Lysosomes

Question 1

Describe lysosomes?

Answer 1

Found in all mammalian cells except red blood cells and comprise 1% of the cell volume
Typically roughly spherical membrane bound organelles of 200-400 nm in diameter, filled with soluble hydrolytic enzymes
Often have a dense protein rich core visible with electron microscopy and histochemistry
The biogenesis of the lysosome is controlled by the transcription factor TFEB
It binds and triggers expression of lysosomal genes
They tend to localise at the plasma membrane when nutrients are available

Question 2

What are the cellular functions of a lysosome?

Answer 2

The main role of lysosomes is as a degradative compartment in which macromolecules are hydrolysed into their constituent components
Lysosomes also perform other functions, including:
Role in apoptosis
They can repair plasma membrane damage
Within immune cells they can act as a secretory organelle

Question 3

What is the relationship with lysosomes in a plant?

Answer 3

Within a plant, the vacuole is related to the animal lysosome as it contains hydrolytic enzymes
However, is more diverse in function e.g. Storage of nutrients/waste, degradative compartment and provides osmotic pressure to keep the plant turgid

Question 4

Describe the function of degradation of macromolecules?

Answer 4

It acts as a cellular recycling centre
Material from inside and outside of the cell is delivered by endocytosis and autophagy
Hydrolases break down this material into its constituent parts e.g. glycoproteins into amino acids and sugars
These digestion products are transported into the cytosol by membrane transporters within the membrane of the lysosome, for use in cellular metabolic pathways

Question 5

Describe lysosomal hydrolyases?

Answer 5

The lysosomes lumen contains >45 different hydrolases
Hydrolases break covalent bonds by hydrolysis (the addition of H2O)
Lysosomal hydrolases digest macromolecules into their component parts
The different classes of hydrolases have different substrates

Question 6

How is the lysosomal pH maintained?

Answer 6

Lysosomal hydrolases have acidic pH optima
The lysosome lumen is maintained at pH 4.5 - 5 (cytosol pH = 7.2)
The acidic pH of the lysosome lumen is maintained by the Vacuolar proton pump (vATPase)
The vATPase pumps protons (H+) into the lysosome lumen and H+ transport is energised by ATP hydrolysis

Question 7

How do lysosomes transport products of degradation into the cytosol?

Answer 7

The membrane of the lysosome has transporters for water soluble molecules generated by lysosomal hydrolases e.g. amino acids, sugars, nucleosides
These lysosomal membrane proteins (transporters) are highly glycosylated - to protect from lysosomal proteases

These molecules are transported into the cytosol where they can enter cellular metabolic pathways
Typically the transporters are secondary active transporters that couple transport of their substrate with that of H+
Co-transport with H+ (moves down it’s concentration gradient)

Question 8

What are the methods of delivery to the lysosome?

Answer 8

Endocytosis: extracellular material and plasma membrane proteins
Arrive bound to the proteins on the cell surface or carried through a non-specific manner

Autophagy: material from the cytosol and whole cytoplasmic organelles

Phagocytosis: large extracellular particulate species such as microorganisms

Question 9

What is autophagy?

Answer 9

Autophagy involves the removal/degradation of whole cytoplasmic components - engulfed in autophagosomes for degradation in lysosomes
Dynamic process - carried out by several functional units consisting of autophagy-related proteins
ULK1 protein kinase complex, the transmembrane protein ATG9, the phosphoinositide 3-kinase (PI3K) complex, and a proteins that carry out PTM of Atg8 homologs

Autophagy is a continual process, but enhanced by the starvation of cells, enabling the cell to recycle nutrients and promote cell survival
Large portions of the cytosol are non-selectively captured into the autophagosome
Therefore during starvation it provided the cell with energy and building blocks to retain cellular functions from this material

It has also been shown to target bacteria to support the viability of host cells

Question 10

How is a autophagosome generated?

Answer 10

Autophagosome - 1um, is generated upon induction by stresses such as starvation
Double membrane structure, containing a cytoplasmic fraction

1. Phagophore (isolation membrane) is generated at the autophagosome formation site
   This is generated from an extended ER membrane structure - omegasome (crescent structure)
   Formation supported by actin
2. Elongation and closure of the edge of the membranes to form sealed double membrane autophagosome
3. Fusion with lysosomes containing various hydrolases = mature to autolysosomes

The fusion is mediated by binding of LC3 with FYVE coiled domain and the tethering of kinesin and microtubules facilitating + end direction of movement
Autolysosomes have a single membrane structure with a high electron density

Question 11

What are the types of autophagy?

Answer 11

Macroautophagy - most common
Chaperone mediated autophagy - they interact with misfolded proteins
Microautophagy - the membrane of the lysosome itself invaginates around the thing needed to be degraded

Question 12

Describe macroautophagy?

Answer 12

This removes old organelles, aggregates of proteins and long-lived proteins
Macroautophagy involves the envelopment of cytoplasmic material by an autophagosome
The cytoplasmic components are surrounded by a double membrane that may be donated the plasma membrane, ER and/or mitochondria
The autophagosome fuses with the lysosome delivering the cytoplasmic components to the lysosome for degradation
Material needed to be broken down is encapsulated by the autophagosome

Question 13

What are some hereditary diseases of autophagy?

Answer 13

SENDA
Vici syndrome
Joubert syndrome
Tumorigenesis

Question 14

What are some other roles of lysosomes?

Answer 14

Apoptosis
Plasma membrane repair

Immune response - Lysosomes in mature dendritic cells are also important for the transport of peptide-loaded MHC-II molecules to the plasma membrane and for the killing of virally infected or tumorous cells by cytotoxic T lymphocytes and natural killer cells

Cholesterol homeostasis - Lysosomes take in cholesterol through the LDL receptor by endocytosis and is transferred to other intracellular destinations (contact sites): ER, mitochondria or peroxisomes

Question 15

Describe apoptosis in relation to lysosomes?

Answer 15

Apoptosis involves the activation of cytosolic proteases, caspases, which act on cellular targets to induce cell death
There is increased permeability of the lysosomal membrane in apoptotic cells
= release of lysosomal proteases (cathepsins) into the cytosol
Lysosomotropic detergents damage the lysosomal membrane inducing lysosomal membrane permeabilisation
LMP can also be caused by sphingosine and phospholipase A2

Cathepsins (not at pH optimum) can cleave proteins at cytosolic pH and can trigger the mitochondrial/intrinsic pathway of apoptosis potentially via Bid
Lysosomes are not essential for apoptosis - they amplify this pathway

Question 16

Describe plasma membrane repair in relation to lysosomes?

Answer 16

AKA ESCRT-mediated repair
Plasma membrane can be perforated by mechanical damage - within mechanically active tissues e.g. Epithelial cells

Lysosomes act as a reserve of membrane to repair these holes
Damaging a cultured epithelial cell with a needle induces the exocytosis of lysosomes to the damaged site
This is triggered by an influx of Ca2+ into the cell from the extracellular fluid, which is detected by the lysosomal membrane protein synaptotagmin 7
The lysosome will then move along microtubules and fuse with the membrane in order to fill the hole

Question 17

Give an overview of lysosomal storage diseases?

Answer 17

A group of >50 different genetic disorders, in which there are defects in the degradative function of lysosomes
They are often associated with the abnormal accumulation (storage) of molecules within the lysosome
This is poorly understood but we think autophagy is often disrupted

Lysosomal storage diseases have a variety of causes:
Defective trafficking of hydrolases to lysosomes
Deficiencies in specific lysosomal hydrolases
Deficiencies in lysosomal transporters

Question 18

Name some lysosomal storage disorders?

Answer 18

I-cell disease
Pompe disease
Fabry Disease 
Infantile sialic acid storage disease (ISASD)
Salla disease

Question 19

Describe I-cell disease?

Answer 19

Inclusion cell disease AKA mucolipidosis type II - an autosomal-recessive disorder
This is a mutation in the gene (GNPTA) which encodes N-acetylglucosimine-1-phosphotransferase
It is called I-cell disease as there is formation of intracellular inclusions
As undigested substrates accumulate in lysosomes

Clinical features include: facial and skeletal abnormalities, severe psychomotor retardation and heart failure usually occurs in 1st decade
There is no cure and treatment is limited to reducing the symptoms

Question 20

What is the molecular basis of I-cell disease?

Answer 20

Cells have reduced intracellular levels of lysosomal hydrolases, but the enzymes get secreted by the cell into the culture medium
N-acetylglucosamine-1 phosphotransferase is an enzyme localised to the cis-Golgi and is involved in the modification of mannosylated glycans attached to newly synthesised lysosomal hydrolases to produce the mannose-6-phosphate (M6P) tag
If the hydrolases are not modified by N-acetylglucosamine-1 phosphotransferase they are not recognised by M6P receptors and are secreted by the cell

M6 tag is formed in the Golgi network
The M6P recognises the tag to be placed into endosomes
If this doesn’t work - we can form and secrete these enzymes but they don’t make there way to lysosomes

Question 21

Describe Pompe disease?

Answer 21

AKA glycogen storage disease type II
Caused by autosomal recessive mutations - within a gene encoding lysosomal hydrolase a-D-glucosidase

Clinical symptoms: Progressive cardiac and skeletal myopathy and within infantile onset this disease death usually occurs within 1st year of life due to cardiorespiratory failure
This is disease of the muscle

Question 22

What is the molecular basis of Pompe disease?

Answer 22

The vast majority of glycogen is stored and hydrolysed in the cytosol, but a small % enters into the lysosome by a poorly understood mechanism
Lysosomal ⍺-D-glucosidase cleaves glycogen into glucose that can then transported to the cytosol

The role of lysosomal glycogen hydrolysis is poorly understood, but a deficiency in lysosomal ⍺-D-glucosidase causes the abnormal accumulation of glycogen in cells and the resultant symptoms of the disease
Therefore unable to degrade glycogen

Question 23

What is the treatment of Pompe disease?

Answer 23

Can be treated by with enzyme replacement therapy (ERT)
Intravenous infusions of mannose-6-phosphate modified ⍺-D-glucosidase are given to patients
The addition of the tag means the enzyme isn’t just floating around in the blood stream and they can be taken up into the endocytic pathway and arrive at lysosomes

The enzyme is taken up by cell surface mannose-phosphate receptors and delivered to lysosomes
ERT is most effective for cardiac muscle

Question 24

Describe Fabry disease?

Answer 24

X-linked disorder that results from mutations in ⍺-galactosidase
This is a lipid storage disorder - sphingolipidoses

Symptoms include: facial abnormalities, a wide range of non-specific effects that include renal and cardiac problems due to problems in the vasculature
Progressive organ and tissue damage reduces life expectancy to 40 years - increased to 50 years with treatment for renal failure

Symptoms result from the deposition of glycolipid Gb3 in the walls of capillaries, kidney tubule and glomerular cells, nerves and dorsal root ganglia

Question 25

What is the molecular basis of Fabry disease?

Answer 25

⍺-galactosidase removes terminal galactose from the glycolipid globotriaosylceramide(Gb3)
Lack of ⍺-galactosidase activity results in accumulation of Gb3 and in lysosomes and lipid droplets in many cell types

Question 26

What is the treatment for Fabry disease?

Answer 26

Enzyme replacement therapy - administration of a mannose-6-phosphate modified form of the enzyme
Migalastat
Up to 50% of subjects with Fabry disease have mutations in the enzyme that cause it to become degraded by ERAD
Migalastat binds to these ⍺-D-glucosidase mutants in the ER and stabilizes these such that they can traffic to lysosomes
In the lysosome the drug dissociates (tag on the modified enzyme) due to the low pH and the enzyme can now function

Question 27

Describe Infantile sialic acid storage disease (ISASD) and Salla disease?

Answer 27

ISASD
Caused by autosomal recessive mutations in the sialin gene
reatment is limited to controlling the symptoms, which include facial abnormalities, mental retardation and enlarged heart, liver and spleen
Subjects typically die within first 2 years of life

Salla disease
Caused by mutations in sialin and is found in the Finnish population
Salla disease is less severe than ISASD, but results in physical and mental impairment and life expectancy is reduced to 50 years
Treatment for both diseases is limited to managing symptoms

Question 28

What is the molecular basis of ISASD and Salla?

Answer 28

Sialin is a lysosomal membrane transporter that transports sialic acids from the lysosome into the cytosol
Sialic acids are 9 carbon monosaccharides that are breakdown products of glycolipids, glycoproteins and glycosaminoglycans

The loss of sialin transport activity results in the accumulation of sialic acids in the lysosomes
ISASD mutations either cause the protein to be degraded by ERAD or abolish transport activity
The principal Salla mutation results in expression of a protein with 15% of wild type activity

Question 29

Give a summary of the lysosomal storage diseases?

Answer 29

Defect in trafficking of enzymes to lysosomes - I-cell disease

Missing lysosomal hydrolase activity - Fabry disease and Pompe disease

Missing lysosomal membrane transporter - Infantile sialic acid storage disease and Salla disease