inflammatory diet.

Bad fats increase LDLs and lower HDLs, good fat increase HDLs and lower LDLs

Front 54

High vs low glycemic index

Back 54

High glycemic: simple carbohydrates (white bread, sugary foods, etc) promote pro-inflammatory diet

Low glycemic: diet rich in fruits and vegetables, complex carbs, fiber. Promote anti-inflammatory diet

Front 55

Anti vs pro-inflammatory diets

Back 55

Pro-inflammatory: high levels of TNF-α, low adiponectin. TNF-α is an inflammatory cytokine that can inhibit insulin signaling and lead to insulin resistance.

Anti-inflammatory: high adiponectin, low TNF-α. Increased adiponectin maximizes AMPK signaling which results in increased insulin sensitivity (we want this).

Front 56

Identify the 3 mechanisms that account for phosphorylation of AMPK causing a net increase in ATP synthesis

Back 56

Fatty acid Oxidation

Oxidative Phosphorylation

Glycolysis