Download Peroxisomes: family of versatile organelles

Course:
Molecular, cell biological and genetic aspects
of diseases (743665S)
Lecture:
Peroxisomes
Faculty of Biochemistry
and Molecular Medicine
Lecturer:
Tuomo Glumoff, Ph.D.
University Lecturer
Acknowledgment:
some materials on these lecture handouts are
courtesy of Dr. Vasily Antonenkov
Contents:
1. What are peroxisomes?
2. Functions associated with peroxisomes
3. Proliferation, biogenesis and maintenance of peroxisomes
4. Protein import into peroxisomes
5. Interplay of peroxisomes with other organelles
Topical reading for those who are especially interested in peroxisomes:
http://www.sciencedirect.com/science/journal/01674889/1863/5
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Volume 1863, Issue 5, Pages 787-1070 (May 2016)
Assembly, Maintenance and Dynamics of Peroxisomes
Edited by Ralf Erdmann
Topics:
Characterization, prediction and evolution of plant peroxisomal targeting signals type 1 (PTS1s)
Structural biology of the import pathways of peroxisomal matrix proteins
The first minutes in the life of a peroxisomal matrix protein
Peroxisomal protein import pores
Role of AAA+-proteins in peroxisome biogenesis and function
Regulation of peroxisomal matrix protein import by ubiquitination
Peroxisome biogenesis, protein targeting mechanisms and PEX gene functions in plants
Towards the molecular mechanism of the integration of peroxisomal membrane proteins
Targeting and insertion of peroxisomal membrane proteins: ER trafficking versus direct delivery to peroxisomes
Multiple paths to peroxisomes: Mechanism of peroxisome maintenance in mammals
De novo peroxisome biogenesis: Evolving concepts and conundrums
The birth of yeast peroxisomes
A role for mRNA trafficking and localized translation in peroxisome biogenesis and function?
Human disorders of peroxisome metabolism and biogenesis
Peroxisomes in brain development and function
Hepatic dysfunction in peroxisomal disorders
Proliferation and fission of peroxisomes — An update
Peroxisome homeostasis: Mechanisms of division and selective degradation of peroxisomes in mammals
Pexophagy in yeasts
Pexophagy and peroxisomal protein turnover in plants
Small GTPases in peroxisome dynamics
Sharing with your children: Mechanisms of peroxisome inheritance
Why do peroxisomes associate with the cytoskeleton?
Regulation of peroxisome dynamics by phosphorylation
Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites
Peroxisome biogenesis in mammalian cells: The impact of genes and environment
No peroxisome is an island — Peroxisome contact sites
1. What are peroxisomes?
microbodies
peroxisomes
- liver
- kidney
microperoxisomes
- other tissues
glyoxysomes
glycosomes
- glyoxylate cycle
enzymes (in
addition)
- glycolytic
pathway
enzymes
(Trypanosoma)
Woronin
bodies
(mushrooms)
mitochondrion
peroxisome
Med InFoZ (medinfoz.com)
• diameter: peroxisomes 0.5µm; microperoxisomes 0.1-0.2µm
• single membrane
• no own DNA
Microperoxisomes
•
Most peroxisomes in plants and yeasts
are with size around 0.5 µm. In
contrast, peroxisomes in mammalian
tissues (except for liver and kidney) are
small (0.1-0.2 µm) 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.
•
Rat parotid exocrine glands (photo).
Why such name?
•
First enzymes discovered in the particles: catalase and H2O2-producing
oxidases (urate oxidase, D-amino acid oxidase and glycolate oxidase);
•
•
Catalase is able to catalyze two reactions: dismutation of H2O2:
2H2O2 = 2H2O + O2 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 organelles: peroxisomes,
i.e. particles were hydrogen peroxide is metabolized.
Main metabolic pathways in peroxisomes
- fatty-acyl β-oxidation: can handle different substrates, which are imported by
different ABCD transporters
- fatty-acyl α-oxidation of phytanic acid
- plasmalogen synthesis
Waterham et al. Biochim Biophys Acta. 2016
- bile acid synthesis
- glyoxylate detoxification
Main metabolic pathways in peroxisomes.
Peroxisomal proteins involved in the different pathways are indicated by their respective
gene names and boxed.
Substrates and products are unboxed.
The main enzymes involved in the peroxisomal fatty acid beta-oxidation pathway are
indicated in light orange. The peroxisomal fatty acid beta-oxidation pathway can handle
different substrates (indicated by distinct colors), including very long chain fatty acids
(sVLCFA, unVLCFA (green)), dicarboxylic acids (DCA (blue)), the bile intermediates DHCA
and THCA (dark red), and pristanic acid (magenta), which are imported into peroxisomes by
the different ABCD transporters as indicated. The peroxisomal enzymes involved in alphaoxidation of phytanic acid (purple) are indicated in yellow.
The peroxisomal enzymes involved in plasmalogen synthesis (black) are indicated in purple.
The peroxisomal enzymes involved in bile acid synthesis (dark red) are indicated in bright
and light orange. The peroxisomal enzyme involved in glyoxylate detoxification (brown) is
indicated in red and catalase required for H2O2 degradation in green.
Abbreviations:
ACAA1 = 3-ketoacyl-CoA thiolase
ACOX1 = Acyl-CoA oxidase 1
ACOX2 = acyl-CoA oxidase 2
ABCD1 = ABC transporter D1
ABCD2 = ABC transporter D2
ABCD3 = ABC transporter D3
AGPS = alkyl-dihydroxyacetonephosphate synthase
AGT = alanine-glyoxylate aminotransferase
AMACR = 2-methylacyl-CoA racemace
BAAT = bile acid–CoA:amino acid N-acyltransferase
brAcyl = branched-acyl
CA = cholic acid
CAT = catalase
CDCA = chenodeoxycholic acid
DBP = D-bifunctional protein
DCA = dicarboxylic acids
DHAP = dihydroxyacetone phosphate
DHCA = dihydroxycholestanoic acid
FAR1 = fatty acyl reductase 1
GNPAT = dihydroxyacetonephosphate acyltransferase
HACL1 = 2-hydroxyphytanoyl-CoA lyase
LBP = L-bifunctional protein
PHYH = phytanoyl-CoA 2-hydoxylase
PrDH = pristanal dehydrogenase
SCPx = SCPx
sVLCFA = saturated very long chain fatty acids
THCA = trihydroxycholestanoic acid
unVLCFA = unsaturated very long chain fatty acids.
2. Functions associated with peroxisomes
•
The main function of mammalian
peroxisomes is beta-oxidation of
long- and very long-chain fatty acids
and a side chain of bile-acid
precursors. Dual localization of the
beta-oxidation in mammals
(peroxisomes and mitochondria).
•
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
down to acetyl group.
•
•
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.
Catabolism of purines - another example how peroxisomes
participate in a complex metabolic pathway.
Further functions of peroxisomes
The main function of peroxisomes is
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) chain fatty acids, bile acids,
some amino acids, etc. Some non-lipid
molecules with poor solubility in water:
purines and oxalates, are oxidized also
in peroxisomes.
Further functions of peroxisomes
•
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.
•
Role of peroxisomes: prevent
formation of H2O2 and glyoxylate in
mitochondria and in chloroplasts
Glyoxysomes in plant seeds
•
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 glyoxylate cycle.
3. Proliferation, biogenesis and maintenance of
peroxisomes
•
Some amphipathic compounds increase
amount and size of peroxisomes in liver,
kidney and intestine of rodents (mice,
rats), and, in less extent, 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.
•
Proliferation of peroxisomes is an
adaptation mechanism to excessive
consumption of lipids. It is part of more
complex system for regulation of lipid
metabolism.
Mitochondrial beta oxidation is also increased, but at the lower level.
•
•
Receptor concept: the cytosolic receptor binds ligand (peroxisome proliferator) and
delivers it to 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 regulation of other pathways in lipid metabolism and in cell growth
and differentiation.
After ligand binding the PPAR forms
a heterodimer with another nuclear
receptor, the retinoid-X receptor
(RXR), that binds retinoic acid
(related to vitamin A);
Next, the PPAR/RXR complex binds
to DNA sequences containing
repeats of hexanucleotide: AGGTCA,
known as peroxisome proliferator
response element (PPRE). These
repeats were found in the promoter
regions of PPAR target genes. Some
additional proteins (co-activators) are
involved in the binding.
(next page)
Lee & Kim PPAR Research 2015
•
Biogenesis of peroxisomes is a complex
process requiring more than 30 different
proteins called Pex proteins;
•
Peroxisomes have no own DNA, therefore
matrix and membrane proteins are
synthesized on free (poly)ribosomes in
cytoplasm and imported into peroxisomes
by means of special mechanisms;
•
•
Several steps in biogenesis:
1. Budding of a new peroxisome from
special subdomain of endoplasmic
reticulum
2. Growth and maturation of the
peroxisome that involves delivery of the
newly synthesized proteins into the
particles
3. Division of the matured peroxisome into
daughter peroxisomes.
•
•
model for de novo
biogenesis
Hua & Kim Biochim Biophys Acta. 2016
model for the maintenance of peroxisomes:
•
growth and division from pre-existing
peroxisomes
•
de novo from other organelles, like ER
Hua & Kim Biochim Biophys Acta. 2016
Agrawal & Subramani Biochim Biophys Acta. 2016
Schematic representation of
peroxisome biogenesis pathways.
1] PMPs are both translated in the
cytosol on free ribosomes or on the
ER-associated ribosomes and
incorporated post-translationally or
cotranslationally in the ERmembrane. The ER-translocon,
Sec61, is important for the PMP
incorporation process. Similarly,
TA-proteins (tail-anchored) are
imported into the ER-membrane
via the GET pathway.
2] Subsequently, an intra-ER
sorting process targets the PMPs
to respective pER domains.
3] The PMPs are exported from the
ER in vesicular carriers and require
Pex19. Pex16 is also important for
the exit of Pex3 and other PMPs
from the ER in mammalian cells.
4] The vesicular carriers containing
complementary sets of PMPs fuse
to assemble the importomer
complex. The fusion process
requires peroxins Pex1 and Pex6.
5] This assembly enables the
nascent peroxisome to import
matrix proteins and become a
metabolically active organelle.
6] Type II PMPs are imported
directly into the peroxisome
membrane with the assistance of
Pex3 and Pex19 (inset).
7] The de novo route involving the
ER also contributes to the cellular
peroxisome population, thus
sustaining the growth and division
pathway and substituting for it
when it is blocked or impaired.
Using this backup pathway, in
mutant cells (such as pex3∆ and
pex19∆ cells) lacking functional
pre-existing peroxisomes,
reintroduction of the missing gene
will form peroxisomes de novo and
the new peroxisomes that are
generated will restart growth and
division pathway.
Agrawal & Subramani Biochim Biophys Acta. 2016
Waterham et al. Biochim Biophys Acta. 2016
Signalling pathways that trigger peroxisome proliferation in mammals. (A) Extra- or
intracellular signalling molecules such as growth factors, hypolipidemic compounds, ROS or
fatty acids bind to specific nuclear receptors (e.g. PPARα) leading to PPAR-dependent and
potentially to PPAR-independent signalling pathways.
Schrader et al. Biochim Biophys Acta. 2016
(B) In the PPAR-dependent pathway, binding of activating ligands to PPARs and to its
partner RXR induce conformational changes of the receptors resulting in transcriptional
complex formation. Activation of the transcriptional complex occurs upon transcriptional
cofactor binding, which then allows binding to PPREs in peroxisomal genes to initiate gene
expression. PPAR-independent mechanisms have also been described which may signal
through as yet unidentified receptors (indicated by “?”)
Schrader et al. Biochim Biophys Acta. 2016
(C) Expression of peroxisomal biogenesis factors (e.g. Pex11) and/or β-oxidation enzymes
can promote peroxisome proliferation and/or increased fatty acid metabolism. Peroxisome
proliferation is initiated by membrane remodelling followed by a multi-step process of
membrane elongation (growth), constriction and final membrane scission. These processes
are mediated by division proteins (e.g. Pex11β, Fis1, Mff and DLP1)
Schrader et al. Biochim Biophys Acta. 2016
Model of peroxisome division in mammalian cells. DHA facilitates the oligomerization of
Pex11β, which leads to the formation of Pex11β-rich regions and initiates peroxisome
elongation (step 1). Peroxisomes elongate there in one direction (step 2). Mff and Fis1 are
localized to peroxisomes (step 3), where Mff then recruits DLP1 and the Mff–DLP1 complex
translocates to the membrane-constricted regions of elongated peroxisomes.
Honsho et al. Biochim Biophys Acta. 2016
The functional complex comprising Pex11β, Mff, and DLP1 promotes Mff-mediated fission
during peroxisomal division (step 4). The complex may include Fis1 that also interacts with
DLP1. The activated DLP1 hydrolyzes GTP, by which peroxisomal membranes are cleaved,
thereby giving rise to peroxisomal fission (step 5), followed by translocation of daughter
peroxisomes via microtubules (MT) (step 6).
4. Protein import into peroxisomes
•
Transport of folded, cofactor-bound
and oligomeric proteins by shuttling
receptors pex5 (PTS1) and
pex7(PTS2);
•
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);
•
After delivering of proteins into
peroxisomes, the receptors move back
to cytosol.
•
•
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, it forms temporal channel which allows
matrix proteins penetrate the membrane.
Model of peroxisomal matrix protein import.
(I) Proteins harboring a peroxisomal targeting signal of type 1 (PTS1) are
recognized and bound by the import receptor Pex5 in the cytosol;
(II) The cargo-loaded receptor is directed to the peroxisomal membrane and
binds to the docking complex (Pex13/Pex14/Pex17);
(III) The import receptor assembles with Pex14 to form a transient pore;
(IV) Cargo proteins are transported into the peroxisomal matrix in an unknown
manner. Cargo release might involve the function of Pex8 or Pex14;
(V) The import receptor is monoubiquitinated at a conserved cysteine by the E2enzyme complex Pex4/Pex22 in tandem with E3-ligases of the RINGcomplex (Pex2, Pex10, Pex12);
(VI) The ubiquitinated receptor is released from the peroxisomal membrane in an
ATP-dependent manner by the AAA-peroxins Pex1 and Pex6, which are
anchored to the peroxisomal membrane via Pex15. As the last step of the
cycle, the ubiquitin moiety is removed and the receptor enters a new round of
import. The designation is based on the yeast nomenclature.
Emmanouilidis et al. Biochim Biophys Acta. 2016
Import pathways of peroxisomal matrix proteins. The import of peroxisomal matrix proteins
occurs via two pathways, based on the presence of distinct peroxisomal targeting signal (PTS)
sequences in the cargo protein. Cargo proteins with a PTS1 motif exhibit a C-terminal tripeptide
(S/A/C)–(K/R/L)–(L/M) (PTS1, left) and bind to the Pex5 receptor in the PTS1 import pathway.
Cargo proteins harboring an N-terminal PTS2 motif with a consensus sequence
(R/K)-(L/V/I)-X5-(H/Q)-(L/A) are recognized by Pex7/Pex21 and imported via the PTS2 pathway
(right).
(A) Newly made cargo proteins and peroxins
are released from polyribosomes into the
cytosol. (B) In the cytosol, PTS1 cargo is
recognized by the soluble Pex5 receptor,
while PTS2 cargo is recognized by a
complex of the Pex7/Pex21 receptors
leading to the formation of receptor–cargo
complexes. Note, that the coreceptor of
Pex7 differs depending on the species.
In human a long splice isoform of Pex5 acts
as a coreceptor. In yeast species
Pex21/Pex18 and Pex20, act as coreceptors
(C) The cargo–receptor complexes bind to a docking subcomplex comprising Pex14 and Pex13
at the peroxisomal membrane. (D) Subsequently, the receptor–cargo complexes translocate the
matrix protein into the peroxisome. Note, that — although the basic steps along the pathways
are similar — the peroxisomal proteins involved and their specific roles vary between organisms.
Peroxisomal protein import. Schematic view of the human import machinery for
peroxisomal matrix proteins and membrane proteins highlighting the roles of the different
PEX proteins (peroxins).
Waterham et al. Biochim Biophys Acta. 2016
(note the difference to the previous slides)
PTS2 cargo recognition by the
Pex7–Pex21 receptor.
(A) Cartoon and surface
representation of the PTS2 cargo–
receptor complex. The Pex7WD
domain (yellow) and the C-terminal
domain of the coreceptor Pex21
(cyan) together recognize the
helical PTS2 signal peptide of
Fox3N (red) fused to MBP (gray).
(B) Zoomed view of the PTS2 helix
with side chains depicted as sticks.
Emmanouilidis et al. Biochim Biophys Acta. 2016
Peroxisomal protein import receptor cycle: (1) Cargo recognition in the cytosol. (2) Docking
of the receptor–cargo complex to the peroxisomal membrane via the docking complex. (3)
Pore assembly and protein translocation. (4) Receptor ubiquitination. (5) Receptor export
and recycling. Abbreviations: ubiquitin (Ub).
Meinecke et al. Biochim Biophys Acta. 2016
5. Interplay of peroxisomes with other organelles
No peroxisome is an island
In order to optimize their multiple cellular functions, peroxisomes must collaborate and
communicate with the surrounding organelles. A common way of communication between
organelles is through physical membrane contact sites where membranes of two organelles are
tethered, facilitating exchange of small molecules and intracellular signaling. In addition contact
sites are important for controlling processes such as metabolism, organelle trafficking,
inheritance and division.
How peroxisomes rely on
contact sites for their various
cellular activities is only
recently starting to be
appreciated and explored
and the extent of
peroxisomal communication,
their contact sites and their
functions are less
characterized.
Shai et al. Biochim Biophys Acta. 2016
Peroxisome–mitochondria contact site. Peroxisomes can be localized adjacent to a
specific mitochondrial niche near the ER-mitochondria contact site proximal to where
the pyruvate dehydrogenase (PDH) complex is found in the mitochondria matrix. The
proximity to the ER-mitochondria contacts may suggest a function of a three way organelle
junction. The peroxisome-mitochondria tether has been suggested to be mediated
by the interaction between Pex11, a key protein involved in peroxisome proliferation,
and Mdm34, one of the proteins creating the ER-mitochondria tether (ERMES).
Schrader et al. Biochim Biophys Acta. 2016
There is emerging evidence that the functional relationship between peroxisomes and
mitochondria is the result of an organellar co-evolution originating in the early ancestors of
all eukaryotes. This fundamental interconnection between peroxisomes and mitochondria is
reflected by an increasing number of cooperative functions, such as fatty acid β-oxidation,
innate immune response, maintenance of ROS homeostasis or even regulation of apoptosis
and cell survival.
Furthermore, both organelles share a significant number of proteins linked to the cellular
processes.
Peroxisome–lipid droplet contact site. In the budding yeast, peroxisomes stably adhere
to lipid droplets (LDs) thereby stimulating the breakdown and transfer of various
lipids across these organellar boundaries. Peroxisome protrusions, termed pexopodia,
have been seen to invade the LD core as a result of hemifusion of the single leaflet of the
lipid droplet membrane and the outer leaflet of the peroxisomal membrane. Pexopodia
are believed to facilitate the flux of fatty acids into peroxisomes and the transfer of
peroxisomal oxidation enzymes into LDs.
Peroxisome–lysosome contact site. It was recently identified that a peroxisome–lysosome
contact site occurs (termed LPMC) in human cells. The LPMC is dynamic and cholesterol
dependent. The tether holding the two organelles together is the integral lysosomal membrane
protein, Synaptotagmin VII (Syt7), through binding to the lipid PI(4,5)P2 on the peroxisomal
membrane. This contact site facilitates the transfer of cholesterol from lysosomes to
peroxisomes.
Peroxisome–peroxisome interaction. In mammalian cells, peroxisomes are engaged
in close self-interactions. Although such interactions are transient, peroxisomes are able to
re-contact rapidly creating long-term contacts.
The interaction between peroxisomes has three possible roles:
(A) Movement of peroxisome populations in the cell.
(B) Fusion between peroxisomes.
(C) Creation of functional units to exchange metabolites or lipids.
Three hypotheses as to how peroxisomes maintain such diverse contacts with other organelles:
(A) Random encounters: peroxisome may contain all the necessary proteins, lipids and
molecules to interact with any other organelle and even with multiple organelles at once.
Random encounters enable the creation of contact sites between peroxisomes and other
organelles.
(B) Conditional contact sites: peroxisome contact sites are condition dependent hence forming
specifically with certain organelles for specific functions. Changes in contact site partners are
enabled by specific signals for the regulation of the tether/proteins/lipids.
(C) Specialized peroxisomes: peroxisome sub-populations exist in the cell, each tailored to
interact with a specific organelle. Each sub-population could contain a unique proteome and
has the ability to interact with a specific organelle for a different function.
Attachment of peroxisomes to cytoskeleton and movement along microtubular filaments and
actin cables are essential and highly regulated processes enabling metabolic efficiency,
biogenesis, maintenance and inheritance of this dynamic cellular compartment. Several
peroxisome-associated proteins have been identified, which mediate interaction with motor
proteins, adaptor proteins or other constituents of the cytoskeleton. It appears that there is a
species-specific complexity of protein–protein interactions required to control directional
movement and arresting. An open question is why some proteins with a specific role in
peroxisomal protein import have an additional function in the regulation of cytoskeleton
binding and motility of peroxisomes.
Neuhaus et al. Biochim Biophys Acta. 2016