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1

Specific targets of apoptosis

Thymocytes that recognize self antigens

Virus infected cells

Defective cells

Unnecessary cells during development: webbing between digits

Excess cells like neurons that fail to make appropriate connections

Cells that have exceeded their desired lifespan

Cancer cells (via therapeutic treatment)

2

Syndactyly

Two or more digits are fused together, can be soft tissue or even bones

Failure of apoptosis to remove the webbed digits that develop during fetal development

3

Morphology of Apoptosis

Chromatin condensation and DNA fragmentation by endonucleases to form a laddered appearance in electrophoresis

Progressive cell shrinkage by cytoskeleton degradation

Plasma membrane blebbing

Apoptotic bodies: membrane bound cell fragments, doesn't result in inflammation, phagocytes recognize the DPPS to eat them

4

Diseases linked with excessive apoptosis

AIDS: progressive loss of T lymphocytes due to apoptosis

Alzheimer's: some of the proteins in the amyloid plaques can trigger capases

Parkinson's: mutation for inhibitor of apoptosis is linked to Parkinson's

Stroke or ischemic injury

Toxic-induced diseases: alcohol can induce apoptosis in neurons and hepatocytes

5

Diseases linked with suppression of apoptosis

Autoimmune disorders: don't remove self-reactive immune cells

Cancer: tumor growth can be stimulated by cell cycle proliferation or suppression of apoptosis

6

Three pathways of apoptosis

Intrinsic (mitochondrial dependent): triggered by cellular stress

Extrinsic (death receptor mediated): triggered by soluble factors

Granzyme B: triggered by lymphocyte recognition

7

Bcl-2 families

24 different family members that can be pro or anti-apoptotic, many regulator proteins to respond to different types of cellular stress, which is the primary trigger for the intrinsic pathway

BH1-4: anti-apoptotic domain (Bcl-2)
BH1-3: pro-apoptosis domain (Bax family)
BH3: pro-apoptosis (BH3-only family)

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Bcl-2 Pathway

1. Pro-apoptosis BH3-only family proteins are activated by different types of cellular stress: regulated by transcriptional and post-translational mechanisms, anoikis is cell death due to detachment from substrate

2. Activation results in release of BH3-only proteins from sequestration: can be attached to cytoskeletal proteins, Bad is sequestered to protein 14-3-3 when phosphorylated, Bid is inactive until cleaved by granzyme B or caspase-B

3. BH3-only proteins can bind to pro-survival proteins like Bcl-2 and keeps them from binding/inactivating Bax family proteins (Bax and Bak)

4. Free Bax and Bak create channels in the mitochondrial outer membrane that allow cytochrome C to leak out: 7 molecules of Apaf-1 combine with 7 cytochrome C molecules to form the apoptosome (wheel of death), recruits and activates procaspase-9

9

Caspases

Cysteine-dependent aspartyl-directed proteases

Synthesized as zymogens so need to be cleaved for activation

Initiators: activate other caspases in a cascade (2,8,9,10)

Effector/Executioners: do damage to the cellular structures that result in apoptosis (3,6,7)

Other 7 caspases involved in inflammation control, processing of cytokines, and not involved in apoptosis

10

Other apoptosis regulatory proteins

Inhibitors of Apoptosis (IAP): found in cytoplasm

Smac (DIABLO): promotes apoptosis, mitochondrial protein that is released with cytochrome C, binds to and inactivates IAPs

11

Non-caspase-mediated death

AIF: apoptosis-inducing factor

Located in the intermembrane space of mitochondria, released when mitochondria permeabilized by Bax/Bak

Travels to nucleus, induces nuclear chromatin condensation and DNA fragmentation

12

Extrinsic apoptosis pathway

Ligand binding to death receptors causes the cytoplasmic tails to bind the Fas-associated death domain (FADD), death receptors are part of the tumor necrosis factor (TNF) family of receptors

Death-inducing signaling complex (DISC): receptor tail, FADD, and procaspases 8 and 10

FADD has a death effector domain (DED) that recruits procaspase 8 and allows for its activation to caspase 8

Caspase 8 allows for cross talk between intrinsic and extrinsic pathways, is an initiator for caspases 3,6, and 7

13

Granzyme B

Serine protease that is released by cytotoxic T cells and NK cells, causes apoptosis of virally infected cells

Released with perforin, which helps it enter infected cells

Activates the BH3-only protein Bid by cleaving it, also directly activates executioner caspase 3 and initiator caspase 8

14

Tumor suppressor p53

Transcription factor that is upregulated in response to multiple types of cell damage like hypoxia

Upregulates transcription of BH3-only pro-apoptosis proteins like Bax to trigger the intrinsic apoptosis pathway

Interacts with Bax/Bak to promote their oligimerization and cause mitochondria permeability

Promotes transcription of various death receptors for the extrinsic apoptosis pathway

Upregulates TFs that are secreted by the cell and bind to survival cytokines, blocking the survival cytokine pathway and triggering apoptosis

15

Heteroplasmy

The differences in the ration of normal and abnormal mtDNA among cells in a particular tissue/organ

Threshold effect: certain ratio of normal:abnormal mitochondria must be crossed in order for symptoms to develop

Mitochondria dive and even fuse together, do so independent of host cell replication

16

Myoclonic Epilepsy Ragged-Red Fibers
(MERRF)

Defective respiratory enzyme function and ATP production

Mutation in tRNA Lys

Myoclonic Epilepsy with short stature, hearing loss, lactic acidosis

Ragged-red fibers present in muscle biopsies

17

3 functions of cristae

Part of intermembrane space that project into the matrix

1. Perform redox reactions of the ETC, enzymes project into matrix

2. Synthesize ATP via ATP synthase

3. Regulate transport of metabolites into/out matrix

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Intermembrane Space

Contains enzymes like creating kinase, adenylate kinase (converts ATP and AMP to 2 ADP), and cytochrome C

19

Mitochondrial Damage

Multiple etiologies: trophies factor withdrawal, protein misfolding, DNA damage from radiation/ROS/toxins, drugs, anoxia

Multiple Disease states: psychiatric disorders, dementias, strokes, heart diseases, autoimmune disorders

20

Cytochrome c Oxidase

Complex IV

Site of cyanide, azide (NaN3), and CO toxicity

Cyanide and azide bind to Fe3+ in the heme a subunits

CO binds to the Fe2+ in the heme a3 subunits

Prevents the transport of electrons in the ETC, reduction of ETC transporters and loss of oxidized forms, loss of H+ gradient needed for ATP synthase

21

Na+/K+ Pump

1. Binding of cytoplasmic Na+ stimulates phosphorylation by ATP

2. Phosphorylation causes the protein to change its conformation and face outside the cell

3. Na+ expelled and 2 K+ binds

4. K+ binding triggers release of phosphate group

5. Loss of phosphate restores original conformation facing the cytoplasm

6. K+ is released and Na+ can bind again

22

Cell Swelling

Lumen diameter is smaller in swollen cells for the kidney tubules

Swollen cells have clear finely stained cytoplasm, normal cells have denser pink (eosinophilic) stain

Normal cells have central nucleus, swollen cells have peripheral nucleus

Proximal convoluted tubules have more mitochondria so more susceptible to hypoxia injury and swelling, while DCT and glomerulus don't swell as easy

Mitochondria and rER can swell also

Due to mitochondrial damage that leads to decreased Na/K pump activity, Na+ and water come in while K+ leaves

23

4 Mechanisms of Intracellular Accumulations

1. Abnormal metabolism: inadequate removal of a normal substance due to impaired packaging and intracellular transport

Fatty liver or steatosis

2. Impaired protein folding: can be acquired or inherited via mutation

Leads to defects in protein packaging, intracellular/extracellular transport, and/or exocytosis

Alpha1-antitrypsin

3. Inherited enzyme deficiencies: failure to degrade a metabolite due to enzyme deficiency

Lysosomal storage diseases

4. Deposition and accumulation of an exogenous substance: cells lack enzymatic capability to degrade or transport the exogenous substance

Accumulation of silica or carbon in occupational exposures

24

Steatosis

Normal liver is brown to dark red but fatty liver is enlarged and yellow

Hepatocytes have clear cytoplasmic vacuoles that look like soap bubbles, contain triglycerides

Caused by too much fat synthesis or inadequate transport (protein) leads to steatosis

Nucleus pushed to side of cell

Variably sized vacuoles due to merging

H&E stain gives clear vacuoles as an artifact, Oil Red O stain has red stain that binds to lipids

25

Accumulation of cholesterol

Intracellular accumulation of cholesterol is common in macrophages in patients with xanthomas, atherosclerosis, and inflammation

Xanthomas: fatty deposits under joints, hypercholesterolemia (hyperlipidemia) creates yellowish nodules under tendons of the heel/knee

Tissue macrophages actively take up LDL cholesterol and store it, foamy macrophages

26

Lysosomal Storage Diseases

Genetic mutation that alters the function of a lysosomal enzyme in a catabolic pathway

Accumulation of the substrate or accumulation of degradation intermediaries, which may be toxic to cells

Decreased production of the pathway's product

27

Gaucher Disease

Sphingolipid storage disorder resulting from glucocerebrosidase deficiency

Deposition of glucocerbrosides in macrophages in the liver, spleen, lymph nodes, or bone marrow

Lipid-laden macrophages look like wrinkled-tissue paper, not foamy appearance

28

Intracellular Accumulation of Proteins

Excess protein reabsorption in renal tubular cells: disorder with heavy protein leakage across glomerulus, tubular cells have increased protein reabsorption

Excess Protein Synthesis: excess synthesis of antibodies by plasma (Mott) cells can outpace secretion, creates dilated endosomes called Russell bodies

Defective transport and secretion from gene mutation: alpha 1 - antitrypsin deficiency is recessive, misfolded proteins build up in liver ER and not secreted, causes emphysema in lungs

Aggregates of cytoskeleton proteins: keratin filaments in alcoholic liver disease, Mallory bodies, also in Alzheimer's

Intracellular accumulation of pigments: endogenous and exogenous

29

Pompe's Disease

Normal blood sugar levels

Severe cardiomegaly

Glycogen accumulation in lysosomes

Normal glycogen structure

Recessive, deficiency of alpha-glucosidase in lysosome, converts glycogen to glucose

30

Intracellular accumulation of endogenous pigments

Lipofuscin: collection of lipids and proteins that can't be metabolized, multiple small golden brown pigment granules in myocytes like in the heart and skeletal muscle, normal wear and tear from cells that are post mitotic or don't divide frequently, due to oxidation and can also impair other degradation pathways

Hemosiderin: aggregate of ferritin micelles, yellow brown pigments
Accumulates due to higher iron absorption, lower iron use, hemolytic anemia, and transfusions
Can be in the liver, use Prussian blue stain

31

Intracellular accumulation of exogenous pigments

Anthracosis: accumulation of exogenous carbon by alveolar macrophages

Common in smokers and urban people, no apparent cell injury

Transferred from lungs to lymph nodes

Appear as black spots

Also tattoos

32

Shapiro-Will Test

Null Hypothesis: data are normally distributed

P<0.05 means data are NOT normally distributed

33

Hemolytic Anemia and PPP

Glutathione peroxide reduces hydrogen peroxide, regenerate reduced glutathione via NADPH in glutathione reductase

Deficiency of glucose-6-P dehydrogenase (first enzyme in PPP) reduces NADH, reduced glutathione, and integrity of red blood cells

RBCs carry oxygen so need antioxidant

34

Location of the PPP

Cytoplasm of tissues that need NADPH for FA and steroid synthesis or detoxification

Also in RBCs since need glutathione to protect from oxidative damage

Adrenal gland, liver, tested, adipose tissue, ovaries, mammary glands, and RBCs

35

2 Key Phases of the PPP

1. Oxidative Phase-
Substrate: glucose 6-P and NADP+
Products: NADPH and ribulose 5-P
Controlling Enzyme: glucose 6-P dehydrogenase
Regulation: inhibition by NADPH

2. Non-Oxidative Phase-
Substrate: glyceraldehyde 3-P and Fructose 6-P
Products: Ribose 5-P
Controlling Enzyme: none
Regulation: levels of ribose 5-P

36

Oxidative Phase of the PPP

1. Glu 6-P oxidized to 6-phosphogluconolactone and makes NADPH

Glucose 6-P dehydrogenase (rate limiting), inhibited by NADPH and fatty acyl-CoA

2. Lactone hydrolyzed to 6-phosphogluconate by lactonase

3. 6-phosphogluconate decarboxylated to ribulose 5-P and makes NADPH by 6-phosphogluconate dehydrogenase

37

Non-Oxidative Phase of the PPP

Isomerization phase: Ribulose 5-P can isomerize to xyulose 5-P by Ribulose 5-P epimerase or to ribose 5-P by ribulose 5-P isomerase

Rearrangement phase: transketolases and transaldolase form 3-7 C sugars, form Fru 6-P and glyceraldehyde 3-P for glycolysis, also form erythrose 4-P for aromatic AAs

Transketolases translocates 2 C and transaldolases move 3 C

38

Purine Synthesis

1. Form 5-phosphoribosyl-pyrophosphate (PRPP) from ribose 5-P via PRPP synthetase using a pyrophosphate from ATP and Mg

Activated by inorganic P and inhibited by purine ribonucleotides that are mono or di

Used by Salvage and synthesis pathways

2. Synthesis of 5'-phosphoribosylamine from PRPP by removing the PP for an amine, amino from Gln and it turns into Glu, first committed step in purine synthesis since PRPP can become pyrimidine

3. Remaining Steps: add Gly, add carbon from THF, an amine from another Gln, closing first ring, adding carboxyl from CO2, adding Asp, loss of fumarate, another carbon from THF, and closing the second ring to form IMP

Overall: 5 ATP, 2 Gln, 1 Gly, 1 CO2, 1 Asp, and 2 formate

39

From IMP to Beyond

AMP: add GTP and Asp for amine then remove fumarate

GMP: oxidation to XMP then add amine from Gln (requires PP and gives off Glu)

ATP and GTP from monophosphate: use base specific kinase like Adenylate kinase to make 2 ADP from ATP and AMP, next use a nucleoside diphosphate kinase that works for any nucleoside di and triphosphates

40

Regulation of Purine Synthesis

When one nucleotide is high it inhibits its formation and stimulates the other to form, ATP stimulates IMP to form GTP and inhibits ATP formation

AMP pathway needs GTP and GMP pathway needs ATP so balance purine levels

PRPP synthetase inhibited by AMP, ADP, GMP, GDP, and IMP while it's activated by PP

Second enzyme in purine synthesis (glutamine PRPP aminotransferase) is inhibited by AMP, GMP, and IMP

From IMP ATP and GTP stimulate production of the other monophosphate

41

Purine Catabolism

AMP-
1. Amino removed to form IMP then hydrolyzed to inosine
2. AMP hydrolyzed to adenosine then deaminated to inosine

Inosine converted to hypoxanthine by removing ribose, oxidized by xanthine oxidase to xanthine, xanthine oxidized to uric acid while involving hydrogen peroxide

Uric acid is insoluble and causes gout

GMP-
Hydrolyzed to guanosine then form guanine from hydrolysis, deaminated to xanthine

42

Purine Salvage Pathway

Convert base to nucleoside monophosphate, important for cells that can't do de novo synthesis

Take base and use phosphoribosyltransferase to add PRPP, one for adenosine and another for hypoxanthine/guanine to share

43

Severe Combined Immunodeficiency

Caused by Adenosine Deaminase deficiency, which converts adenosine to inosine

Get high levels of dATP which inhibits ribonucleotide reductase to prevent formation of dNDP (and later dNTP) from NDP

Need dNTP for DNA synthesis like in actively proliferating cells like T and B lymphocytes

Defective T and B cells lead to severe combined immunodeficiency

44

Gout

Xanthine oxidase converts hypoxanthine to xanthine to uric acid

Formation of uric acid crystals in extremities instead of urine

Caused by partial HGPRT deficiency or overreactive PRPP synthetase

Allopurinol structurally similar to hypoxanthine and is a suicide inhibitor of xanthine oxidase

45

Lesch-Nyah's Syndrome

Defect in production or activity of HGTPT to cause increased hypoxanthine and guanine, results in increased uric acid

Also increased PRPP stimulates production of purines to create vicious cycle

Gout-like symptoms but also neurological abnormalities like aggression and self-mutilation

46

Pyrimidine Synthesis

Start with simple materials then add PRPP

1. Form Carbamoyl Phosphate: ATP, Gln, and CO2 form Carbamoyl Phosphate via Carbamoyl phosphate synthetase II (committed step, similar to first enzyme but in cytosol not mitochondria)

2-3. Form Dihydroorate: carbamoyl phosphate condenses with Asp to form carbamoyl aspartate, then lose water to get dihydroorate

4-6. Form UTP: dihydroorate oxidized to orotic acid, combined with PRPP to form oritidine monophosphate, decarboxylated to UMP

Use UMP kinase and then nucleoside disphosphate kinase to get UTP

7. Form CTP: UTP combined with Gln and ATP to form CTP, can't form other cytosine phosphate from other uridine phosphates directly

47

Pyrimidine Catabolism

Occurs in liver, open rings to form soluble end products

Cytidine forms beta-alanine

Thymine forms beta-aminoisobutyrate, NH3, and CO2

48

Pyrimidine Salvage Pathways

Can't do efficiently so use kinases

Uridine-cytidine kinase forms UMP or CMP and ADP from nucleoside and ATP

Deoxycytidine kinase

Thymidine kinase

49

Synthesis of Deoxyribonucleotides

Need to make NDP first, Ribonucleotide Reductase catalyze conversion to dNDP with help of NADPH

RNR works for ADP, CDP, GDP, and UDP

dUTP goes to dUMP then dTMP and eventually to dTTP

50

Formation of dTMP

dUMP converted to dTMP by thymidylate synthase

Methyl group donated by methylene THF, becomes dihydrofolate (DHF)

Need to regenerate THF from DHF via dihydrofolate reductase (DHFR), ideal target for chemotherapeutic agents

51

Nucleotide Synthesis Drugs

Hydroxyurea: Targets ribonucleotide reductase

Methotrexate: Targets dihydrofolate reductase

Fluourouracil: Targets thymidylate synthase