Lysosomes/Proteosomes (16)

Constitutive Secretory Pathway (default)

a. Proteins secreted as fast as they are synthesized (ex. Ach-R pathway in RER to Golgi to lysosome, no storage/deposits)

b. Synthesis and secretion are sensitive to protein synthesis inhibitors (When inhibitor inhibits synthesis, secretion immediately stops because nothing is stored)

c. small electron lucent vesicles

Purpose is to maintain equilibrium in extracellular environment (in eukaryotic cells)

Regulated Pathway

a. Proteins are stored for a long period of time

b. Not immediately sensitive to protein synthesis inhibitors [ex, Insulin is stored waiting for ligand-receptor binding to trigger release]

c. Large electron dense vesicles

Purpose - Large bursts of protein secreted due to receptor activation (in eukaryotic cells)

Christian de Duve has been credited with discovering lysosomes. Which technique below

was critical to his discovery?

a. MALDI-TOF

b. Plastic thin sectioning electron microscopy

c. Freeze fracture electron microscopy

d. Two photon microscopy

e. None of the above

e, The critical technique he used was differential centrifugation

He ended up with a pellet, performed assays (not microscopy):

If intact,

• Noticed respiratory activity caused by mitochondria

If lysed (injured)

• Noticed respiratory activity

• Noticed hydrolytic activity due to lysosomes

Which of the following about lysosomes and lysosomal enzymes is false?

a. Works optimally at a pH of 8.3

b. Is surrounded by a double membrane

c. Are formed through the fusion of the plasma membrane and the rough

endoplasmic reticulum

d. Can break down most molecules except for phospholipids.

e. All of the above are false

e, all are false

Lysosomes:

• Works optimally at pH of 4.5-5.0

• Single membrane bound

• Has to be able to be stained with acid phosphatase

• Contain 50± hydrolytic enzymes (ex, proteases, phospholipases, glycosidases)

Categories:

- Heterophagy

- Autophagy

- Specialized

Rudolph Virchow and Metchnikoff

a. Developed many of the modern corneal transplant techniques

b. Were pioneers in the area of heterophagy

c. Showed that autophagy plays a key role in cancer cells

d. Were two famous Russian scientists who identified the role of Vitamin C in

collagen synthesis

e. Played with the Dave Matthews band in the mid-1990s

b, pioneers in heterophagy that were studying puss (basis of the infection)

Rudolf VIrchow saw/concluded:

• “A WBC giving birth to a RBC

• WBCs were trying to spread disease

Elie Metchnikoff saw same thing, but concluded that:

• The WBC was phagocytosing (eating-cell) the RBC

• WBCs were trying to prevent disease

• Concluded this in 1880’s, won Noble Prize in 1908

You are studying the formation and trafficking of phagosomes in cells. What general area

best matches this research arena?

a. Heterophagy

b. Mitochondrial mediated apoptosis

c. Autophagy

d. Duchenne Muscular Dystrophy

e. Invasion of cancer cells through basal laminas

a, Heterophagy is a vital cellular process that enables cells to phagocytose microorganisms or extracellular particles.

Consists of:

Primary lysosome - Before fusion with a phagosome

Secondary lysosome - After fusion with a phagosome

Phagocytosis/Fc receptors zipping up:

Antibodies bind bacterial surface proteins (Fab region binds antigen; Fc region sticks out).

• Leukocyte Fc receptors recognize and bind the Fc region of antibodies (opsonization).

• Actin cytoskeleton assembles, forming a “zipper” around the bacterium to engulf it.

• Bacterium is enclosed in a phagosome, which fuses with lysosomes for degradation.

Myasthenia gravis is a neuromuscular disease (auto-immune). The molecular basis for this disease is

which of the following?

a. Too little serotonin is released from the pre-synaptic nerve terminals

b. Too little acetylcholine is released from the pre-synaptic nerve terminals

c. The axonal microtubules disassemble over time causing axons to shorten

d. There are too few nicotinic acetylcholine receptors on the post synaptic cell

e. None of the above

d

- MG patients are sensitive to curare (antagonizes/blocks nicotine acetylcholine receptors) but symptoms can be fixed with eserine (inhibits acetylcholinease, which is responsible for breaking down Ach and recycling it). Due to two possibilities:

• Due to too few Ach-R

• Due to too little Ach being released

MEPPs - Records the membrane potential change in the fusion of a single Ach vesicle

• Healthy: Had potential of 2mV

• MG: Half the potential of healthy patient (1mV)

Ultrastructural Autoradiography, Dan Drachman

• Dan performed autoradiography on ³H-alpha bungarotoxin

In Normal patients - Healthy amount of Ach-R connected to muscle

In MG - Less Ach-R

How the basis of myasthenia gravis was elucidated is an interesting story with one of the most critical steps involving the neurobiologists who used the electroplax of the Torpedo Ray as a source of material to generate antibodies. How did this work contribute to our understanding of this disease?

a. It indicated that myasthenia gravis may be an autoimmune disease

b. It indicated that myasthenia gravis was due to the inability of the post-synaptic

cell to produce an action potential

c. It indicated that the cause of the disease was due to high concentrations of

circulating LDL in the blood

d. It indicated that the issue was a defect in the pre-synaptic voltage gated calcium

channels

e. None of the above

a, Researchers used the electroplax (organ), which contains a lot of neurons that secrete AChRs, from a Torpedo Ray to create mAbs for Ach.

They injected this into mice which then showed MG symptoms leading to the question, is it an autoimmune disease?

Basis of disease:

1. anti- AcH-R block binding of Ach

2. Immunoprecipitation of Ach-R leading to an immune response

Vyvgart binds to the Fc receptors on what type of cell which is critical to its function?

a. Human capillary endothelial cells

b. Human fibroblasts

c. Human T cells

d. Human B cells

e. Human intestinal epithelial cells

a, Vyvgart is the FC fragment of an IgG antibody that competes against all IgGs in binding with FcRns receptors on capillary endothelial cells that can treat myasthenia gravis

• All antibodies including anti-Ach IgGs have a shorter half-life

• Harmful AchR antibodies are destroyed

Yoshinori Ohsumi was recently awarded the Nobel Prize for his work in which area?

a. Lysosomal protein trafficking

b. Heterophagy

c. Autophagy

d. Carbon fixation

e. Photorespiration

c,

Autophagy is the process where cells degrade and recycle their own components — like damaged organelles or misfolded proteins — using lysosomes.

• Ohsumi used yeast cells to identify genes and mechanisms controlling autophagy

You are in a laboratory that is dedicated to understanding the site of the development and regulation of the phagophore nucleation site. What area below best matches your research activity?

a. Integrin receptor function

b. Autophagy

c. Heterophagy

d. Mechanism of action of the MPTP

e. Photoinhibition

b

• Autophagosomes originate from the phagophore nucleation site at the ER. The phagophore expands and closes to form the autophagosome, which fuses with lysosomes for degradation (autolysosome).

• Drugs like chloroquine block autophagy by accumulating in lysosomes, which may sensitize cancer cells to chemotherapy but can harm normal cells too.

• Cisplatin is a chemotherapy drug that causes DNA damage and increases mitochondrial reactive oxygen species (mtROS), leading to apoptosis. Combining cisplatin with chloroquine increases treatment effectiveness but may increase kidney toxicity due to higher ROS levels.

Specialized acrosomes:

Sperm are specialized lysosomes that release enzymes to help penetrate the egg during fertilization.

Vacuoles (in plant cells) serve lysosome-like functions by storing digestive enzymes to break down waste and recycle cellular components.

Non-hereditary (environmental)

Silicosis miners disease

• Due to: silica entering lysosomes of lung cells —> lysosomes leak —> cell death —> fibrosis (collagen secretion) in lungs

• Less gas exchange and less lung flexibility

Chloroquine (prevents lysosomal activity by changing pH) myopathy

• Chloroquine accumulates in lysosomes, raises pH, and disrupts enzyme activity leading to buildup of undigested material

• This causes muscle weakness (myopathy) or vision loss (retinopathy)

Hereditary (lysosomal storage disease)

Conditions:

• Failure to synthesize a lysosomal enzyme

• Failure to add m6p to lysosomal enzymes

• 70 different diseases

Tay-Sachs Disease

• Deficiency in hexosaminodase A, causes build up of gangliosides (used by neurons alot)

• This deficiency impacts brain (blind, deaf, seizure, muscular atrophy, startled reflex)

• No cure/enzyme replacement therapy

Hurler Cell Syndrome (mucopolysaccharidosis type 1)

• Children born with this have facial features like gargoyles

• Deficiency in alpha-L-iduronidase

• Experiment:

  • Co-culture (Hurler/Normal cells) shareing medium

  • Over time, Hurler cells correct themselves over time (due to constitutive pathway)

• Aldrazyme used as a cure, very expensive

I Cell Disease (I for inclusions)

• Observations:

  • Lysosomes have few lysosomal enzymes

  • lots of lysosomal enzymes in extracellular space

  • I cell will endocytose normal cell hydrolase (m6p tag working)

  • Fibroblasts will not endocytose I cell hydrolase because no m60 tag

Gaucher’s Disease

• Deficiency in glucocerebrosidase

• leads to enlargement of spleen and erosion of the long bones due to accumulations of glucocerebroside

• Cerezyme (1st enzyme replacement therapy)

Pompe’s Disease

• Deficiency in acid alpha-glucosidase

• Giant lysosomes contain large glycogen build up in skeletal muscle

• Myozyme is ERT used to cure

Proteosomes

• Not an organelle, but a macromolecular (large) machine

• Degrades ubiquitin labelled proteins (damaged/misfolded)

• 30000 proteosomes per cell

Q) Can proteosomes be targeted for cancer?

• Proteasomes can be targeted for cancer by using proteasome inhibitors (like Bortezomib or Velcade) to block enzymatic activity in proteosome that cleave proteins. This causes toxic protein buildup, ER stress, and apoptosis in cancer cells, making them more vulnerable to treatment.

• Cocktail of drugs needed to deal with cancer, can’t be done with Velcade alone