Download Lysosomes: Death by Enzyme Malfunction

Lysosomes: Death by Enzyme Malfunction
Lecture Outline
• Introduction
• The Lysosome: Structure
• The Formation of Lysosomes
• Lysosomal Enzymes Digest Everything
• Functions of Lysosomes
• Why doesn't the lysosome digest itself?
• Lysosome & Digit Formation
• Lysosomes in the Disease Process
• Occupational Diseases: Silicosis
• Abnormalities of Mucopolysaccharide Metabolism
• Background to Tay Sachs: Glycosphingolipids
• Tay Sachs Disease
Introduction
The primary lysosome looks like one of the simplest cellular organelles. Basically it is a bag of digestive
enzymes surrounded by a single biomembrane. But looks can be deceiving. Lysosomal enzymes are capable
of digesting essentially every type of biological molecule. For this reason, the lysosome was originally
considered only to be involved in digesting materials that the cell ingested through phagocytosis or
pinocytosis. With more research it has become clear lysosomes have many more cellular responsibilities.
This was dramatically emphasized when it was shown that the absence of a single lysosomal enzyme in
humans can lead to serious abnormalities, dementia and death. Since then lysosomes have been linked to
numerous human diseases.
The Lysosome: Structure
• A newly formed lysosome that has not yet been engaged in any cellular activity is called a primary
lysosome; recent research suggests this organelle may be more theoretical than real in humans
• All others are historically classed as secondary lysosomes (e.g., digestive vacuole, residual vacuole,
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autophagic vacuole, etc.)
A single biomembrane surrounds enzyme-rich matrix
Over 2 dozen lysosomal membrane proteins have been identified
Lysosomal membrane proteins are heavily glycosylated
Matrix varies in density: it is relatively homogeneous in primary lysosomes; in secondary lysosomes the
matrix contains various inclusions (e.g., partially digested organelles or bacteria, etc.)
Over 60 luminal proteins have been identified
Matrix consists of many different hydrolytic enzymes
Enzymes can digest every cell component
Acid Phosphatase = classic, marker enzyme; used to demonstrate the presence of lysosomes in animal
tissues
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• Proteomic analyses suggest there are more membrane and matrix proteins than have previously been
identified (Lubke et al, 2009. Biochimica et Biophysica Acta 1793: 625-635).
Lysosomal Biogenesis: The Formation of Lysosomes
Lysosomal enzymes are synthesized on the rough endoplasmic reticulum (rer) and packaged into
prelysosomal vesicles by the Golgi.
Rough Endoplasmic Reticulum: Stacks or Singles
Golgi
The enzymes are glycosylated in the rough endoplasmic reticulum and a mannose group is phosphorylated in
the Golgi to target them to lysosomal vesicles as discussed in a future lecture. In the classic view of
lysosomal biogenesis, prelysosomal vesicles that bud directly from the Golgi fuse to form mature, primary
lysosomes.
Recently it has been shown there are different routes to forming lysosomes. It is clear our understanding of
the details of endosomal events and lysosome biogenesis are still in their infancy (Van Meel and
Klumperman, 2008. Histochem. Cell Biol. 129: 253-266). The endolysosomal system is considered to be a
vast interconnecting network of tubules, vacuoles and vesicles. Components are shuttled to their specific
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areas through protein and vesicular targeting, topics covered in future lectures. The topics of endosome
formation and lysosomal digestion are also detailed in our tutorial sessions on receptor-mediated
endocytosis. Here we will examine the way primary lysosomes are formed, they enzymes they contain and
start on an examination of Tay Sachs disease.
The Lysosomal Enzymes
The following picture shows the diversity of lysosomes enzymes that are capable of digesting almost all
biological molecules.
Lysosomes & Cell Function
The next diagram is a composite showing essentially all of the known functions of lysosomes. We've divided
these up into 6 functional groups that are summarized below. These are not the only functions of lysosomes
as new responsibilities for this organelle are being discovered including metal ion homeostasis and cell
membrane repair.
1. Digestion of Ingested Materials - - Cells ingest materials by various endocytotic means including the
classic phagocytosis ("cell eating") and pinocytosis ("cell drinking"). Inside the cell, the material that is taken
up is enclosed in an endosome (phagosome or pinosome, respectively). Inside the cell the endosome fuses
with a primary lysosome to form a digestive vacuole. In the digestive vacuole the hydrolases of the lysosome
will act on the ingested material to break it down. After digestion is complete, the vacuole is called a residual
vacuole because it is full of residual, indigestible components. The contents of the residual vacuole are
released outside of the cell by exocytosis.
2. Cell Death - Lysosomes mediate events in the controlled or programmed death of cells called apoptosis.
This is discussed below. They also come into play during necrosis, the pathologic death of cells and tissues.
For example, meat becomes tenderized after the death of the animal because the lysosomes break down
releasing their enzymes into the muscle causing the digestion of the contractile and other muscle proteins.
3. Autophagy - The survival of cells requires that cellular constituents are constantly turning over. New
molecules and structures are made while old unnecessary or worn out components are removed. During
starvation, cells use autophagy to break down cellular components to provide energy for their survival. In the
case of organelles, the mitochondrion, for example, is separated from other cellular constituents by an
isolation membrane to become an autophagosome. The autophagosome fuses with primary lysosomes to
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form an autophagic vacuole within which the mitochondrion is digested. The resulting residual vacuole is
exocytosed. The following picture shows the sequence of events in digesting mitochondria.
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It is still not known whether the double-membrane isolation membrane that forms the autophagic vacuole is
formed de novo or from existing membrane structures such as the endoplasmic reticulum (for more see:
Juhasz & Neufeld, 2006. Autophagy: A 40 year search for a missing membrane source. PLoS Biology 4:
0161-0164.)
4. Protein Turnover - In this situation, molecules are digested by lysosomal enzymes. The exact ways in
which the different types of molecular turnover occur are under active investigation. But this process
removes old, abnormal or unnecessary molecules allowing cells to alter their physiology or behaviour. Some
of the molecules enter the digestive pathway via receptor mediated endocytosis as mentioned below and
discussed in detail in a future lecture.
5. Extracellular Functions - Lysosomal enzymes have responsibilities that lie outside of the cell as well.
For example they can digest extracellular components or modify the cell surface. For example, high levels of
secretion of glycosidases are linked to some of the changes in cell adhesion molecules that underlie the
behaviour of some cancer cells.
6. Receptor-Mediated Endocytosis - Lysosomes play an important role in the uptake and modification of
critical molecules such as cholesterol. They also mediate events of receptor recycling and the shutting down
of events of cell communication. These topics are detailed in future lectures.
Why doesn't the lysosome digest itself?
Since the lysosome is full of digestive enzymes that can digest essentially all cellular components, why
doesn't the lysosome digest itself? This is because the inner leaflet of the lysosomal membrane is coated with
an extensive glycocalyx (like that present in the intestinal epithelium to prevent its digestion; see lecture on
"The Cell Membrane"). The integral and peripheral membrane proteins on the inner surface are highly Nglycosylated glycoproteins containing poly-lactosamine which prevents access by the digestive enzymes.
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The figure above shows the expression of GFP-LC3 (LC3 is a marker protein for autophagy) in mouse heart muscle cells revealing
the presence of autophagosomes.
The major lysosomal membrane proteins are the lysosome-associated membrane proteins (LAMP-1 and -2).
These proteins not only play roles in the structural integrity of the lysosome, they mediate various other
functions as well (e.g., chaperone-mediated autophagy). For a review on how lyosomes digest other
membranes but not their own see: Kolter & Sandhoff, 2005. Ann. Rev. Cell Dev. Biol. 21: 81-103.
Occupational Diseases: Silicosis
Material made of Silica: Rose quartz, glass, digital watches, onyx, porcelain, beach sand, agate
Inhaled silica (silicon dioxide) dust enters lungs
Macrophage ingest & dust enter 2o lysosomes
Can't be digested
Lysis & release of enzymes
Sets up inflammatory response in lung tissue
Can lead to Tuberculosis and failure of respiratory system
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Lysosomes in the Disease Process
Over 50 monogenic human genetic diseases are known that are primarily lysosomal storage diseases (Lubke
et al, 2009. Biochimica et Biophysica Acta 1793: 625-635). Here’s a short list of some of them:
Protein
LAMP2
NPC1
Acid ceramidase
Alpha-glucosidase
Alpha-L-iduronidase
Beta-hexosaminidase A
Beta-hexosaminidase B
Glucocerebrocidase
Disease
Danon Disease
Niemann-Pick Disease (type C1)
Farber Disease
Pompe disease
Hurler Syndrome
Tay Sachs
Sandhoff Disease
Gaucher Disease
As with Tay Sachs disease as we detail below, many of these diseases are due to the inability to process
certain components which leads to the build up of vacuoles packed full of indigestible contents. This buildup
then affects cell function. As seen in the following pictures, for Danon Disease the buildup of
autophagosomes in skeletal and heart muscle is the primary problem.
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Figure 3 from Malicdan et al, 2008. Neuromuscular Disorders 18: 521-529.
Insight into these often rare diseases is growing due to such new approaches as the Niemann-Pick Type C
Disease Gene Database (Runz et al, 2008. Human Mutation 29: 345-350).
Abnormalities of Mucopolysaccharide Metabolism
• Genetic Defect = Absence of 1 Enzyme (e.g., alpha-fucosidase, alpha-mannosidase, etc.)
• Tay Sachs Disease, Hurler's Syndrome, Gargoylism, etc.
• Often called Glycosphingolipid (GSL) lysosomal storage diseases: because they involve problems with
digestion of GSLs
• Detection: Amniocentesis & enzyme analysis or genetic screening
• Possible Medical Intervention: Genetic Engineering; Pharmacological; Enzyme replacement therapy
These enzymes are injected into the blood on a regular basis with moderate success. Using recombinant
technologies will help to generate enzymes with specific targeting as well as, in the future, the ability to
cross the blood-brain barrier. At the moment, where available most these therapies are very expensive—on
the order of $500,000/per year per person!
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Background to Tay Sachs: Glycosphingolipids
Glycosphingolipids are commonly found at the surfaces of eukaryotic cells. They are comprised of a
ceramide moiety that inserts in the cell membrane plus an oligosaccharide chain. As can be seen in the
picture below the ceramide portion consists of fatty acid chains like the phospholipids discussed in the
lecture on cell membrane structure. Attached to these are sugar moieties (sialic acid residues) that orient to
the outside of the cell. When the cell membrane components are recycled, normal digestion occurs by the
stepwise removal of monosaccharides producing fatty acid chains that can be released from the lysosome.
Tay Sachs Disease
• Occurrence in Jewish People of Ashkenazic (Central European) Descent
• Due to missing Hexosaminidase A (also called N-Acetylglucosaminidase A)-an enzyme that removes
acetylglucosamine residues from polysaccharides
• Neurologic Disease: Build up in secondary lysosomes constrict nerve axons
• Leads to blindness, dementia & paralysis
• Evident by 6 mo.; Death 2-5 years of age
• Quebec: Lineage with similar disease; different gene defect
Let's look in a bit more detail about how the loss of the single Hexosaminidase A enzyme can have such a
devastating effect.
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In normal cells the turnover of ganglioside GM2 occurs regularly. Once inside digestive vacuoles the normal
complement of enzymes breaks it down and the contents of the residual vacuole are exocytosed. In Tay
Sachs, the absence of Hexosaminidase A prevents complete digestion of the ganglioside GM2 because
acetylglucosamine residues cannot be cleaved off. This results in the inability of the residual vacuoles to be
exocytosed. Thus they continue to accumulate in the cytoplasm of the cell causing it to swell up. We'll look
at this issue in more detail when we discuss the topic of protein targeting in cells.
LROs: Lysosome-Related Organelles
Some cells possess organelles with similarities to lysosomes and late endosomes but are morphologically,
structurally or compositionally distinct (Raposo et al, 2007. Curr. Opin. Cell Biol. 19: 394-401). Thus these
LROs possess some of the enzymatic content of lysosomes but have other distinct functions essential to the
functioning of the cells in which they are contained. Melanosomes (pigment granules) are one example. They
are found in melanocytes (the basis of tanning and of melanoma) in the skin and synthesize melanin in
response to UV light (Wasmeier et al, 2009. J. Cell Sci. 121: 3995-3999). They are also present in epithelial
cells of the retina and iris (eye colour). The acrosome of the sperm cell is an LRO. It is a bag of hydrolytic
enzymes at the tip of the sperm cell specially designed for penetration of the egg during fertilization.
©Copyright 1998-2009 Danton H. O'Day
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