Peroxisomes: family of versatile organelles

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Kontinkangas, L101A
Biochemistry of cellular organelles
Lectures: 1. Membrane channels;
2. Membrane transporters;
3. Soluble lipid/metabolite-transfer proteins;
4. Mitochondria as cellular organelles;
Seminar: Isolation of subcellular organelles;
5. Mitochondrial inheritance;
6. Mitochondria in health and disease;
7. Endoplasmic Reticulum (ER) and lipids;
8. Structure and function of peroxisomes;
Seminar: Mitochondria in cellular life.
Dr. Vasily Antonenkov, Visiting professor
Dept. Biochemistry, Oulu University
Oulu, Finland
Web site:
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Lecture 8: Structure and function of
peroxisomes
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8.
History and terminology;
Morphology;
Function of peroxisomes in mammals;
Induction of peroxisomes by fibrates and lipids;
Diversity of peroxisomal functions;
Biogenesis of peroxisomes;
Peroxisomal disorders;
Channels and transporters in peroxisomes.
History and terminology
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First enzymes discovered in the particles: catalase and H202-producing
oxidases (urate oxidase, D-amino acid oxidase and glycolate oxidases);
Catalase is able to catalyze two reactions: dismutation of H202:
2H202=H20 + 02 and peroxidation (if substrate is available) of some
compounds (methanol, ethanol, certain phenols, formaldehyde, formic
acid, and the nitrite ion):
The oxidases produce H202 that can be used by catalase to oxidize
corresponding substrates.
The proposed chain of reactions gives the name to the organelles:
peroxisomes, i.e. particles were hydrogen peroxide is metabolized.
Terminology
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Peroxisomes and their relatives form family of organelles called
microbodies. In addition to peroxisomes this family contains:
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Plant seeds: Glyoxisomes. These particles contain in addition
to catalase and oxidases the enzymes involved in glyoxylate cycle;
Trypanosome species (parasites in blood causing some diseases
like sleeping disease): Glycosomes. The particles contain
glycolytic pathway enzymes side by side with proteins characteristic
for other microbodies;
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• Microperoxisomes: small peroxisomes detected mainly
morphologically in all mammalian tissues except liver and kidney
where large peroxisomes are present;
• Woronin bodies: special type of microbodies in fungi
(mushrooms) dealing with repearing of wounded roots.
Morphology
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Mammalian liver or kidney peroxisomes are globular structures
approximately 0.5 micrometers in diameter, surrounded by a single
membrane.
Liver and kidney peroxisomes frequently contain electron dense
structure called nucleoid – natural crystal of the enzyme urate oxidase.
• Rat liver peroxisomes (two different stainings):
Microperoxisomes
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Most peroxisomes in plants and yeasts
are with size around 0.5 mkm. In
contrast, peroxisomes in mammalian
tissues (except for liver and kidney) are
small (0.1-0.2 mkm) and without
nucleoid. It is difficult to recognize them
on tissue slices using common
transmission electron microscopy.
•
Method has been developed (Graham,
Karnovsky staining) to detect catalase
in peroxisomes by its peroxidase
activity with diaminobenzidine. The
product of this reaction forms an
insoluble sediment which can be seen
under EM.
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Rat parotid exocrine glands (photo).
Function of peroxisomes (I)
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The main function of
peroxisomes is an oxidative
degradation of compounds with
poor (low) solubility in water and
in lipids as well. Most of these
compounds are amphipathic
molecules (mostly lipids)
containing a charged group and
a large hydrophobic part, such
as long- (C16-C20) and very
long- (C22 and longer) fatty
acids, bile acids, some amino
acids, etc. Some non-lipid
molecules with poor solubility in
water: purines and oxalates, are
also oxidized in peroxisomes .
Function of mammalian peroxisomes (II)
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One example how mammalian peroxisomes can participate in a
complex degradation pathway: formation of bile acids from
cholesterol.
Function of mammalian peroxisomes (III)
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The main function of mammalian
peroxisomes is the beta-oxidation
of long- and very long-chain fatty
acids and the side chain of bileacid precursors. Dual localization
of the beta-oxidation in mammals
(peroxisomes and mitochondria).
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The peroxisomal beta-oxidation of
fatty acids does not proceed to
completion, i.e., complete
degradation of fatty acids to
acetyl (C2) groups. Instead, it
produces chain-shortened fatty
acids (C6-C14) which can be
used inside or outside
peroxisomes for synthesis of
some compounds or exported to
mitochondria for further oxidation.
Function of mammalian peroxisomes (IV)
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Alpha-oxidation of branchedchain fatty acids derived from
phytol – constituent of plant
chloroplast;
Oxidation of glycolic acid, main
source of it is plant leafs and
other green staff.
Function of mammalian peroxisomes (V)
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Catabolism of purines - another
example how peroxisomes
participate in a complex metabolic
pathway.
Proliferation of mammalian peroxisomes (I)
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Some amphipathic compounds increase
amount and size of peroxisomes in liver,
kidney and intestine of rodents (mice,
rats), and, in a less extend, in humans.
Proliferation of peroxisomes leads to
activation of peroxisomal beta-oxidation
of long chain fatty acids in the liver for
more than 10 times (rodents).
•
The natural peroxisome proliferators are
most probably some long chain fatty
acids, especially unsaturated acids.
•
The proliferation of peroxisomes is an
adaptation mechanism to excessive
consumption of lipids. It is a part of
more complex system for regulation of
lipid metabolism.
Proliferation of mammalian peroxisomes (II)
Mitochondrial beta oxidation is also
increased, but at the lower level.
Proliferation of mammalian peroxisomes (III)
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Receptor conception: cytosolic receptor binds ligand (peroxisome proliferator) and
deliver it to the nucleus where the complex interacts with DNA and activates
transcription of peroxisomal genes. Peroxisome proliferator-activated receptor
family of proteins - PPAR’s. Only PPAR alpha is responsible for proliferation of
peroxisomes, other members of the family are involved in different steps of
regulation of the lipid metabolism and in cells growth and differentiation.
Proliferation of mammalian peroxisomes (IV)
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After ligand binding the PPAR’s
heterodimerize with another
nuclear receptor, the retinoid-X
receptor (RXR), that binds
retinoic acid (related to vitamin
A), and next, the PPAR/RXR
complex binds to DNA
sequences containing direct
repeats of the hexanucleotide
AGGTCA, known as direct repeat
1 (DR-1) response element.
These repeats are present in the
promoter regions of PPAR target
genes. Some additional proteins
(co-activators) are involved in the
binding.
Diversity of peroxisomal functions (I)
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Peroxisomes in plant leafs
participate in photorespiration
producing link between
chloroplasts and mitochondria.
•
Photorespiration is a lightdependent uptake of O2 and
release of CO2. Photorespiration
regulates efficiency of
photosynthesis that is important
at high level of illumination.
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Role of peroxisomes: prevent
formation of H2O2 and
glyoxylate in mitochondria and in
chloroplasts
Glyoxysomes in plant seeds
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Plants can convert lipids to sugars using glyoxylate cycle that is short
variant of the citric acid cycle. Animal cells can not carry out the net
synthesis of carbohydrate from fat because they have no the
glyoxylate cycle.
Woronin bodies
Like plants, fungi (mushrooms), contain a
network of roots that are named hyphae.
These roots are separated into sections that
connected to each other by pore. In the
case of membrane damage in the one
section, the pore is sealed by Woronin
bodies (like stopper seals bottle) that leads
to separation of hyphae sections preventing
gross leakage of cytoplasm.
Woronin bodies are close relatives to
peroxisomes. Both particles have the same
biogenesis mechanism, similar morphology
and share homologous proteins. However,
Woronin bodies contain the only few
proteins.
Biogenesis of peroxisomes
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Biogenesis of peroxisomes is a complex
process in which participate of more than
30 different proteins called Pex proteins.
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Peroxisomes have no its own DNA,
therefore matrix and membrane proteins
are synthesized on free (poly)ribosomes in
cytoplasm and imported into peroxisomes
by special mechanisms.
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Several steps in biogenesis: 1. Budding of
a new peroxisome from special subdomain
of endoplasmic reticulum; 2. Growth and
maturation of the peroxisome which
involves delivery of the newly synthesized
proteins into the particles; 3. Division of the
matured peroxisome into daughter
peroxisomes
Protein import into peroxisomal matrix
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Transport of folded, co-factor-bound
and oligomeric proteins by shuttling
receptors pex5 (PTS1) and
pex7(PTS2).
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Receptors recognize newly
synthesized proteins in the cytosol
using peroxisomal targeting signals
(PTS): PTS1 is at the C-terminus with
a consensus sequence
(S/A/C)(K/R/H)(L/I); PTS2 is near the
N-terminus with a consensus
sequence (R/K)(L/V/I)-xxxxx(H/Q)(L/A) (only few proteins).
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After delivering of proteins into
peroxisomes, the receptors move back
to cytosol.
Transfer of proteins across the peroxisomal membrane
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The problem is to deliver folded and even oligomeric protein across
the membrane into the matrix.
Hypothesis: Transient pore model. When Pex 5 receptor protein
together with cargo protein reach the peroxisomal membrane, the
Pex 5 forms temporal channel which allows matrix proteins
penetrate the membrane.
Peroxisomal disorders
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Group of inherited diseases in humans caused by malfunctioning of
peroxisomes.
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Two groups: peroxisome biogenesis disorders caused by malfunctioning of
the Pex proteins (example: Zellweger syndrome, cerebro-hepato-renal
syndrome caused by deficiency of the pex5 receptor) and the single
peroxisomal enzyme deficiencies (example: Refsum disease caused by
mutation in the gene coding for phytanoyl-CoA hydroxilase, the key enzyme
in alpha oxidation of branched chain fatty acids).
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Zellweger syndrome is lethal at early age. Most peroxisomal matrix
enzymes are not imported into the particles and degraded in the cytosol
after the synthesis or ineffective in their function. The biochemical
aberrations observed in Zellweger patients includes accumulation of longand very long fatty acids, branched fatty acids, precursors of bile acids and
decrease in formation of bile acids and plasmalogenes.
Mechanism of solute transfer across peroxisomal membrane is
quite unusual
a
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Contrary to other biological membranes
which are open or closed for all solutes, the
peroxisomal membrane is freely permeable
to small solutes but has very low
permeability to molecules comparable by
size with cofactors and ATP (‘bulky’
solutes);
The peroxisomal membrane may contain
non-selective channels (porins) side by side
with transporters specific for ‘bulky’ solutes
like ATP and NAD transporters.
NADH
ATP
Uric acid
Ps
Glycine
Cyt
Transporter
Peroxisomes contain their own pool of
cofactors but share common pool of small
solutes with surrounding cytoplasm.
Channel
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Transport of lipids (fatty acids and bile
acids precursors) into peroxisomes is
catalyzed by ABC transporters: three
proteins are known, defect of one of
them causes an inherited disease – Xlinked adrenoleukodystrophy when very
long-chain fatty acids are accumulated
in the organism.
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Transporters form homodimers and
accept CoA derivatives of different lipids
as substrates. However, they transfer
free lipids (without CoA). The lipids are
activated back to CoA derivatives inside
peroxisomes by action of acyl-CoA
synthase.
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How lipid products of peroxisomal
metabolism (bile acids, long-chain fatty
acids and others) are exported from the
particles is unclear. It might be that
some ABC transporters work as
exporters or some still undiscovered
transporters present in the membrane.
Peroxisomal lipid
transporters
Peroxisomal ATP/AMP antiporter
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Homologous to mitochondrial ATP/ADP
antiporter.
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Mice with deleted ATP transporter show
no phenotype at normal conditions, but
sick after eating phytol – branched-chain
alcohol.
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Antiporter participates in oxidation of
branched chain fatty acids by providing
ATP for synthesis of acyl-CoA
derivatives by acyl-CoA synthase
located inside peroxisomes.
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Transporters specific for cofactors (NAD,
FAD, CoA) have been recently
discovered in mammalian and plant
peroxisomes. They are homologous to
mitochondrial carriers.
Properties of peroxisomal membrane channels
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All peroxisomal membrane channels
have been described in Oulu;
The channel proteins belong to two
families: Pxmp2p and Pex11p,
respectively;
In mammals up to four different channel
types may be present in the same
peroxisomal membrane. Why so many?
– is not clear;
It seems that all channels are nonselective with a pore diameter up to 1.5
nm;
The channels function as a sizeselective filters with an exclusion limit of
about 400-500 Da for hydrophilic
solutes;
Pex11 proteins in addition to their
channel-forming activity, are involved in
biogenesis of peroxisomes
Predictions based on the properties of peroxisomal channels
B
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The channels allow function of the
shuttle mechanism for oxidation of
NADH and reduction of NADP+ inside
peroxisomes. Due to presence of the
channels the peroxisomal membrane
do not require transporters specific for
shuttle molecules like in the case of
mitochondria;
Presence of channels may explain
function of nudix hydrolases localized
inside peroxisomes. These enzymes
cleave cofactors (NAD/P, CoA) exactly
in the middle of the molecule producing
smaller compounds which are able to
penetrate the membrane through
peroxisomal channels.
Cofactors are delivered into
peroxisomes by specific transporters.
Ps
Cyt
+
NAD
-oxidation
Pyruvate,
Dihydroxyacetone phosphate
NADH
LDH,
G-3-PDH
NAD
Lactate,
Glycerol-3phosphate
+
C
Ps
Cyt
+
NAD(P)
Nudix
hydrolases
CoA
Overview of the transport machinery in
peroxisomal membrane
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ABC transporters – import of lipids in peroxisomes;
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ATP/AMP antiporter – import of ATP into peroxisomes, export of
AMP (?);
Cofactor transporters - import of cofactors into peroxisomes;
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Non-selective channels – transfer of small solutes in- and out of
peroxisomes;
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Channels + nudix hydrolases – removing of cofactors out of
peroxisomes;
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Still not known:
- export of lipids out of peroxisomes (carnitine transporter?);
Suggested questions
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What organelles comprise family of microbodies?
Describe shortly morphology of mammalian peroxisomes and microperoxisomes;
Degradation of what kind of molecules takes place mainly in peroxisomes? Some
examples;
Describe shortly the main metabolic functions of mammalian peroxisomes;
Beta-oxidation of fatty acids in mammalian peroxisomes;
What is the difference between alpha- and beta-oxidation of fatty acids?
Mechanism of proliferation of mammalian peroxisomes;
What receptors are involved in the proliferation of peroxisomes? How they act?
What is the main function of peroxisomes in plant leaves and in glyoxysomes in plant
seeds?
Woronin bodies – what is it?
Describe shortly biogenesis of peroxisomes;
What is the role of Pex5 and Pex7 proteins in biogenesis of peroxisomes;
Two types of peroxisomal disorders – describe shortly;
What is the difference in transfer of small solutes and cofactors across peroxisomal
membrane?
Lipid transporters in peroxisomes;
Properties and functional role of peroxisomal membrane channels.
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