9. Lysosomes

Exam 3

Christian de Duve

• differential centrifugation

• developed in 1934

• won the 1974 Nobel prize for discovering lysosomes (in 1955)

Lysosomes

    a. 50+ hydrolytic enzymes (ex: proteins, nucleases)

    b. enzymatic activity is optimum @ pH 5

    c. "marker enzymes" --> acid phosphatase

Why have an optimum activity pH of 5 for lysosomes?

The wayward lysosomal enzymes that are transported out of the cell via the constitutive pathway are at a pH of 5, so their activity is compromised by up to 100%

categories of lysosomes 

1. heterophagy

2. autophagy

3. sepcialized 

Heterophagy

• Discovered by Rudolph Virchow

• white blood cells were giving birth to red blood cells

Ellie Metchnikoff

• 1884 - "phagocyrosis"

• phagoon - eat

• kytos - cavity

• suggested "probiotics"

• Gerontology 

Optimization

enhancing the delivery of bacteria or therapeutic agents to the lysosome and modulating the lysosomal environment and activity

Myasthenia Gravis (MG)

• Autoimmune Disease

• patients are very sensitive to curare, but symptoms can be ameliorated with Eserine (Physostigmine)

  • patients improved but some had too little ACH release, while others had few ACH-R

• 1960s - minute edd plate, potentials = one vesicle

Dan Drachman

• 3H α-bungarotoxin - binds to the ACH-R

• used electroplax (that specialized in electric organs of the Torpedo Ray) to count ACH-R

mAb agents ACH-R

a. injected into mouse --> MG-like symptoms

b. blood from MG patients --> injected into normal mice - MG again - therefore heterophogy of the ACH-R --> decrease number of ACH-R

Vyvgart

• is the FC fragment of an IgG antibodies 

• Capillary endothelial cells bind all IgG molecules and help them to recycle increasing their half life in the blood

• competes for these FcRns receptors with all IgGs thus causing all circulating IgG molecules, including the anti-Ach IgGs, to have a much shorter half life

• FDA approved in 2021 to treat myasthenia gravis

Autophagy

• discovered by Yoshinori Ohsumi (nobel prize - 2016)

• self-eating cell to break down and recycle old or damaged parts

ex: mitochondria 5 to 6 days

      peroxisomes 1 to 2 days

• starts when the membrane structure (phagophore) forms from the RER

• encloses the material to be degraded, forming an autophagosome, which is tagged by the protein LC3

• lysosomes binds to the autophagosome, breaking down its contents

• Certain drugs, such as bafilomycin A1 and chloroquine, can interfere with this essential recycling process

Chloroquine

• a lysosomotropic agent and accumulates preferentially in lysosomes

• May sensitize cancer cells to chemotherapy agents, but harm normal cells too

non-inherited Lysosomal diseases

• caused by environment

ex: silicosis (aka miner’s diseases)

chloroquinie

• silica particles inhaled into the lungs are taken up by cells, but can't be properly broken down by the lysosomes

• An outside agent enters a cell, which can then cause the lysosome inside to become leaky

•  releasing its destructive contents into the cell and leading to inflammation and cell death

inherited Lysosomal diseases

• illnesses related to problems with the cell's recycling centers, the lysosomes

• known as lysosomal storage diseases, which means they are hereditary—passed down through families

• involve the lysosomes failing to break down certain substances, causing them to build up and be "stored" inside the cell

• leads to damage and disease

Tay-Sachs Diseases

• causes by a defeat in the Hexosaminidase A (HEXA) gene

• leads to the body being unable to properly break down a fatty substance called gangliosides

• causes a toxic accumulation of gangliosides particularly in the brain, leading to progressive neurological damage

• no Enzyme Replacement Therapy (ERT) available to treat this disease

• occurs in approximately 1 out of every 5,000 births 

Hurler Syndrome - MPS 1:

• caused by a defect in the alpha-L-iduronidase enzyme, leading to the accumulation of waste inside cells

• using a co-cell culture experiment:

  • when defective Hurler cells are grown in the same dish as normal cells

  • the normal cells release the missing functional enzyme into the surrounding fluid

  • then taken up by the Hurler cells, thereby "correcting the Hurler cell" and clearing the accumulated waste

• relates to the existence of treatments like Aldurazyme, a form of enzyme replacement therapy

I(inclusion)-cell Disease

• inherited condition and type of lysosomal storage disorder

•  the cell's waste disposal units (lysosomes) are empty because they lack the necessary lysosomal enzymes 

• the missing enzymes are found in large amounts outside the cell

• key to this problem lies in a defect that occurs in the cis-Golgi apparatus

  • the cell fails to add a crucial shipping label, the M6P (Mannose-6-Phosphate) "zip code," to the enzymes

  • Without this M6P tag, the enzymes are incorrectly shipped out of the cell instead of being delivered to the lysosomes

• can import normal, correctly-tagged enzymes from outside

• but normal cells cannot import the mis-tagged I-cell enzymes proving the M6P tag is essential for proper enzyme delivery and uptake.

Gaucher Disease

• Deficiency in Glucocerebrosidase

• Leads to enlargement of the Spleen

• Erosion of the Long Bones due to
  accumulations of Glucocerebroside

• Cerezyme was the first ERT for Lysosomal Diseases - 1991

Pompe Disease 

• a lysosomal storage disease that can be treated with a medication called Myozyme

• caused by a deficiency in the essential enzyme acid alpha-glucosidase (GAA)

  • the lysosomes need to break down a type of sugar molecule

• Myozyme is a form of Enzyme Replacement Therapy that is given to patients to replace the missing GAA enzyme

Proteasome

• are large protein complexes responsible for hydrolyzing (breaking down) proteins only, acting as the cell's main protein shredders

•  vital for the cell, evidenced by the fact that yeast mutants with defective ___ died

• are massive structures (750,000 Daltons), are involved in managing cellular stress through the Unfolded Protein Response (UPR)

Effects of proteasome inhibitors

• Increase ER Stress

• Trigger Apoptosis

• Turn off cell survival pathways

• Affect mitochondrial function

• Influence epigenetic changes

• Influence DNA Repair

Proteasome Inhibitors and Cancer

• important class of drugs used in cancer treatment

• receiving regulatory approval:

  • bortezomib (Velcade)

  •  carfilzomib

  • ixazomib

• drugs work by entering a cancer cell and blocking the action of the proteasome (the cell's protein shredder)

• Specifically, bortezomib inhibits the proteasome's protein-cleaving sites, 

• leading to the accumulation of damaged and regulatory proteins inside the cancer cell

• This buildup of proteins triggers cell stress and ultimately causes the cancer cell to die, making it an effective targeted therapy

But why are cancer cells more sensitive to proteasome inhibitors than normal cells 

Cancer cells overproduce normal proteins and generate mutated proteins as well, so cancer cells rely heavily on proteasomes to protect them from proteotoxicity – the failure to clear these proteins from the cell. This imbalance triggers apoptosis in cancer cells