Lysosomes and proteasomes

Question 1

Christian de Duve

Answer 1

Was awarded the Nobel prize in 1974 for discovering lysosomes. Used differential centrifugation and observed that mitochondria and lysosomes co-sediment

Question 2

Why do lysosomes have a low pH?

Answer 2

The pH of lysosomes is maintained at 5. This pH is necessary for optimal function of hydrolytic enzymes in the lysosomes, such as proteases. The cytosolic pH is generally more basic at 7 in order to have ideal protein function there. A V-class ATPase maintains the proton gradient between the two areas.

Question 3

Acid phosphatase

Answer 3

A marker enzyme in lysosomes- it functions at a low pH.

Question 4

Lysosomes and transmission electron microscopy

Answer 4

Endocytosed membrane proteins that are to be degraded are also delivered to the lysosomes and can enter the organelle. Early evidence that membranes can be delivered to the interior of a membrane bound compartment came from electron micrographs that showed membrane vesicles and fragments of membranes in endosomes and lysosomes

Question 5

How are resident lysosomal proteins delivered?

Answer 5

Resident lysosomal proteins, like proton pumps, will remain in the lysosome. They are delivered to the lysosome in transport vesicles that bud off of the trans-golgi network or the endosomes.

Question 6

What is the main function of lysosomes?

Answer 6

To degrade extracellular materials taken up by the cell and to degrade intracellular components under specific conditions

Question 7

Function of autophagy

Answer 7

Materials that need to be degraded have to be delivered to the lysosome so they can be digested by the enzymes. Autophagy is the pathway that is used to deliver cytosolic materials to the lysosomal lumen for degradation. When cells are under stressful conditions, they use the lysosome to recycle macromolecules for nutrients (autophagy).

Question 8

What occurs in the autophagic pathway?

Answer 8

It begins with the formation of a cup-shaped membrane structure that envelops a region of the cytosol or an entire organelle, forming an autophagosome. The autophagosome’s outer membrane fuses with the lysosome to deliver a large vesicle bound by a lipid bilayer to the lysosome’s interior. The autophagosome and its contents are then degraded to their molecular components.

Question 9

Heterophagy

Answer 9

The transport of materials from the extracellular medium into the interior of the cell by endocytosis, and the subsequent digestion of the contents of endocytotic vacuoles by lysosomal enzymes. The process consists of endocytosis (early and late endosomes) or phagocytosis (like with bacteria).

Question 10

Endocytic pathway

Answer 10

Extracellular materials are internalized from the plasma membrane. Early endosomes, carrying endocytosed plasma membrane proteins, and vesicles from the trans-Golgi network carrying lysosomal membrane proteins fuse with the late endosome. When these vesicle fuse with the late endosome, their membrane proteins are transferred into the endosomal membrane. Proteins that need to be degraded are put into vesicles that bud into the interior of the late endosome. This forms a multivesicular endosome.

Question 11

Multivesicular endosome

Answer 11

An endosome that contains many internal vesicles. Proteins that will be brought to the lysosome to be degraded are put into multivesicular endosomes. The endosome fuses with the lysosome and releases the vesicles into the lumen of the lysosome, where they can be degraded.

Question 12

Rudolph Virchow

Answer 12

History of heterophagy- with pus, you can observe cells in the microscope. Concluded that WBCs were giving birth to RBCs, although we know now that this isn’t true

Question 13

Elie Metchnikoff

Answer 13

History of heterophagy discovered phagocytosis (eat, cavity in Greek). Found that WBCs were fighting disease

Question 14

Phagocytosis

Answer 14

The uptake of large particles- this is how WBCs remove pathogens.

Question 15

Fc receptors

Answer 15

Antibodies have a region called the Fab domain that binds specifically to its antigen. As antibodies coat the pathogen during interactions with their Fab domain and the antigen on the cell surface, a second antibody domain (the Fc domain) is exposed. WBCs have an Fc receptor on their surface that binds to the pathogen-bound antibody. This begins a process to engulf (phagocytose) and kill the pathogen.

Question 16

Myasthenia gravis

Answer 16

An autoimmune disease where acetylcholine receptors in the neuromuscular junction are attacked by the immune system. This causes symptoms such as muscle weakness and drooping eyelids. Lysosomes internalize and degrade acetylcholine receptors, so lysosomal dysfunction could be a contributing factor to the disease. Lysosomes could be a potential therapeutic target for MG.

Question 17

Curare

Answer 17

Curare is a neuromuscular blocking agent. It binds to the acetylcholine receptor and prevents action potentials from stimulating skeletal muscles. It was first used as a muscle relaxant in anesthesia. Myasthenia gravis patients are sensitive to curare and curare tests can be used to diagnose MG.

Question 18

Eserine

Answer 18

Can be used to relieve the symptoms of myasthenia gravis. It allows acetylcholine to accumulate in the synapse by inhibiting the enzyme acetylcholinesterase, which breaks down acetylcholine.

Question 19

Dan Drachman

Answer 19

He first showed that myasthenia gravis is an autoimmune condition. Using botulism toxin many years before it was introduced for clinical use, he demonstrated that muscles wasted without motor neuron stimulation, a principle important in the pathogenesis of diseases such as ALS and Duchenne muscular dystrophy.

Question 20

3H-bungarotoxin

Answer 20

Antagonist of acetylcholine receptors (interferes with their function). Used in Dan Drachman’s research

Question 21

Yoshinori Ohsumi

Answer 21

Japanese cell biologist known for his work in elucidating the mechanisms of autophagy- won the Nobel prize in physiology in 2016. Ohsumi’s research played a key role in helping to uncover the critical physiological activities of autophagy, including its function in helping cells adapt to various types of stress, in contributing to embryo development, and in eliminating damaged proteins.

Question 22

The role of autophagy in cancer

Answer 22

In cancer cells, autophagy suppresses tumorigenesis by inhibiting cancer-cell survival and inducing cell death, but it also facilitates tumorigenesis by promoting cancer-cell proliferation and tumor growth

Question 23

Chloroquine

Answer 23

A lysosomotropic agent that accumulates preferentially in lysosomes. May sensitize cancer cells to chemotherapy agents
but harm normal cells too. Chloroquine can be used in combination with chemotherapy, making both the cancer cells and normal cells, like kidney cells, more sensitive to the chemotherapy. This is how normal cells are damaged.

Question 24

Acrosome

Answer 24

Specialized lysosome. The acrosome is a unique organelle located over the sperm nucleus. This acidic vacuole contains a number of hydrolytic enzymes that, when secreted, help the sperm penetrate the egg’s coats.

Question 25

Plant vacuoles

Answer 25

Similar to lysosomes, plant vacuoles have degradative enzymes in their lumen and have an acidic pH. Also found in green algae and fungi. Vacuoles likely have the same degradative function that lysosomes do.

Question 26

Silicosis

Answer 26

Miner’s disease- caused by inhaling silica dust over a long period of time. Silica particles are sharp, so lysosomes leak and cells die. Results in decreased ability in CO2 and oxygen exchange, as well as fibrosis due to collagen production from the injury (limits lung elasticity).

Question 27

Drug induced lysosomal diseases

Answer 27

Chloroquine has many applications for rheumatoid arthritis and other diseases. However, prolonged use can cause chloroquine myopathy (muscle weakness, fatigue) or chloroquine retinopathy (blindness)

Question 28

Replacement therapies for lysosomal diseases

Answer 28

There are a variety of replacement therapies, such as bone marrow transplant, enzyme replacement therapy, stem cell therapy, gene therapy, and others.

Question 29

Genzyme Sanofi

Answer 29

A Cambridge MA company that pioneered
Replacement Enzyme Therapy for treating lysosomal storage
diseases

Question 30

Tay-Sachs disease

Answer 30

The change in the HEXA gene that causes Tay-Sachs disease results in a deficiency of the enzyme beta-hexosaminidase A. This enzyme is required to break down the fatty substance GM2 ganglioside. The buildup of fatty substances damages nerve cells in the brain and spinal cord. Symptoms- cherry red spot in the retina, blindness, deafness, muscle atrophy, seizures, startled reflex to loud noises.

Question 31

Tay-Sachs gene therapy trial

Answer 31

An early stage gene therapy trial in Barcelona found that there had been a very modest increase in HexA bioactivity in both
patients (less than two percent). More encouragingly, the second patient treated showed signs of increased myelination and a plateau in disease development.
The data presented marked the first reported evidence for potential
disease modification in Tay-Sachs disease, and suggests an opportunity for gene replacement therapy to improve outcomes for children with this condition.

Question 32

Hurler syndrome

Answer 32

Originally called gargoylism. It is an inherited lysosomal disorder caused by the absence of alpha-L-iduronidase, an enzyme responsible for the degradation of glycosaminoglycans (GAGs or mucopolysaccharides). GAGs are polysaccharides which have widespread functions in the body. They will accumulate in the cells and tissues and eventually cause death.

Question 33

Zebra bodies

Answer 33

Hurler cells have zebra bodies- tissues will exhibit a black and white striped pattern when examined under a microscope. This is due to the accumulation of phospholipids, which could be a consequence of secondary lysosomal disturbance and contribute to cardiac failure.

Question 34

In terms of enzyme synthesis, how are Hurler cells different from normal cells?

Answer 34

In a normal cell, lysosomal enzymes are synthesized in the endoplasmic reticulum. After they are synthesized, they either migrate to the lysosomes or they leave the cell and are then recaptured and brought back into the cell. Hurler cells are unable to synthesize their own ALI enzyme, but they are able to pick it up and store it in their lysosomes if it is supplied from outside. This is a general rule for lysosomal storage diseases.

Question 35

Aldurazyme

Answer 35

Enzyme replacement therapy for Hurler syndrome patients. It restores a level of enzyme activity that is necessary to remove GAGs.

Question 36

Conditioned medium experiment

Answer 36

The experiment demonstrated a mechanism by which ERT could be used to treat disease. Demonstrated Hurler cells have M6P receptors.

Question 37

What is one disadvantage of enzyme replacement therapy?

Answer 37

The therapy is very expensive. The average cost per year is 300 thousand dollars. With a GoodRx coupon, the cost is still 36 thousand dollars

Question 38

I-cell disease

Answer 38

In this disease, multiple necessary enzymes are missing from the lysosomes. This is because affected individuals lack N-acetylglucosamine phosphotransferase that is necessary for the enzymes to be tagged with M6P in the cis-golgi. Therefore, the enzymes are secreted rather than being sorted to the lysosomes as they should be.

Question 39

Mannose 6-phosphate

Answer 39

Formed in the cis-golgi, acts as a sorting signal to direct lysosomal enzymes from the trans golgi network to the late endosome. The enzymes marked with M6P are segregated from secreted and membrane proteins in the trans golgi network.

Question 40

Gaucher’s disease

Answer 40

Deficiency of the lysosomal enzyme glucocerebrosidase results in the accumulation of harmful quantities of lipids, specifically the glycolipid glucocerebroside, throughout the body especially within the bone marrow, spleen and liver. This disease causes enlargement of the spleen and erosion of the long bones.

Question 41

Cerezyme

Answer 41

ERT for Gaucher’s Disease. Cerezyme acts like glucocerebrosidase (the body’s natural enzyme), breaking down GL-1 into its more basic elements, glucose and ceramide, that can be naturally removed from the body.

Question 42

Pompe’s disease

Answer 42

Caused by mutations in a gene that makes a lysosomal enzyme called acid alpha-glucosidase (GAA). The body uses GAA to break down glycogen into glucose (an energy source). In Pompe disease, mutations in the GAA gene reduce or completely eliminate this essential enzyme. Excessive amounts of lysosomal glycogen accumulate everywhere in the body, but the cells of the heart and skeletal muscles are the most seriously affected.

Question 43

Myozyme

Answer 43

ERT for Pompe disease, depicted in the “extraordinary measures” movie. Replaces acid alpha glucosidase to improve symptoms.

Question 44

Proteasomes

Answer 44

Large, protein degrading organelles that influence many cellular functions, including the cell cycle, apoptosis, transcription, and DNA repair.

Question 45

Steps in a proteasome degrading a protein (3)

Answer 45

1. The protein is tagged to target it for proteosomal degradation. Cells control the tagging of proteins and can determine the rate at which a certain protein is degraded.
2. The proteasome binds to the tag on the targeted protein and unfolds the protein as it’s transferred to an internal chamber
3. Protein-cutting subunits of the proteasome degrade the protein into small peptides that are released to the cytosol.

Question 46

Ubiquitin

Answer 46

A polypeptide that marks a protein for degradation in the proteasome

Question 47

Protease inhibitors

Answer 47

Protease inhibitors are promising candidates for anti-cancer agents. Since dysregulated proteolytic activities can contribute to tumor development and metastasis, antagonization of proteases with tailored inhibitors is an encouraging approach. Three proteasome
inhibitors have received regulatory approval and are used routinely in clinical settings, including bortezomib,
carfilzomib and ixazomib

Question 48

Velcade

Answer 48

Velcade a targeted chemotherapy that blocks proteasomes, which causes protein buildup inside of the cell and causes it to die. Disadvantage- very expensive