The Role of Biomolecules and Cell Structures in Gaucher Disease

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The Role of Biomolecules and Cell Structures in
Gaucher Disease

• Goals
• Study a specific disease as a vehicle to
  understand the interrelationships of biological
  molecules and various cell organelles.
• To learn cell function to figure out what causes
  a debilitating disease.

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Gaucher Disease On-Line Resources

• Good overview of Gaucher Disease for the
  layperson
• http//www.gaucherdisease.org
• An in depth scientific resource concerning
  Gaucher disease
• http//www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispm
  im?230800
• Gaucher Disease A Clinical Trial of Gene Therapy
• http//neuro-www2.mgh.harvard.edu/gaucher/genether
  apy.html

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Gaucher Disease

• An inherited condition
• Affects 1 in 60,000 people
• Affects 1 in 500 Jews of Eastern European
  origin
• 1 in 10 are carriers of the gene!
• GD Results in....
• Anemia ? Always tired
• Huge spleen and liver ? Enlarged abdomen
• Weak bones ? Bent or curved legs
• Neurological problems ? Cross-eyed head back
• Premature death

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GD is a genetic disease

• Recently a therapy has been developed
• Costs 100,000 per year.
• The therapy turned 11 year old Brian Berman's
  life around.
• Before therapy
• too weak to walk up stairs.
• anemia threatened his life
• After Therapy
• Hemoglobin count is normal
• Purple belt in karate
• Plays basketball and football
• Reference Biotechnology vol 9, p 600 1991
  B.J.Spalding.

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Gaucher Disease

• Due to the inability of macrophages to digest the
  glycolipid, glucocerebroside(GC).

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The detailed structure of an animal cells plasma
membrane
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Breakdown of GC in Healthy People

• The enzyme, GCase, breaks down glucocerbroside in
  most people.
• GCase Glucocerebrosidase.

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GCase is defective in GD Patients

• Why cant GCase break down GC?

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Why is it a problem if the enzyme doesnt work?

• Accumulation of GC in macrophage cells causes GD
• More GC enters the cell than can be broken down

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Why do GD patients have the defective enzyme?

• They inherited a mutant gene that encodes the
  protein glucocerebrocidase
• a Gene is
• A segments of DNA in a chromosome
• Carries information that can be decoded to
  produce a specific kind of protein
• DNA is a polymer of nucleotides

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DNA Nucleotides

• Four Kinds of nucleotides in DNA
• A Adenine
• T Thymine
• G Guanine
• C Cytosine
• Why is the order of nucleotides in a gene
  Important?
• Central dogma of Biology
• 1. Order of nucleotides in a gene determines the
  order of amino acids in a protein
• 2. Order of amino acids in a protein
  determines..
• A.
• B.

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Nucleotide Structure

• Nucleotides are....
• the building blocks (monomers) of DNA and RNA
• As monomers they transfer energy to power
  almost all processes in all cells
• e.g. ATP

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• 30,000 genes code for proteins that perform all
  life functions

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GD Patients have defective Macrophage Cells
called

• Gaucher cells
• Derived from Macrophages (big eater)
• A kind of WBC that eats dying cells , invading
  microbes, and damaged tissue
• Gaucher cells 10xs larger than normal
• 100 mm vs. 10 mm
• What is accumulating within the lipid bilayer to
  make them so large?
• How does it get there?
• Lets review the functions of cell organelles.

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Eukaryotic Cell Structure

• 1. Nucleus
• Site of DNA, the genetic material
• 2. Smooth Endoplasmic Reticulum
• Makes lipids and cell membranes
• 3. Ribosomes
• Site of protein synthesis

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Eukaryotic Cell Structure

• 4. Rough ER
• Makes proteins found in membrane bound
  organelles exported from the cell
• 5. Golgi Apparatus
• Modifies newly made proteins, lipids, and
  carbohydrates
• 6. Vesicles
• Membrane-bound balloons that transport and
  store substances in cells

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Eukaryotic Cell Structure

• 7. Lysosomes
• Sacs containing enzymes that digest worn out cell
  parts
• 8. Cytoskeleton
• Protein fibers that help a cell maintain its
  shape
• 9. Mitochondria
• Harvest energy from organic molecules (e.g.
  sugars and fats) to produce ATP

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Molecules involved with GD

• What does GCase do?
• Which organelle probably contains
  glucocerebrosidase in most people?
• Where are digestive enzymes?
• How does it get there?
• Starting with the GCase gene in the nucleus,
  trace the pathway of the enzyme to this
  organelle.

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GCase Pathway from GCase gene to lysosome
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GCase Pathway from GCase gene to lysosome
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GCase Pathway

• 1. GCase Gene read to make RNA ?
• 2. RNA goes to Rough E.R ?
• 3. Ribosomes read RNA ? GCase produced ?
• 4. GCase packaged into vesicles ?
• 5. Vesicles transported to Golgi ?
• 6. GCase modified in Golgi ?
• 7. GCase packaged into vesicles ?
• 8. Vesicles fuse with lysosomes

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Ribosome on Rough ER Producing a Protein

• Ribosome reads mRNA to produce a protein
• ID of structures...
• Red ?
• Blue ?
• Brown ?
• Yellow ?

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Rough E.R. to Golgi Apparatus
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Transport from Golgi Apparatus

• Proteins modified by Golgi Apparatus are
  either...
• Used inside cell
• e.g.?
• Or
• Exported from cell
• e.g.?

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Membrane Bound GC

• Glycolipid molecules (GC) are normally found on
  the surface of cells.
• GC helps cells recognize each other

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Synthesis of GC in Cells

• Which organelle synthesizes GC?
• Well, where are lipids made in cell?
• Where are sugars added to newly made
  biochemicals?
• I.e. where do chemical modifications occur?
• Trace the biosynthetic pathway of the glycolipid,
  GC, through the cell to its final home in the
  plasma membrane

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GC Synthesis and Transport
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GC Synthesis and Transport
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Digestion of GC

• Now we've got GCase in a lysosome of macrophage
  cells and GC in the plasma membrane of the cells
  they eat.
• How does GC get into Macrophage lysosomes?
• Lets take a look at this happens.......

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Macrophages Big Eaters
Phagocytosisa macrophage snacking on bacteria

• Eat dead, injured, and foreign cells
• These cells have GC in their membrane
• Engulfed cells transported to lysosome for
  digestion
• ID of ...
• 1
• 2
• 3
• 4
• 5

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The formation and functions of lysosomes (Layer 1)
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The formation and functions of lysosomes (Layer 2)
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The formation and functions of lysosomes (Layer 3)
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Lysosome Fusing with a Vesicle containing a cell

• Lysosomal digestive enzymes digest cell
• Green _______________
• Where is GC?
• GC is in the membrane of_____________________
• What happens to the GC in.
• normal people?
• GD patients?

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Gaucher Cells

• Macrophage with Defective GCase
• Undigested GC builds up in.
• Lysosome membrane
• Affected lysosomes become ____________ in size
• Plasma membrane of macrophage
• Affected macrophages become ________________in
  size and are known as Gaucher Cells.

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Why does the presence of Gaucher cells in the
bone marrow cause the following symptoms?

• Anemia
• Huge spleen and liver
• Weak bones
• Neurological problems
• Cross-eyed head back
• Premature death

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Gaucher Cells before and after therapy
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GD Therapies

• How would the following therapies work at the
  cellular level to overcome disease symptoms?
• 1. Enzyme Therapy
• 2. Cell Therapy
• 3. Gene Therapy

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1. Enzyme Therapy

• What would happen to the enzyme in a pill?
• It would be subjected to degradation in the gut
  just like any other protein.
• So its given by intravenous injection.
• Obtain the enzyme from human placentas or from
  genetic engineering in prokaryotes.
• Need treatment every two weekswhy?

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Problems Involved with Enzyme Therapy

• Even if it got to the bone marrow, where would it
  have to go in the cell?
• Has got to get into the lysosome.
• How do we get it there?
• A special signal needs to be added to proteins
  that are "supposed" to end up in the lysosome.
  This signal is a special sugar (called mannose
  phosphate).
• This signal is recognized by macrophages which
  take it from body fluids and put it into
  lysosomes.

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2. Cell Therapy

• Could transplant bone marrow from well person to
  sick person.
• This workswhy?

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Problems with Cell therapy

• Finding a compatible donor
• 20 risk of fatal complications
• Cell transplantation therapy does work!
  Therefore.....
• the presence of normal cells can reduce the
  severity of the disease.
• How can we make normal cells?

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3. Gene therapy

1. Isolate blood stem cells from GD patient
2. Insert functional gene for GCase into stem cells
   using a viral vector
3. Put these genetically engineered cells back into
   the patient
4. Patients blood stem cells reproduce to form
   normal macrophage cells
5. Gene therapy should work because the enzyme makes
   symptoms go away.

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Genetically modified cells returned to patient
Stem cells reproduce to produce normal macrophage
cells
Cells removed from bone marrow
Genetically modified cells returned to patient
How gene therapy works
Isolate blood stem cells
Virus inserts the normal gene into one of the
stem cells chromosomes
Stem cells infected with virus carrying the
therapeutic gene
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Possible Problems with Gene Therapy

• Virus might start replicating and cause disease
• Immune system might overreact to the virus
• e.g. Jesse Gelsinger (died Fall 1999)
• GC gene might go into patient's DNA in a place
  that causes a mutation that results in cancer or
  disrupt genes needed for life .
• Clinical trials are now being done.