Lysosomes and Proteosomes

lysosome

cell organelle filled with enzymes needed to break down certain materials in the cell

-~50 hydrolytic enzymes (electron-dense)

-optimal pH 5

Christian de Duve

discovered lysosomes

Christian de Duve experiment

discovery of lysosomes

diff centrifugation --> pellet --> leave intact --> measure activity under:

-normal conditions: has respiratory activity (mitochondria)

-"injured" (lysed) conditions: respiratory activity + hydrolytic activity (lysosomes)

requirements for an organelle to be considered a lysosome

single-membrane bound and acid phosphatase

acid phosphatase

one of two requirements for organelle to be a lysosome

-marked by black dye

heterophagic lysosomes

lysosomes containing substances that originated outside the cell

autophagic lysosomes

contains substances derived of intracellular origin

specialized lysosomes

organelles that do not look like lysosomes but demonstrate the two requirements to be a lysosome

-acrosome of sperm (tip), plant vacuoles

Rudolph Virchow vs. Elie Metchnikov

both studied pus under light microscope: red blood cells found within white blood cells

Virchow: assumed WBCs gave birth to RBCs

Matchikov: WBCs phagocytose RBCs

current understanding of heterophagic lysosome pathway

antibody to component on extracellular material (bacteria) to be brought to lysosome --> bivalent Fab arms bind to surface proteins of material, Fc tail binds to Fc receptor of lysosomal cell --> sequential order of binding as material is absorbed --> lysosom transport

myasthenia gravis

a chronic autoimmune disease that affects the neuromuscular junction and produces serious weakness of voluntary muscles (droopy eyelids)

normal neuromuscular junction vs myasthenia gravis neuromuscular junction

NMJ: axon of neuron stores neurochemical vesicles --> action potential --> release of vesicles into synaptic cleft --> acetylcholine binds to nicotinic acetylcholine receptors --> Na influx --> muscle contraction

MGJ: nicotinic acetylcholine receptors internalized and degraded --> impaired muscle contraction

ligand and receptor in neuromuscular junction

ligand: acetylcholine; receptor: nicotinic acetylcholine receptor

early drugs to treat myasthenia gravis

-presumed that myasthenia gravis was neuromuscular disease

curane: plant; blocks Ach-R

eserine: inhibits Achase --> increase in half-life of Ach (decreases symptoms

eserine derivative

physostigmine

-inhibits Achase (early treatment for myasthenia gravis)

miniature endplate potential (MEEP)

tested change in cell potential of normal cells and myasthenia gravis cells at rest (old belief that MG was neuromuscular disease)

control (normal neuron): 2 mV; treatment: 1 mV

possible reasons behind initial understanding of myasthenia gravis

too few Ach release and/or too low Ach-R present --> weakened muscular response

Dan Drachman

myasthenia gravis cells have less Ach-R than do normal muscle cells

-3H-alpha-bungarotoxin

bungarotoxin

a neurotoxin that selectively blocks acetylcholine receptors

-found in venomous snakes

Dan Drachman experiment

observed muscle junctions

added 3H-alpha-bungarotoxin to cells --> binding to Ach-Rs --> more Ach-Rs on normal cells than on myasthenia gravis cells

torpedo ray

has high concentration of nicotinic acetylcholine receptor in electroplax

-concentration of receptors gives shocking ability

mice experiments with myasthenia gravis

mAB against Ach-R --> mouse demonstrated myasthenia gravis-like symptoms

serum from MG patients --> inject into mouse --> MG-like symptoms

conclusion from mice experiment with myasthenia gravis patient serum

myasthenia gravis is an autoimmune disease

pathway of myasthenia gravis

mAB against nicotinic acetylcholine receptor --> bind --> immunoprecipitation --> block activity of Ach-R --> too few Ach-R

neostigmine

a drug that inhibits the activity of acetylcholinesterase

-treatment for myasthenia gravis

prednisone and plasmapherisis

treatments for myasthenia gravis

-decrease mAB count against nicotinic acetylcholine receptor

contemporary treatments for myasthenia gravis

neostigmine, prednisone and plasmapheresis, chloroquine

chloroquine and myasthenia gravis

increases lysosomal pit pH from 5 to 7 --> reduces activity of pit (decreased degradation of Ach-R)

chloroquine with cisplatin

increases sensitization of cancer cells to chemotherapy, but also increases likelihood of harm to normal calls

autophagy of peroxisomes

1/2 life 1-2 days

mitophagy

autophagy selective for degradation of mitochondria

-could be important in organismic aging

lysosomal diseases

Miner's disease (silicosis), chloroquine myopathy and retinopathy; lysosomal storage diseases: Tay-Sachs disease, Hurler syndrome (MPS1), I-cell disease, Gaucher's disease

Miner's disease

fibrosis of the lungs caused by the silica puncturing of lysosomes

-miners breathe in silicon particles from mining (sharp) --> endocytotized easily (gas) --> punctures lysosomes (leak)

chloroquine myopathy and retinopathy

side effects of chloroquine treatment

myopathy: part of striated muscle cells occupied by lysosomes

retinopathy: retinal toxicity (blindness)

Lysosomal storage disease

a hereditary disorder associated with defects in lysosomal enzymes

-failure to synthesize lysosomal enzyme; failure to add M6P to zip code in Golgi

treatments for lysosomal storage diseases

enzyme replacement therapy, pharmacological chaperone therapy, gene therapy

Tay-Sachs disease

lysosomal storage disease:

lysosomes accumulate glucosamine-glycans and ganglioside GM2; defect in hexosaminidase A

children die at 2-5 yo; effects brain, muscle atrophy

hexosaminidase A

deficient enzyme in Tay Sachs

Hurler syndrome (mucopolysaccharidosis (MPS1))

deficient alpha-L-iduronidase

-Gargoylism (facial deformities), Genzyme treatment

Genzyme

replacement enzyme therapy company; Hurler syndrome

Hurler syndrome replacement enzyme therapy experiment

co-culture/conditioned-medium experiment

cell culture insert with normal cells at bottom of basal layer, Hurler's cells at filter layer in apical later --> time --> Hurler cells corrected

co-culture/conditioned-medium experiment for Hurler cells results

Hurler's cells corrected

-normal cells and Hurler cells share medium, but not touching --> must be lysosomal enzymes in medium

I-disease

disease characterized by lysosomes absent of enzymes (lack of M6P zip code)

testing endocytosis of I-cells

I-cell fibroblasts can endocytose normal lysosomal enzymes, but normal cells cannot endocytose I-cell enzymes (missing M6P zip code)

Gaucher disease

defect in glucocerebrosidase --> accumulation of glycogen in lysosomes

-Cerenzyme

Glucocerebrosidase

enzyme responsible for degradation of glucocerebroside; deficiency leads to accumulation and Gaucher disease

Cerenzyme

offers replacement enzyme therapy for Gaucher's disease

proteasomes

breakdown and recycling of damaged or abnormal intracellular proteins