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

Mitochondria is the major source of ATP production, necessaryfor cellular functions and integrity.

Accumulation of mutations in mtDNA canlead to cellular dysfunction by altering oxidative phosphorylation,Ca2+ homeostasis,oxidative stress and protein turnover. Dysfunctionof mitochondria leads to many neuro-degenerative diseases such as Parkinson’sdisease and Alzheimer’s disease. Hence, damaged mitochondria needs to beeliminated by inducing a plethora of stress signals causing programmed celldeath. Mitochondrial homeostasis and quality control is maintained by a selectiveform of autophagy, i.e.

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, mitophagy. Earlier studies have shown that multistepsignalling events of PINK1 (PTEN-induced putative kinase1) and Parkin E3Ubiquitin ligase regulates mammalian mitophagy. Here, we review the complexsignal transduction mechanism of PINK1 and Parkin focusing on pathways thatsequester mitochondria to autophagosome. Also post-translational modification suchas ubiquitinylation and phosphorylation of Ubiquitin and Parkin has added abroader perspective to the understanding of cellular damage.Keywords: Mitophagy, PINK1, Parkin, UbiquitinylationIntroductionMitochondriaare organelles enclosed within a double membrane, which is comprised of theouter mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM)(Fig. 1a). Ample amount of mitochondria are present in most cell types whichoccupies proximately 10–40 % of total cellular volume 1.

The mitochondrialspace between the OMM and IMM is attributed as the intermembrane space (IMS). Mitochondriaare crucial for eukaryotic cells, as it performs a number of critical functions.It plays a pivotal role in generation of cellular energy, regulating lipidmetabolism, cytosolic calcium flux buffering and sequestering the cell deathmachinery. Malfunction of the mechanisms that regulate mitochondrial qualitycontrol have proven to be a major driving force of normal ageing 2.Furthermore, failure of mitochondrial quality control mechanisms, causingelevated oxidative stress, is strongly linked to age-related conditions such asneurodegeneration 3, 4.Mostof the cellular chemical energy is produced in the mitochondrial matrix via theprocess of oxidative phosphorylation (OXPHOS), in the form of adenosinetriphosphate (ATP).

It involves the oxidation of tricarboxylicacid (TCA) cycle component, acetyl-CoA togenerate NADH and FADH2, which transfer electrons to the electrontransport chain components in the inner mitochondrial membrane, terminating inthe reduction of oxygen in the matrix to produce an electrochemical gradientacross the inner mitochondrial membrane that is used to produce ATP 5. Eventually, electrons are transferred to molecularoxygen (O2), reducing it to H2O (fig). However, due to leakage of electrons at complex I or complex III of theelectron transport chain, O2 can be incompletely reduced which leadsto generation the superoxide anion, the precursor to most Reactive oxygenspecies 6. Low levels of deleterious side-product, ROS plays variousphysiological roles, while high and/or prolonged elevations of ROS can causeoxidation of proteins, lipids, and nucleic acids, leading to cellular dysfunctionand programmed cell death 7. To combat high levels of ROS, there arenumerous check points to protect the overall integrity of the mitochondrialnetwork. First, mitochondria contains plenty of anti-oxidants such assuperoxide dismutase and glutathione to prevent ROS-induced damage. Secondly,there is a broad collection of cellular factors that repair or replace damagedmitochondrial components. These factors include mitochondrial chaperones, mitochondrialproteases, DNA repair enzymes and the ubiquitin-proteasomal degradation system.

Lastly, when mitochondrial damage becomes too extensive beyond repair, theentire mitochondrion can be selectively degraded in the lysosome through aprocess referred to as mitophagy. MitophagyMitophagy is the selective degradation of defectiveor dysfunctional mitochondria by autophagy. Mitophagy keeps thecell healthy by preventing the accumulation of dysfunctional mitochondria whichcan lead to cellular degeneration. Mitophagy in yeast is mediated by Atg32 and inmammals it is mediated by PINK1 and Parkin mediated pathway as well asindependent pathway. Besides selectiveremoval of damaged mitochondria, mitophagy  plays a crucial role in adjustingmitochondrial numbers to changing cellular metabolic needs, and during specificcellular developmental stages, such as during cellular differentiation of red blood cells 8.  Revolutionary work bythe Youle laboratory initially coupled Parkin to mitophagy, and subsequentcontributions from other laboratories have defined a central role for PINK1 inregulating Parkin succeeding mitochondrial damage. PINK1-Parkin pathway startsby unravelling the difference between healthy and damaged mitochondria. PTEN-induced kinase1 (PINK1), a 64kDa protein contains amitochondrial targeting sequence (MTS) and is recruited to the mitochondria.

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

In unhealthymitochondria, upon the loss of mitochondrial membrane potential that can beinduced artificially by mitochondrial uncouplers (e.g. carbonyl cyanidem-chlorophenylhydrazone (CCCP)), PINK1 gets stabilised and activated on theouter mitochondrial membrane (OMM) by processes which are not yet fullydiscussed 10,11.

Depolarised mitochondria then recruits cytosolic Parkin withthe help of enzymatic activity of PINK1 12. Parkin exists in a native auto-inhibitedconformation which becomes activated on mitochondrial depolarisation 13. ActivatedPINK1 phosphorylates both Ubiquitin and Parkin at their respective Ser65residues 14, 15, 16, 17.  Detailed structuraland biophysical characterisation by sovereign laboratories illustrated thatphospho-ubiquitin (pUb) binds with high affinity to phosphorylated Parkin. Thisbinding allosterically induce conformational changes that promote recruitmentof its E3 ubiquitin ligase, and initiation of Parkin activity 13–17.

ActiveParkin is proclaimed to ubiquitylate infinite proteins that reside in the OMM,by elongating pre-existing ubiquitin chains attached to OMM proteins or byubiquitylating proteins de novo. Some of these proteins include Mfn1/Mfn2 17. Theubiquitylation of mitochondrial surface proteins recruits mitophagy initiationfactors. Parkin promotes ubiquitin chainlinkages on both K63 and K48.

K48 ubiquitination initiates degradation of theproteins, and could allow for passive mitochondrial degradation. K63ubiquitination is thought to recruit autophagy adaptors LC3/GABARAP which willthen lead to mitophagy. It is still unclear which proteins are necessary andsufficient for mitophagy, and how these proteins, once ubiquitylated, initiatemitophagy.PINK1-Parkin independent pathwayinvolves NIX and its regulator BNIP3