Lysosomes and Proteasomes

What is the classic path that all organelles follow except lysosomes?

1. Identification of the organelle via microscopy

2. Characterization via biochemistry and molecular biology

3. But lysosomes were first discovered through a “biochemistry” approach and not with the microscope

Christian de Duve

• 1974 Nobel Prize discovered lysosomes

• Through velocity sedimentation and density gradient centrifugation techniques

What technique did Christian de Duve follow?

1. Took whole cells and homogenized in sucrose

2. Centrifuged pellet which he then characterized

3. When the pellet was intact, there was respiratory activity that was due to the mitochondria present

4. When pellet was “injured” (lysed), then he found respiratory activity due to mitochondria that were also in the fraction but now detected proteolytic activity due to the presence of lysosomes. He was the first to describe that lysosomes have hydrolytic enzymes.

What is acid phosphatase?

A lysosomal marker enzyme

Over 40 to 60 hydrolytic enzymes can break down nearly every molecule that it hydrolyzes. What pH does lysosomes work optimally at?

pH 4.5 to 5, not pH 7

Why have an optimum activity pH of 5.0 for lysosomes?

The wayward lysosomal enzymes that are transported out of the cell via the constitutive pathway are at a pH of 7 so their activity is compromised by up to 100%

Single membrane delimited organelles are…

very electron dense due to high concentration of these enzymes

How are lysosomes defined?

As a single membrane bound organelle that contains acid phosphatase - a “marker enzyme” that “marked” any organelle that stained positive for acid phosphatase an lysosome

Marker enzyme

• Acid phosphatase

• If a single membrane bound organelles stains positively, then by definition, this is a lysosome

What are the ways that lysosomes can be classified as?

1. Heterophagy - eating others

2. Autophagy - eating self

3. Specialized - other more highly differentiated functions

Ruldolph Virchow

Saw a RBC inside of a WBC and thought they were giving birth. Actually saw phagocytosis.

Elie Metchnikoff

• Believed that phagocytosis is responsible for aging. Gray hair is caused by phagocytes eating hair pigment = WRONG

• Outside Pasteur Institute believed WBC were traitors that spread infections around the body

• Won Noble Prize for introducing the concept of probiotics, gerontology and the cellular basis of immunity

Myasthenia gravis

neuromuscular disease due to over active heterotrophy/autophagy

What two medications were given to patients with MG?

1. Curare - blocks ACH-R on the NMJ by blocking pores

2. Eserine - extends the half-life of ACH thus making the active concentration of ACH higher in the synaptic cleft. One of these drugs, neostigmine, is used today to treat MG.

When the Swedish workers recorded MEPP, what did they find?

• The amplitude is smaller in MG patients compared to normal people

• The 2 mv (miniature endplate potentials) are due to the fusion of one synaptic vesicle

Dan Drachman

• MG patients had less binding of 3H-alpha bungarotoxin (snake venom known to bind strongly to ACH-R)

• Too few receptors in MG patients

Based on what Dan Drachman discovered, the explanation for the fewer receptors in MG is because this is an autoimmune disease. How was this determined?

1. Injection of purified ACH-R from Torpedo Ray into mice caused MG-like symptoms

2. Injection of blood serum from MG patients into normal mice caused MG-like symptoms in the recipients

3. Explanation - Anti ACH-R antibodies “immunoprecipiate” receptors in the post synaptic NMJ

What is Vyvgart?

FDA approved drug designed to treat MG by increasing the degradation rate of all circulating antibodies in the blood which also includes the anti-nicotinic acetylcholine receptors that are the basis of MG

What is the mitochondria half-life found in lysosomes?

5 to 6 days

What is the half-life of peroxisomes that are found in lysosomes?

1 to 2 days

When pentobarbital increases SER,…

lysosomes digest it when this barbiturate is removed

Why is autophagosome and LC3 important to autophagy?

• Autophagosome originates from the RER

• LC3 required for mature autophagosome to bind to and fuse with lysosomes

What is Cisplatin and how does it work?

• Common chemotherapy drug

• Binds to nuclear DNA in cancer and other cells to prevent repair triggering apoptosis

What two drugs can alter lysosomal function?

• Bafilomycin A - poisons the proton pump pumping protons into the lysosome

• Chloroquine - anti-malarial drug that causes the lysosomal pH to go from 4.5 to around 7

Yoshinori Ohsumi

Nobel Prize for autophagy

What is the acrosome?

• Tip of sperm cell

• Highly differentiated lysosome

• Forms from Golgi vesicles such as the lysosome and contains hydrolytic enzymes that break down the outer membrane of the egg

Plant vacuoles serve as

“lysosomes”

Miner’s Disease - Silicosis

1. Silica particles taken up by macrophages into the lung in phagocytotic vacuoles

2. Fuse with lysosomes to make secondary lysosomes leaky

3. Fibrous material builds up and makes the tissue inelastic

4. Mediated locally since it can be mimicked in culture with lung cells/ Cells die while other cells deposit collagen

Chloroquine myopathy

• Patient was injected with chloroquine and this compromised the ability of the lysosomes in muscles to work

• Used to treat malaria in WW2 (still today)

• Lysosomotripic agent and works by increasing the pH of the lysosome

Chloroquine retinopathy

cause irreversible blindness that continues to progress even after discontinuation of chloroquine

What is Genzyme?

• Three person start up that is now part of Genzyme-Sanofi that is a large pharma group

• Developd enzyme replacement therapies to treat a myriad of lysosomal diseases/lysosomal storage diseases

Tay Sachs Disease

• defect in Hexosaminidase A resulting in accumulation of ganglioside GM2

• Accumulation of gangliosides because they can’t be broken down in the lysosomes

• Common in certain populations like 1/5000 French Canadians

• Children die at birth or live until 2-5

• Affects brain/neural tissue because of the high concentration of gangliosides in neural tissue

• No ERT because brain tissue in inaccesible

What happened at UMass Chan Medical School and Auburn University that is associated with Tay Sachs Disease?

• Successfully given to children

• Gene therapy directs brain cells to produce Hexosaminidase A

Hurler’s Disease/Hurler Syndrome (mucopolysaccharidosis Type I)

• Results in big lysosomes accumulating in cells. Manifests itself with many deformities. This is due to the accumulation of glycoaminoglycans

• But when co-cultured with normal cells, the Hurlers cells now appear normal

• This is called a conditioned medium or co-culture experiment

• defect is the inability of Hurlers to produce alpha L-iduronidase

• Basis behind enzyme replacement therapy

• ERT - Aldurazyme

In I-cell disease (Inclusion disease), it was observed that some lysosomes with fewer hydrolases. What is the conclusion?

Either lysosome enzymes aren’t synthesized at all or there might be a problem with Mannose-6-Phosphate (M6P) address tag that is added in the cis-Golgi

In I-cell disease (Inclusion disease), it was observed that they have lots of hydrolases in the extracellular fluid. What is the conclusion?

Hydrolytic enzymes are indeed synthesized

In I-cell disease (Inclusion disease), it was observed I-cell fibroblasts endocytose hydrolases from normal fibroblasts and they accumulate in lysosomes. What is the conclusion?

Cells have normal M6P receptors because they are also on the outer membranes

In I-cell disease (Inclusion disease), it was observed that normal fibroblasts will not endocytose I-cell hydrolases. What is the conclusion>

I cell lysosomal enzymes either lack or have defective M6P address tags

What is the culprit in I-cell disease?

Defective N-acetylglucosaminylphosphotransferase in cis-Golgi

Gaucher’s disease

• Deficiency in glucocerebrosidase

• Most common of all lysosomal diseases in which is 1 in 40,000

• Spleen enlargement, bone deformations

• Can be treated with highly effective enzyme replacement therapy - Cerezyme from Genzyme Corporation for Type I Gaucher’s disease

Pompe’s disease

• No acid alpha-glucosidase which is essential for normal muscle development leading to accumulation of glycogen in lysosomes

• Genzyme developed Myozyme for treatment

• Documented in movie called “Extraordinary Measures”

• Has orphan drug status like many other enzyme replacement therapies

In Pompe’s disease, how is glucose made avaliable to cells from glycogen?

1. Glycogen can be broken down to glucose in the cytoplasm\

2. Glycogen is transported to the lysosome where the acid alpha glucosidase can break it down into glucose that is then pumped out of the lysosome into the cytoplasm where it is used in glycolysis. This is the problem point for Pompe’s disease

What are proteasomes?

very large, protein-degrading macromolecular machines or particles that influence a variety of cell functions that include cell cycle, apoptosis and involved as the final stage in the Unfolding Protein Response system that is designed to detect improperly folded proteins

What is the molecular weight of proteasomes?

750,000 Daltons - large molecule

How many proteasomes are typical in a cell?

30,000

What is a key event in proteasomes?

• Ubiquinitnation of target proteins

• Required for proteasome degradation

• This is where UB tagged misfolded proteins go once they are ejected from the RER as part of the Unfolded Protein Response process

What does recent work on proteasomes inhibitors suggest?

• These drugs may be more useful chemotherapeutic agents

• Studies with mutant years show that if proteasomes are not functional, then the cells die.

• Cancer cells appear to be more sensitive to proteasome inhibitors than normal cells

What is Bortezomib?

• Velcade marketed by Millenium Pharmaceuticals

• Used to treat relapsed multiple myeloma

Different drugs target different parts of the proteasome. So how do they work?

• Cancer cells make lots of mutant proteins as well as overloading the cell with normal proteins so the balance is different

• By inhibiting the proteasomes systemically there is little effect on normal cells because they don’t exhibit “protein overload” but the effects on cancer cells can result in triggering apoptosis

• Combined with other treatments

What are the multiple effects that proteasome inhibitors can have?

• Increase ER stress

• Trigger Apoptosis

• Turn off cell survival pathways

• Affect mitochondrial function

• Influence epigenetic changes

• Influence DNA Repair