Mitochondria are transferred to molecular oxygen (O2), reducing it

Mitochondria is the major source of ATP production, necessary
for cellular functions and integrity. Accumulation of mutations in mtDNA can
lead to cellular dysfunction by altering oxidative phosphorylation,
Ca2+ homeostasis,
oxidative stress and protein turnover. Dysfunction
of mitochondria leads to many neuro-degenerative diseases such as Parkinson’s
disease and Alzheimer’s disease. Hence, damaged mitochondria needs to be
eliminated by inducing a plethora of stress signals causing programmed cell
death. Mitochondrial homeostasis and quality control is maintained by a selective
form of autophagy, i.e., mitophagy. Earlier studies have shown that multistep
signalling events of PINK1 (PTEN-induced putative kinase1) and Parkin E3
Ubiquitin ligase regulates mammalian mitophagy. Here, we review the complex
signal transduction mechanism of PINK1 and Parkin focusing on pathways that
sequester mitochondria to autophagosome. Also post-translational modification such
as ubiquitinylation and phosphorylation of Ubiquitin and Parkin has added a
broader perspective to the understanding of cellular damage.

Keywords: Mitophagy, PINK1, Parkin, Ubiquitinylation

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Introduction

Mitochondria
are organelles enclosed within a double membrane, which is comprised of the
outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM)
(Fig. 1a). Ample amount of mitochondria are present in most cell types which
occupies proximately 10–40 % of total cellular volume 1. The mitochondrial
space between the OMM and IMM is attributed as the intermembrane space (IMS). Mitochondria
are crucial for eukaryotic cells, as it performs a number of critical functions.
It plays a pivotal role in generation of cellular energy, regulating lipid
metabolism, cytosolic calcium flux buffering and sequestering the cell death
machinery. Malfunction of the mechanisms that regulate mitochondrial quality
control have proven to be a major driving force of normal ageing 2.
Furthermore, failure of mitochondrial quality control mechanisms, causing
elevated oxidative stress, is strongly linked to age-related conditions such as
neurodegeneration 3, 4.

Most
of the cellular chemical energy is produced in the mitochondrial matrix via the
process of oxidative phosphorylation (OXPHOS), in the form of adenosine
triphosphate (ATP). It involves the oxidation of tricarboxylic
acid (TCA) cycle component, acetyl-CoA to
generate NADH and FADH2, which transfer electrons to the electron
transport chain components in the inner mitochondrial membrane, terminating in
the reduction of oxygen in the matrix to produce an electrochemical gradient
across the inner mitochondrial membrane that is used to produce ATP 5. Eventually, electrons are transferred to molecular
oxygen (O2), reducing it to H2O (fig). However, due to leakage of electrons at complex I or complex III of the
electron transport chain, O2 can be incompletely reduced which leads
to generation the superoxide anion, the precursor to most Reactive oxygen
species 6. Low levels of deleterious side-product, ROS plays various
physiological roles, while high and/or prolonged elevations of ROS can cause
oxidation of proteins, lipids, and nucleic acids, leading to cellular dysfunction
and programmed cell death 7. To combat high levels of ROS, there are
numerous check points to protect the overall integrity of the mitochondrial
network. First, mitochondria contains plenty of anti-oxidants such as
superoxide dismutase and glutathione to prevent ROS-induced damage. Secondly,
there is a broad collection of cellular factors that repair or replace damaged
mitochondrial components. These factors include mitochondrial chaperones, mitochondrial
proteases, DNA repair enzymes and the ubiquitin-proteasomal degradation system.
Lastly, when mitochondrial damage becomes too extensive beyond repair, the
entire mitochondrion can be selectively degraded in the lysosome through a
process referred to as mitophagy.

 

Mitophagy

Mitophagy is the selective degradation of defective
or dysfunctional mitochondria by autophagy. Mitophagy keeps the
cell healthy by preventing the accumulation of dysfunctional mitochondria which
can lead to cellular degeneration. Mitophagy in yeast is mediated by Atg32 and in
mammals it is mediated by PINK1 and Parkin mediated pathway as well as
independent pathway. Besides selective
removal of damaged mitochondria, mitophagy  plays a crucial role in adjusting
mitochondrial numbers to changing cellular metabolic needs, and during specific
cellular developmental stages, such as during cellular differentiation of red blood cells 8.

 

 

Revolutionary work by
the Youle laboratory initially coupled Parkin to mitophagy, and subsequent
contributions from other laboratories have defined a central role for PINK1 in
regulating Parkin succeeding mitochondrial damage. PINK1-Parkin pathway starts
by unravelling the difference between healthy and damaged mitochondria. PTEN-induced kinase1 (PINK1), a 64kDa protein contains a
mitochondrial targeting sequence (MTS) and is recruited to the mitochondria.

In healthy mitochondria, PINK1 is constitutively imported
through the outer membrane via the TOM complex, and partially through the
inner mitochondrial membrane via the TIM complex. PINK1 then spans the inner
membrane and is cleaved from 64-kDa to 60-kDa. It is then cleaved by inner
mitochondrial membrane associated PARL into 52-kDa which
is regulated by the recently described SPY complex 9.
This new form of PINK1 is degraded by proteases within the mitochondria in order
to keep the PINK1 concentration in check.

In unhealthy
mitochondria, upon the loss of mitochondrial membrane potential that can be
induced artificially by mitochondrial uncouplers (e.g. carbonyl cyanide
m-chlorophenylhydrazone (CCCP)), PINK1 gets stabilised and activated on the
outer mitochondrial membrane (OMM) by processes which are not yet fully
discussed 10,11. Depolarised mitochondria then recruits cytosolic Parkin with
the help of enzymatic activity of PINK1 12. Parkin exists in a native auto-inhibited
conformation which becomes activated on mitochondrial depolarisation 13. Activated
PINK1 phosphorylates both Ubiquitin and Parkin at their respective Ser65
residues 14, 15, 16, 17.

 

 

Detailed structural
and biophysical characterisation by sovereign laboratories illustrated that
phospho-ubiquitin (pUb) binds with high affinity to phosphorylated Parkin. This
binding allosterically induce conformational changes that promote recruitment
of its E3 ubiquitin ligase, and initiation of Parkin activity 13–17. Active
Parkin is proclaimed to ubiquitylate infinite proteins that reside in the OMM,
by elongating pre-existing ubiquitin chains attached to OMM proteins or by
ubiquitylating proteins de novo. Some of these proteins include Mfn1/Mfn2 17. The
ubiquitylation of mitochondrial surface proteins recruits mitophagy initiation
factors. Parkin promotes ubiquitin chain
linkages on both K63 and K48. K48 ubiquitination initiates degradation of the
proteins, and could allow for passive mitochondrial degradation. K63
ubiquitination is thought to recruit autophagy adaptors LC3/GABARAP which will
then lead to mitophagy. It is still unclear which proteins are necessary and
sufficient for mitophagy, and how these proteins, once ubiquitylated, initiate
mitophagy.

PINK1-Parkin independent pathway
involves NIX and its regulator BNIP3