Peroxisomes

Question 1

Describe peroxisomes?

Answer 1

Organisms that contain peroxisomes: virtually all eukaryotic cells
Prominent in the liver, brain and kidney
Peroxisomes are surrounded by a single bilayer membrane and do not contain DNA - therefor proteins are encoded in the nucleus
The matrix is relatively granular
They import things across the cytosol into the boundary membrane
They all contain oxidative enzymes: catalase and urate oxidase

Question 2

Give an overview of the functions of peroxisomes?

Answer 2

Many different metabolic activities which often depend on the organism in question, the tissue type, developmental stage and even environmental conditions

In plants, part of the pathway of photorespiration is in peroxisomes
In blood stream trypansomes glycolysis is in peroxisomes
In the fungus Penicillium chrysogenum part of the pathway of penicillin biosynthesis is in peroxisomes
In other fungi such as Hansenula polymorpha which can use methanol as a carbon source alcohol oxidase needed for methanol utilisation is a peroxisomal enzyme

Peroxisomes are important from a medical, agricultural and biotechnological perspective

Question 3

Describe the main metabolic functions of the peroxisome?

Answer 3

Long and branched chain fatty acid shortening (β oxidation) - degradation
Important for energy metabolism, removing molecules whose build up have toxic effects in the body and metabolism of some fatty acid based signalling molecules

Ether Lipid (plasmalogen) Biosynthesis
Plasmalogens account for only 0.8% of membrane lipids in liver but 23% in nervous tissue (very important)

Bile acid synthesis
Bile acids aid lipid digestion and the absorption of the digestion products also lipid soluble vitamins A,D,E & K

Glyoxylate detoxification
Conversion of fats to sugars - breaking down fatty acid chains into acetyl CoA to form succinic acid (not in animals)
Glyoxylate is transaminated to glycine using AGT and alanine as the amino group donor
If not further metabolised , glyoxylate is oxidised to oxalate which then precipitates as calcium oxalate resulting in renal failure

Question 4

What does catalase do in the peroxisomes?

Answer 4

Catalase uses H2O2 (generated by other enzymes) to oxidise other substances e.g. Phenols, formic acid, formaldehyde and alcohol
This reaction is useful in the liver and kidneys to detoxify various toxic molecules e.g. Ethanol into acetaldehyde
If excess H2O2 forms it is converted into water and O2

Question 5

Describe peroxisomal β-oxidation?

Answer 5

Peroxisomal β-oxidation shortens fatty acids with chains >22 C atoms – then full degradation in mitochondria
β-oxidation shortens the alkyl chains of fatty acids - removing blocks of 2 carbon units
Therefore converting the fatty acids to acetyl CoA to be used for biosynthetic reactions

Three enzymes are required:
1. Acyl-CoA oxidase:
fatty acyl-CoA + O2 → trans-Δ2-enoyl-CoA + H2O2
It also uses FAD as a cofactor
2. Peroxisomal enoyl-CoA hydratase:
C=C bond hydration and dehydrogenation
3. Peroxisomal thiolase: 
removal of acetyl-CoA

Question 6

Give an overview of what happens in peroxisomal β-oxidation?

Answer 6

4 steps - oxidation, hydration, oxidation and thiolytic cleavage
1. Oxidation using acyl-CoA oxidases (ACOX) which are flavoproteins
The electrons directly react with O2 forming H2O2
2. Hydration - water is added
3. Oxidation - catalysed by LBP and DBP (NAD linked dehydrogenases)
Electrons are sent to the electron transport chain and NADH is exported for reoxidation
4. Thiolysis - thiolases cleave the molecule into 2
The CoA molecule is sent to the citric acid cycle whilst the other repeats this cycle until smaller to be degraded in the mitochondria

Question 7

What molecules are involved in β-oxidation?

Answer 7

Pristanic acid
VLCFA
DHCA and THCA

Question 8

How are proteins imported into peroxisomes?

Answer 8

A short signal sequence - (Ser-Lys-Leu) - is located at the C-terminus of most peroxisomal proteins
This functions as an import signal
At least 23 peroxins driven by ATP hydrolysis are involved in the import

1. Peroxisomal protein, synthesised in the cytosol
   This binds to its receptor (PEX5) by a signal called PTS1
2. Receptor docks to protein complex on peroxisome membrane
3. Protein transported into peroxisome
4. Receptor recycled through action of a protein complex on the peroxisome membrane
5. Cycle repeats

Question 9

What are some defects with protein import?

Answer 9

If the docking or recycling complexes are defective
The protein will stay in the cytoplasm and not be imported
The component of the recycling complex could be defective and the receptor can’t be recycled - it accumulates in the membrane = blocks transport machinery = import stops

Question 10

How are peroxisomes detected?

Answer 10

Peroxisomes can be detected in cells using targeted fluorescent proteins as reporters or using antibodies by immunofluorescence
Using fibroblast cells

Question 11

What are the types of disorders that result from malfunctions in peroxisomes?

Answer 11

Peroxisomes biogenesis disorder (the whole organelle isn’t synthesised properly)

Single gene deficiencies

Question 12

What is a biogenesis disorder of the peroxisome?

Answer 12

Deficiency of multiple peroxisome metabolic pathways due to failure to assemble the organelle
They all fall under: Zellweger Syndrome spectrum

Question 13

Describe Zellweger syndrome spectrum?

Answer 13

Peroxisomes can be completely absent or are just empty
Membranes are ‘ghosts’ - the genes needed for peroxisome formation are non functional
When the peroxisome isn’t formed or is defective - enzymes and proteins that normally function are partly or wholly mis-localised to the cytosol - due to environment they don’t work
These patients either completely lack or have deficiencies in all peroxisome metabolic pathways

If the mutation makes the gene only partially functional defective peroxisomes may be formed = spectrum of phenotypes
Mutations in at least 13 different genes can give rise to Zellweger spectrum disorders
Severity increasing: Infantile Refsum disease, neonatal adreno-leukodystrophy and Zellweger syndrome

Question 14

What are some symptoms/prgnosis of Zellweger syndrome?

Answer 14

Zellweger syndrome:
Facial and cranial abnormalities; weak muscle tone (hypotonia); seizures; inability to feed
Almost every organ system is affected
Babies born with ZS fail to develop and usually die within the first year of life

Neonatal adrenoleukodystrophy and Infantile Refsum disease:
Milder forms of ZS, usually because of some residual activity of the mutated gene
Most have hypotonia, some have seizures, degeneration of myelin in the CNS can result in neurological deficits
Survival depends on the severity of the condition

RCDP:
Peroxisomes are present but a subset of peroxisomal proteins are missing
Abnormalities of the CNS and profound growth and psychomotor retardation
Some patients die in the first year of life, others have survived to early adulthood

Question 15

What is the diagnosis and treatment for Zellweger syndrome?

Answer 15

Diagnosis usually relies on looking at biochemical pathways affected e.g.
Accumulation of very long chain fatty acids as their breakdown is impaired
Elevated phytanic/pristanic acid
Reduced plasmalogens
Increased bile acid intermediates
If the defective gene is known then molecular testing can be performed and counselling offered to affected families

Since multiple biochemical pathways are affected a cure isn’t possible at present and treatment is primarily supportive, treating the symptoms

Question 16

What are some single gene deficiency disorders of the peroxisome?

Answer 16

There are many of these which arise from a mutation in a gene that encodes for a specific enzyme activity within peroxisomes (as opposed to a protein that is needed for assembly of the organelle)

X-ALD and PH1

Question 17

Describe X-ALD?

Answer 17

This is a disorder of peroxisomal beta oxidation and is the most common of these disorders frequency -1:17,000 males
The order is X linked
Affected boys inherit the affected gene from their mother
Therefore only affects males, but females can be carriers

Mutation in the ABCD1 gene encodes a membrane protein (ALDP) belonging to the ATP Binding Cassette family that transports very long chain fatty acids into the peroxisome
In patients very long chain fatty acids (C24-C26 and longer) accumulate and this is used for diagnosis

Question 18

What are some symptoms of X-ALD?

Answer 18

There are different forms of X-ALD and there isn’t a strong correlation between the causal mutation and the clinical presentation e.g. Same mutation but different signs/symptoms
Presentations of the disease: Childhood Cerebral ALD (CCALD) and Adrenomyeloneuroathy (AMN)

CCALD (most serious)
CCALD manifests at 3-10 years of age
There is inflammatory demyelination of neurones in the brain and progressive deterioration leading to disability and death within 3 years

Adrenomyeloneuroathy (AMN)
AMN is later onset (late 20’s normally) and slower progressing
Symptoms include muscle weakness and often impaired adrenocortical function
Some patients may have brain involvement

Question 19

What are some treatments of X-ALD?

Answer 19

Many patients have impaired steroid hormone synthesis due to toxic effects of the long chain fatty acids on the adrenal glands so adrenal hormone therapy given
Fats in the diet are restricted
Bone marrow transplants have been successful in slowing or halting progression of the cerebral form of ALD
However it’s a high risk treatment as in some patients it can cause the disease to progress more rapidly
Gene therapy is also being explored as a treatment and shows promise but the long term prognosis is unclear as this is a very recent experimental treatment
Diagnosis and counselling is offered to affected families

Question 20

Describe PH1?

Answer 20

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive condition caused by defects in Alanine Glyoxylate Aminotransferase, a peroxisomal liver enzyme
Frequency varies between populations 1 in 100,000- 1 in a million
AGT converts glyoxylate to glycine
If glyoxylate accumulates as a result of AGT deficiency oxalate builds up
Oxalate (very soluble) binds Ca2+ ions and precipitates, leading to kidney stones and renal failure

Can be caused by AGT mis-targeting
Most PH1 suffers lack the AGT enzyme completely however a some have most or all of the enzyme mis localised in mitochondria
P11L mutation creates a mitochondrial targeting signal, while G170R creates a protein conformation that is more readily imported by mitochondria

Question 21

What are some treatments and therapies for PH1?

Answer 21

50% of PH1 patients suffer renal failure by age 15, 80% by age 30
High doses of vitamin B6 can normalise oxalate levels in some patients
Patients are advised to avoid foods with high levels of oxalate (e.g. rhubarb, chocolate) and to drink plenty of water
Since AGT is a liver enzyme a liver transplant provides active and correctly localised AGT enzyme
If the kidneys have already been damaged a kidney transplant is required as well
Prognosis depends on early detection. Following transplants some people with PH1 have lived near normal lifespans and women have had healthy babies

Question 22

What is the role of the peroxisome in fungi and plant disease?

Answer 22

In some plant pathogenic fungi such as rice blast fungus and Anthracnose fungi peroxisomes are essential for pathogenicity
Infection requires formation of specialised structures, appressoria which have thick melanised walls
Peroxisomal beta-oxidation of lipids provide energy, osmolites for the development of turgor pressure and precursors for melanin production
Peroxisome biogenesis mutants (e.g. fam1) show much reduced pathogenicity