9/6/17 Flashcards
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)
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
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
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
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
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
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)
Bcl-2 Pathway
- 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
- 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
- BH3-only proteins can bind to pro-survival proteins like Bcl-2 and keeps them from binding/inactivating Bax family proteins (Bax and Bak)
- 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
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
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
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
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
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
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
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
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
3 functions of cristae
Part of intermembrane space that project into the matrix
- Perform redox reactions of the ETC, enzymes project into matrix
- Synthesize ATP via ATP synthase
- Regulate transport of metabolites into/out matrix
Intermembrane Space
Contains enzymes like creating kinase, adenylate kinase (converts ATP and AMP to 2 ADP), and cytochrome C
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
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
Na+/K+ Pump
- Binding of cytoplasmic Na+ stimulates phosphorylation by ATP
- Phosphorylation causes the protein to change its conformation and face outside the cell
- Na+ expelled and 2 K+ binds
- K+ binding triggers release of phosphate group
- Loss of phosphate restores original conformation facing the cytoplasm
- K+ is released and Na+ can bind again
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
4 Mechanisms of Intracellular Accumulations
- Abnormal metabolism: inadequate removal of a normal substance due to impaired packaging and intracellular transport
Fatty liver or steatosis
- Impaired protein folding: can be acquired or inherited via mutation
Leads to defects in protein packaging, intracellular/extracellular transport, and/or exocytosis
Alpha1-antitrypsin
- Inherited enzyme deficiencies: failure to degrade a metabolite due to enzyme deficiency
Lysosomal storage diseases
- 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
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
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
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
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
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
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
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
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
Shapiro-Will Test
Null Hypothesis: data are normally distributed
P<0.05 means data are NOT normally distributed
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
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
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
Oxidative Phase of the PPP
- Glu 6-P oxidized to 6-phosphogluconolactone and makes NADPH
Glucose 6-P dehydrogenase (rate limiting), inhibited by NADPH and fatty acyl-CoA
- Lactone hydrolyzed to 6-phosphogluconate by lactonase
- 6-phosphogluconate decarboxylated to ribulose 5-P and makes NADPH by 6-phosphogluconate dehydrogenase
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
Purine Synthesis
- 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
- 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
- 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
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
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
Purine Catabolism
AMP-
- Amino removed to form IMP then hydrolyzed to inosine
- 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
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
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
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
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
Pyrimidine Synthesis
Start with simple materials then add PRPP
- 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
- Form CTP: UTP combined with Gln and ATP to form CTP, can’t form other cytosine phosphate from other uridine phosphates directly
Pyrimidine Catabolism
Occurs in liver, open rings to form soluble end products
Cytidine forms beta-alanine
Thymine forms beta-aminoisobutyrate, NH3, and CO2
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
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
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
Nucleotide Synthesis Drugs
Hydroxyurea: Targets ribonucleotide reductase
Methotrexate: Targets dihydrofolate reductase
Fluourouracil: Targets thymidylate synthase
