CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW

1.1 THE MITOCHONDRIA

Mitochondria, otherwise known as the powerhouse of the cell is a sub-compartment of the

cell bound by a double membrane structure uses aerobic respiration to generate adenosine
triphosphate (ATP). Mitochondria are intimately involved in cellular homeostasis. Among
other functions, they play a part in intracellular signaling and apoptosis, intermediary
metabolism and in the metabolism of amino acids, lipids, cholesterol, steroids and
nucleotides (Richards and Archibald,2011). Mitochondria have a fundamental role in cellular
energy metabolism. This includes fatty acid, β-oxidation, the urea cycle, and the final
common pathway for ATP production-the respiratory chain.

The mitochondria respiratory chain is a group of five enzyme complexes situated on the inner
mitochondrial membrane. Each complex is composed of multiple subunits, the largest being
complex I with over 40 polypeptide components. Reduced cofactors (NADH and FADH2)
generated from the intermediary metabolism of carbohydrates, proteins, and fats donate
electrons to complex I and complex II. These electrons flow between the complexes down an
electrochemical gradient, shuttled by complexes III and IV and by two mobile electron
carriers, ubiquinone (ubiquinol, coenzymeQ10) and cytochrome C.

According to the generally accepted endosymbiotic theory (Zimmer,2009) mitochondria

originated from symbiosis of an oxidative a -proteobacterium with an anaerobic pre-
eukaryotic host cell 1.5–2 billion years ago. The resulting eukaryotic cell generates ATP
mainly by oxidative phosphorylation in the mitochondria, which yields about 15 times more
ATP from glucose compared to glycolysis under anaerobic conditions. During evolution most
of the genetic material of the ancestral oxidative a-proteobacterium was transferred to the
nucleus of the host cell. Coupled to oxygen consumption in the mitochondria, energy is
released in the respiratory chain in three steps and transiently stored as an electrochemical
proton gradient across the inner membrane. The gradient is dissipated by the ATP synthase
(complex V) to generate ATP (Rees et al.,2009) , or converted into heat by passive proton
leakage (Jastroch et al.,2010) , or through uncoupling proteins.

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 MITOCHONDRIA

Figure 1.1 Schematic diagram showing the mitochondria compartment

Source: Science Fact

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1.2 STRUCTURE OF MITOCHONDRIA

Mitochondria are bounded by a double-membrane system, consisting of inner and outer

membranes. Folds of the inner membrane (cristae) extend into the matrix. The matrix
contains the mitochondrial genetic system as well as the enzymes responsible for the central
reactions of oxidative metabolism (Sun et al., 2005). As discussed by (Dickson et al.,2006),
the oxidative breakdown of glucose and fatty acids is the principal source of metabolic
energy in animal cells. The initial stages of glucose metabolism (glycolysis) occurs in the
cytosol, where glucose is converted to pyruvate. Pyruvate is then transported into
mitochondria, where its complete oxidation to CO 2 yields the bulk of usable energy (ATP)
obtained from glucose metabolism. This involves the initial oxidation of pyruvate to acetyl
CoA, which is then broken down to CO2 via the citric acid cycle. The oxidation of fatty acids
also yields acetyl CoA, which is similarly metabolized by the citric acid cycle in
mitochondria. The enzymes of the citric acid cycle (located in the matrix of mitochondria)
thus are central players in the oxidative breakdown of both carbohydrates and fatty acids
(Dickson et al., 2006).

1.2.1 CRISTAE

Cristae are invaginations protruding into the mitochondrial matrix (Vogel et al.,2006)
connected via narrow tubular or slit-like structures, known as crista junctions (CJs). In recent
years, studies show that components of the electron transport chain (ETC) are confined to the
lateral surfaces of the cristae rather than equally distributed along the IMM (Wilkens et al.,
2013). Moreover, dimers of F1F0 ATP Synthase assemble in rows along the edges of the
cristae (Davies et al., 2011). The CJs can be kept in a closed state by oligomers of the inner-
membrane dynamin-like GTPase, OPA1 (Pham et al.,2016), as well as components of the
mitochondrial contact site and cristae organizing system (MICOS complex) (Glytsou et al.,
2016). Cristae are electrically insulated allowing individual cristae within any given
mitochondria to have different membrane potentials, remains polarized despite depolarization
of neighboring ones (Barrera et al., 2016).

According to (Wollweber et al., 2017),cristae has the ability to change their shapes in
response to the changes in the cell’s environment. When the cell is under stress, the cristae
can become shorter and wider, this change in shape is important in mitochondria adapting to
stress and functioning properly. Cristae plays a role in apoptosis or programmed cell death
(Zerbes et al., 2012).

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1.2.2 MATRIX

The matrix contains the mitochondrial genetic system as well as the enzymes responsible for
the central reactions of oxidative metabolism, it is the fluid-filled space inside the
mitochondria (between the inner and outer mitochondria membrane). The matrix is really
important for the function of mitochondria, it contains the enzymes and cofactors that are
needed for the citric acid cycle and oxidative phosphorylation (Calvo et al.,2017).

The matrix contains the mitochondrial DNA(mtDNA) which contains some of the proteins
that are involved in oxidative phosphorylation. It plays a vital role in the citric acid cycle and
is basically the control center for the cycle, it uses energy from the citric acid cycle to make
ATP by making ATP from the energy generated from the electron transport chain. These
enzymes are called ATP synthases (Shiota et al.,2015).

1.3 FUNCTIONS OF MITOCHONDRIA

Mitochondria, the power house of the cell is one of the most important source of energy
in the form of ATP, the energy rich compound that drives fundamental cell functions. These
functions includes; force generation(in muscle contraction and cell division),the biosynthesis,
folding and degradation of proteins and the generation and maintenance of membrane
potentials(Schmidt et al.,2010).

Apart from cellular respiration and ATP synthesis, mitochondria has numerous other
essential functions, including the production of NADH AND GTP in the citric acid cycle, the
biosynthesis of amino acids, heme groups and iron-sulfur clusters or the synthesis of
phospholipids for membrane biogenesis(Davies et al.,2011). They also act in calcium
signaling, stress responses and generally as cellular signaling hub. Not surprisingly,
mitochondria plays a vital role in human health, mitochondria dysfunction is the cause of
severe often maternally inherited diseases and are also deeply implicated in apoptosis and
ageing(Bratic and Larsson,2013).

1.3.1 MITOCHONDRIA AND APOPTOSIS

Apoptosis or programmed cell death, is a cellular self-destruction mechanism involved in

a variety of biological events. Excessive or insufficient apoptosis contributes to various
diseases related to ischemia, neurodegeneration, autoimmunity, and viral infections, and is

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involved in the growth and regression of tumors (McBride et al.,2006). There are multiple
cellular pathways triggering apoptosis, two of which, the extrinsic and intrinsic pathways, are
better characterized; Intrinsic, for which mitochondrion is the central organelle governed by
pro- and anti-apoptotic proteins interact and determine cell fates and Extrinsic, activated by
cell surface death receptors (TRAIL death receptors, etc.), their inhibitory counterparts and
cytoplasmic adapter or death inhibitory molecules (e.g., FADD or FLIP) (Ferri and
Kroemer,2001).

The apoptotic process is executed by a family of cysteine proteases which specifically

cleave their substrates at aspartic acid residues. These proteases, known as caspases, are
activated through extrinsic and/or intrinsic pathways. The extrinsic pathway is activated by
cell surface death receptors, while the intrinsic pathway is initiated by formation of the
cytosolic apoptosome composed of Apaf-1, procaspase 9, and the cytochrome c released from
mitochondria (Adams,2004).

1.3.2 ELECTRON TRANSPORT CHAIN

The electron transport chain is a series of redox reactions that are carried out by protein
complexes in the inner membrane of the mitochondria. It is one of the key part as to how
mitochondria produces ATP as it establishes the proton gradient across the inner membrane
of the mitochondria, the proton is what powers the production of ATP by ATP synthase
(González-Cabo et al.,2005). The electron transport chain consists of five complexes;

1.3.2.1 COMPLEX I

In mammalian mitochondria, Complex I(NADH ubiquinone oxidoreductase) catalyzes

the oxidation of reduced NADH by CoQ. Complex I is the largest complex in the oxidative
phosphorylation system, consisting of 45 subunits in mammals, for a total molecular mass of
1 MDa (Carrol et al.,2006). Seven subunits are encoded by the mitochondrial genome, the
remaining 38 by nuclear DNA (nDNA) genes. The seven mtDNA encoded subunits and
seven nuclear-encoded subunits (NDUFV1, NDUFV2, NDUFS1, NDUFS2, NDUFS3,
NDUFS7, NDUFS8) form the catalytic “core” structure of the complex (Koopman et
al.,2010) , conserved in virtually all organisms that contain a Complex I, including Thermus
thermophilus . The remaining nuclear-DNA encoded subunits are possibly involved in
Complex I assembly, stability, and regulation of activity.

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 Ultrastructural studies of purified Complex I revealed an “L” shaped object, consisting of
two arms, a hydrophilic “peripheral arm” protruding into the matrix, and a hydrophobic
“membrane” arm, embedded in the mitochondrial inner membrane, with an angle between the
two arms of about 100°. The value of the angle can slightly vary, since the two arms change
their reciprocal positions during the catalytic cycle of the complex (Janssen et al.,2006) . The
two arms harbor three functional modules:

1. the N module, in the peripheral arm, contains the dehydrogenase site, formed by a fl
avin-mononucleotide (FMN) moiety, responsible for the oxidation of NADH to
NAD+(Vogel et al., 2007).
2. the Q module, in the hinge region between the two arms, contains the CoQ reduction
site(Vogel et al., 2007).
3. the P module, or proton translocation module, constitutes the membrane arm, and
includes the seven mtDNA-encoded ND subunits (Vogel et al., 2007).

1.3.2.2 COMPLEX II

Complex II (succinate dehydrogenase ubiquinone–ubiquinol reductase) is the only

membrane-bound member of the tricarboxylic acid (TCA) cycle, where it functions as a
succinate dehydrogenase (SDH), catalyzing the oxidation and dehydration of succinate to
fumarate. Complex II takes part in the mitochondrial respiratory chain(MRC) by coupling
this reaction to the reduction of ubiquinone to ubiquinol, that in turn funnels electrons to
Complex III (Bianchi et al.,2004) . Complex II is the smallest MRC complex (123 kDa),
consisting of four subunits, encoded by SDHA, SDHB, SDHC and SDHD nuclear genes.
SDHA contains a FAD moiety, whereas three iron-sulfur(Fe–S)centers are bound to SDHB.
These two hydrophilic subunits are linked to SDHC and SDHD, two small, hydrophobic
polypeptides that contain a heme b moiety and anchor the complex to the inner
mitochondrial membrane (Sun et al.,2005) . The crystal structure of porcine heart
Complex II consists of a hydrophilic head protruding into the matrix, a hydrophobic tail
embedded in the inner membrane, and a short segment projecting into the intermembrane
space(Sun et al.,2005).

Mitochondrial Complex II shows close homology with a number of bacterial succinate

ubiquinone reductases (SQRs), especially those of a- proteobacteria, from which
mitochondria are supposed to derive. The aminoacid sequences of the flavin and Fe–S
binding domains of Complex II are highly conserved. The membrane domain is less

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conserved, although a four-helix bundle motif is ubiquitously present across
species(Yankovskaya et al.,2003).

1.3.2.3 COMPLEX III

Complex III (ubiquinol–cytochrome C reductase) catalyzes the electron transfer from

reduced CoQH2, (ubiquinol) to cytochrome C. Complex III is made up of 11 subunits
(Fernandez-Vizarra et al.,2007), only one (cytochrome B, cyt b)is being encoded by mtDNA.
Nuclear genes encode the remaining subunits: apo-cytochrome C1, the Rieske iron–sulfur
protein (RISP or UQCRFS1), two relatively large “core” subunits, Core1(UQCRC1) and
Core 2(UQCRC2), and six additional, smaller proteins (UQCR611), the functions of which
are unknown. In addition to the protein backbone, prosthetic groups of Complex III include
the two Fe-containing heme moieties of cyt.B and C1 , and the Fe–S cluster of RISP; these
three subunits form the catalytic redox core of Complex III. The Complex III monomer is
likely a transient form, which quickly converts into a stable, catalytically active homodimer
(Wittig et al.,2006).

1.3.2.4 COMPLEX IV

Complex IV (cytochrome C oxidase), the terminal component of mitochondria respiratory

complex(MRC),transfers electrons from reduced cytochrome C to molecular oxygen. This
reaction is coupled to proton pumping across the inner mitochondrial membrane. Mammalian
COX is composed of 13 subunits (Massa et al.,2008),the three largest being encoded by
mtDNA genes (MTCO1, MTCO2, MTCO3), whereas the remaining subunits are encoded by
nuclear genes. The redox catalytic core is composed of subunits MTCO1 and MTCO2, which
harbor two iron-containing heme moieties and two copper centers (CuA and CuB),
responsible for the electron transfer (Stiburek et al.,2006).

1.3.2.5 COMPLEX V

Complex V, otherwise known as the ATP synthase dissipates the proton electrochemical
gradient generated by the respiratory chain to produce ATP. It comprises an integral
membrane cylindrical rotor-like structure, the F0 particle, and a peripheral matrix facing F1
particle, the catalytic ATP synthase domain (Houstek et al.,2009).

F1 has a bulblike shape, formed by six “cloves”. F0 and F1 are physically connected to
each other by two additional structures: a centrally located, asymmetrical stalk and an

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externally tethered stator. F0 is a rotor harboring a proton channel. Following the
electrochemical gradient, protons flow through the channel guided by the stator, impressing a
rotary motion to F0, which is transmitted to the catalytic head (F1) by the centrally projecting
stalk. The sequential tilt of the cloves, impressed by the asymmetrical stalk during each F1
rotation cycle, drives the condensation of ADP and Pi to form three ATP molecules for each
cycle (Devenish et al., 2008).

All five subunits of F1 and most of the F0 subunits are nuclear encoded (Mulkidjanian et
al.,2007). Dimeric and higher oligomeric forms of ATP synthase seem critical to maintain the
shape of mitochondria by promoting the formation of the inner membrane cristae (Paumard
et al.,2002).

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Figure 1.2 Schematic diagram showing the Electron Transport Chain

Source: Science Fact

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1.3.3 KREB’S CYCLE

The tricarboxylic acid (TCA) cycle, also known as the Krebs cycle or citric acid cycle, is
an important cell's metabolic hub. It is composed of eight enzymes, all of which are within
the mitochondrial matrix except the outlier succinate dehydrogenase, which is related to the
respiratory chain on the inner mitochondrial membrane (Calvacanti et al.,2014). The cycle
serves as a gateway for aerobic metabolism for molecules that can convert to an acetyl group
or dicarboxylic acid. Regulation of the TCA cycle occurs at three distinct points that include
the three following enzymes: citrate synthase, isocitrate dehydrogenase, and alpha-
ketoglutarate dehydrogenase. The cycle also plays a role in replenishing precursors for the
storage form of fuels such as amino acids and cholesterol (Haddad and Mohiuddin,2022).
The Kreb’s cycle functions includes;

1. Energy Production (ATP Generation): The main function of the Krebs cycle is to
extract high-energy electrons from acetyl-CoA molecules derived from carbohydrates,
fats, and proteins. These electrons are transported to the electron transport chain
(ETC) embedded in the inner mitochondrial membrane(Akram,2014).
2. NADH and FADH2 Production: The Krebs cycle generates NADH and FADH2 by
oxidizing acetyl-CoA and reducing NAD+ (nicotinamide adenine dinucleotide) and
FAD (flavin adenine dinucleotide). These electron carriers (NADH and FADH2) are
crucial in the subsequent steps of oxidative phosphorylation, where they transfer
electrons to the electron transport chain, leading to ATP synthesis (Fisher-Wellman et
al.,2015).
3. Intermediates for Biosynthesis: Several intermediates produced in the Krebs cycle
serve as precursors for the biosynthesis of various molecules essential for cellular
function. For instance, oxaloacetate, a key intermediate in the cycle, can be used for
the synthesis of amino acids, including aspartate and pyrimidines (components of
DNA and RNA) (Rahman and Thorburn,2015).
4. Regeneration of Coenzymes: The Krebs cycle helps regenerate coenzymes,
particularly coenzyme A (CoA) and oxaloacetate, which are required for the
continued oxidation of acetyl-CoA in the cycle(Phillips et al.,2015).

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Fig 1.3 Enzymes, reactions and integration in cell metabolic pathways of the Krebs
cycle.

Source:

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1.4 DISEASES OF THE MITOCHONDRIA

Disorders related to mitochondrial dysfunction can result from mutations in the

mitochondrial DNA or nuclear DNA affecting mitochondrial function. These disorders can
have a wide range of effects on different systems in the body. Mitochondrial disease
ultimately reflects a defect of oxidative phosphorylation within a cell, but the pattern of
cellular involvement will determine the clinical features of the disease. Aside from genetic
mutations, mitochondrial dysfunction can also be caused by deficiency in the electron
transport chain complexes and error in the Krebs cycle (Rahman and Thorburn,2015).

1.4.1 HUMAN DISEASES ASSOCIATED WITH COMPLEX I DEFICIENCY

Isolated complex I deficiency is frequent in mitochondrial disorders (Distelmaier et

al.,2009) being responsible for about one-third of all cases (Janssen et al.,2006). The primary
genetic defect may be either in mtDNA or in nuclear DNA genes. Given the complexity of
this huge enzyme, its dual genetic origin, clinical presentations are so heterogeneous,
including, for children, Leigh syndrome (LS), neonatal cardiomyopathy with lactic acidosis,
fatal infantile lactic acidosis (FILA), macrocystic leukoencephalopathy, or pure myopathy
(Bugiani et al.,2004) .

Complex I defects or complex I-related mutations, particularly in mtDNA, includes;

Leber’s hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalopathy with
lactic acidosis and stroke-like episodes), or MELAS/LHON overlap syndromes (Lenaz et
al.,2004) . Several additional, poorly defined, syndromes include neurological signs, such as
ophthalmoparesis, optic atrophy, epilepsy, ataxia and dystonia; gastrointestinal problems,
such as pseudo-occlusion and gastroparesis; hepatopathy; cardiomyopathy; and renal
dysfunctions, such as tubulopathy or proteinuria (Janssen et al.,2006). Others includes;
Mitochondrial neurogastrointestinal encephalopathy(MNGIE), Mitochondrial DNA depletion
syndrome(MDS), Pearson disease(a rare disorder that can cause anemia, liver disease and
other health problems.

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1.5 MITOCHONDRIAL MEMBRANE PERMEABILITY TRANSITION PORE

The mitochondrial permeability transition (mPT) describes a highly-reproducible, Ca2+-

dependent increase of inner mitochondrial membrane permeability to ions and solutes. It is
mediated by a putative channel, the mitochondrial permeability transition pore (mPTP),
which has also been recorded electrophysiologically and called the mitochondrial mega-
channel or multi-conductance channel (Bauer and Murphy, 2020). Depending on the specific
conductance state, mPTP properties may vary from a small nonselective ion channel to a
large pore that allows diffusion of molecules up to 1.5kDa in size. Sustained mPTP opening
causes disruption of mitochondrial energy metabolism and necrotic cell death (Morciano et
al.,2017). Furthermore, mPTP activation can lead to osmotic dysregulation of mitochondria
leading to mitochondrial swelling. This may rupture the outer mitochondrial membrane
causing release of cytochrome c from the intermembrane space into the cytosol which will
initiate apoptotic cell death if cellular ATP levels can be maintained (Patel et al.,2021). These
mPTP-mediated cell death mechanisms play a key role in the pathogenesis of numerous,
aging-associated human diseases including, but not limited to, ischemic heart and brain
disease, liver and kidney failure, cancer, and degenerative diseases (Mithal and
Chandel,2020; Jia and Du,2021).

However, transient mPTP opening, perhaps involving different sub-conductance states,

may exert several physiological roles such as transient rapid alterations in mitochondrial
bioenergetics, reactive oxygen species (ROS) production, Ca2+ efflux and metabolic
regulation (Kwong and Molkentin,2015; Mnatsakanyan et al.,2017). High levels of matrix
Ca2+, inorganic phosphate, cyclophilin D (CypD) and oxidative stress activate mPTP
opening, while divalent and trivalent cations such as Mg2+, Ba 2+, Gd 3+, in addition to H+,
adenine nucleotides and cyclosporine A (CsA), inhibit the opening (Sullivan et al.,2015). The
exact pore-forming structure of the mPT remains controversial. The voltage-dependent anion
channel (VDAC), proapoptotic Bcl-2 family members (Bax and Bak) (Patel et al.,2021;Karch
et al.,2013), the phosphate carrier (PiC)and the translocator protein (TSPO) (Kwong et
al.,2014)had been proposed to contribute to the channel of the mPT. However, subsequent
studies have shown that PiC more likely regulates the probability of mPTP opening, rather
than constituting the CsA-sensitive pore of mPT,( Gutierrez-Aguilar et al.,2014) while
VDAC together with pro-apoptotic Bcl-2 molecules likely facilitate permeation across the
outer membrane or modulate interactions between the inner and outer mitochondrial
membranes that can influence mPT activity. None of these proteins serves as the pore

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(channel) of mPT, and the effect of ligands of TSPO can probably be explained by their
binding to subunit OSCP of ATP synthase(Cleary et al.,2007). The adenine nucleotide
translocator (ANT) may contribute a smaller conductance activity than that of the largest
amplitude conductance of mPTP and may also play a role in the opening of mPTP (Bround et
al.,2020).

1.5.1 MMPTP AND ITS ROLE IN CELL DEATH

Contribution of the MMPTP to necrotic and apoptotic death pathways: The MMPTP plays
key roles in cell death pathways, especially in necrotic cell death, but its role in canonical
apoptosis is still controversial. Classically Bcl-2 mediated death does not require CypD
dependent MMPTP activation, although disruption of inner membrane activities including
impairment of electron transport, occurs during apoptotic events (Hardwick and Soane,2013).
Work by many groups led to the discovery of apoptosis and the recognition that growth-
factor withdrawal dependent cell death was related to proapoptotic Bcl-2 family proteins such
as Bax(Adams and Cory,2007).These proteins may also modulate mPTP formation through
interactions at contact sites between the inner and outer mitochondrial membrane and
changes in mitochondrial morphology(Halestrap et al.,2015). While MMPTP opening itself
may play a role in apoptosis as discussed further below, there exist many other recognized
forms of cell death, in addition to canonical apoptosis and necrotic death (Green and
Llambi,2015).

Apoptosis is characterized by cell shrinkage, membrane blebbing, condensation of the

chromatin (pyknosis) (Galluzzi et al.,2012), and activation of caspase proteases(Kist and
Vucic,2021).Intrinsic apoptotic cell death is caused by stress experienced autonomously by a
cell such as mediated by oncogenes, direct DNA damage, hypoxia, and survival factor
deprivation. Mitochondria regulate caspase dependent cell death through pro-apoptotic Bcl-2
family members such as Bax/Bak which mediate mitochondrial outer membrane
permeabilization (MOMP), leading to formation of the apoptosome and release of
cytochrome c which activates the caspase-cascade(Hardwick and Youle,2009).MOMP is
preventable by high levels of anti-apoptotic proteins such as BclxL (Chipuk and
Green,2008)and the balance between pro-and anti-apoptotic members of the family
contributes to the commitment point on the road to apoptotic cell death(Kroemer et al.,2007).
However, it has also been shown that in intact cells cytochrome c release may also occur after

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acute or prolonged increases in intracellular Ca2+ and mitochondrial Ca2+ overload in a
Bax/Bak independent manner(Quarato et al.,2022). Swelling of mitochondria in response to
MMPTP opening may lead to cytochrome c release by outer membrane rupture, but also by
increasing cytochrome c availability through the Bax/Bak pathway after cristae remodeling.
This may stimulate apoptosis in the presence of preserved ATP levels. In severely damaged
cells necrotic cell death is unavoidable and is characterized by cell swelling, plasma
membrane rupture, and loss of organellar structure without chromatin condensation(Zhu and
Sun,2018;Kist and Vucic,2021) .

Although necrotic death is not genetically regulated during severe injury, in many cells
that survive the initial insult, a programmed set of pathways is activated that is quite different
from apoptotic death. This death, called necroptosis, is highly regulated, caspase independent,
and resembles only morphologically some aspects of necrosis. The main molecular player for
necroptosis is protein RIP kinase 3(Zhang et al.,2016) and its substrate the Mixed Lineage
Kinase Domain-like Protein (MLKL), the latter of which forms the executioner by
permeabilizing the plasma membrane. Necroptosis is usually initiated extrinsically to the cell
by high levels of death signals in the extracellular space, such as TNF, which are typically
found in certain severe acute, degenerative or inflammatory states. The complex necroptotic
pathway, including activation of RIP kinases, has many decision-making forks downstream
of TNF receptor activation that can lead, variously, to survival, apoptosis (which is immune
silent) or activation of cell lysis and pro-inflammatory programs. Although the link between
apoptotic signaling and mPTP activation may at times be direct as outlined in the preceding
section, the mechanism of activation of mPTP during necroptotic cell death is not yet clear,
but a convincing case has been made for ischemia- and oxidative stressinduced myocardial
necroptosis via CaMKII(Quarato et al.,2022). It should be noted that this CamKII mediated
cell death is independent of MLKL, thus may not be defined as necroptosis according to
consensus in the cell death field. The MLKL protein is an executioner of necroptosis and its
N-terminal helix bundle can bind the mitochondria-specific phospholipid,cardiolipin
(Dondelinger et al.,2014). This effect causes permeabilization of the mitochondrial inner
membrane resembling mPT, accompanied by cell and organelle swelling. Indeed, cells
lacking CypD and therefore resistant to MMPTP are resistant to necroptotic cell death
induced by TNFα and caspase inhibition(Wang et al.,2014).

However, in genetic model systems, It was shown that CypD is dispensable for
necroptosis (Karch et al.,2015). Moreover, other studies showed that activated MLKL

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translocates uniquely to the plasma membrane, where it interacts with PIP phospholipids
during the process of cell death (Dovey et al.,2018;Samson et al.,2020). MLKL lacks a
mitochondrial targeting sequence, and forcibly targeting MLKL to mitochondria with the use
of selective tags does not induce cell death (Quarato et al.,2016). Necroptotic signaling
factors can also interact with metabolic enzymes, hampering ATP production, cause loss of
NAD through DNA damage pathways, activate ROS production and precipitate loss of
mitochondrial and plasma membrane potential, all of which may activate mPT. The late
events in necroptosis also involve rapid activation of calpains, Bax and release of pro-
apoptotic factors into the cytosol(Moubarak et al.,2007). However, unlike apoptosis,
mitochondrial depletion does not compromise the ability of a cell to undergo necroptosis,
suggesting that mitochondria plays a regulatory role in this cell death pathway (Tait et
al.,2013).

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Figure 1.4 Role of the mitochondria in apoptosis and necrosis. An increase in the
permeability of the outer mitochondrial membrane is crucial for apoptosis to occur and is
regulated by multidomain pro-apoptotic members of the Bcl-2 family (Bax and Bak),
resulting in the release of several apoptogenic factors into the cytoplasm. In contrast, the Cyp
D-dependent MPT involves an increase in the permeability of both the outer and inner
mitochondrial membranes, and leads to necrosis induced by Ca2+ overload and oxidative
stress. Both types of mitochondrial membrane permeability change are inhibited by anti-
apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-xL).

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1.5.2 MMPTP AS A Ca2+ RELEASE CHANNEL

In respiring mitochondria, Ca2+ influx is primarily mediated by the MCU while Ca2+ efflux
is largely carried out by Na+/Ca2+/Li+ exchange (NCLX). Collectively, through
sophisticated coordination, these pathways maintain steady state mitochondrial free Ca2+
concentrations ([Ca2+]m) in the physiological range of 0.1–1µM. This is despite the ΔΨm
being about −180mV, which is a huge driving force that unopposed would cause massive
accumulation of Ca2+ within the matrix. The rate of Ca2+ uptake increases very steeply with
extramitochondrial [Ca2+], reaching a Vmax as high as 1400nmol Ca2+ ×mg protein−1
xmin−1; while the maximal rate of operation of the antiporter is not faster than about 20nmol
Ca2+ ×mg protein−1 ×min−1(Agarwal et al.,2017) This difference could expose
mitochondria to the risk of Ca2+ overload. However, under physiological conditions,
mitochondrial Ca2+ uptake is relatively small during the rapid oscillatory Ca2+ transients
that occur, for example, in beating heart. In non-excitable cells or when frequencies of Ca2+
transients through GPCR mediated Ca2+ release from IP3 receptors are low, NCLX along
with high matrix Ca2+ buffering capacities can maintain cytosolic and mitochondrial Ca2+
homeostasis globally. Meanwhile, the existence of stochastic, infrequent, and localized
transient mitochondrial membrane depolarization with matching transient mitochondrial
Ca2+ release due to flickering of mPTP under physiological conditions has been widely
recognized(Wang et al.,2016;Lu et al.,2016). In neurons these events occur constantly during
normal physiological activities such as accompanying action potential-induced plasma
membrane depolarization and during synaptic transmission. Therefore neuronal mPT is likely
to be well-adapted for Ca2+ release.These localized events appear to provide a convenient
mechanism for t-mPT to serve as a safety valve to rapidly discharge excessive Ca2+
accumulation in a small subset of mitochondria (Bernadi and von Stockum,2012). An
interesting question is why nature would design a non-selective ion channel for mitochondrial
Ca2+ release. The answer is probably that the very low permeability of the inner membrane,
which is a requisite for energy conservation, would prevent rapid Ca2+ release even after a
full depolarization of ΔΨm (Bernadi and von Stockum,2012).. Furthermore, since the
MMPTP is a multi-conductance channel, the low frequency of t-mPT may be associated with
a low-conductance mode and thus might allow for maximal Ca2+ flux without the need for
persistent membrane depolarization. This arrangement in turn permits fast Ca2+ release even
for very small [Ca2+] gradients, and is consistent with the protective effect of pore flickering
in several disease paradigms(Elrod et al.,2010). Another important role for the “physiological

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mPTP” may be to alter cell metabolism in favor of aerobic glycolysis over oxidative
phosphorylation. The metabolic shift away from aerobic glycolysis occurs in a time-
dependent manner at various key points in development (Fame et al.,2019). One example is
in neuronal synapse development, where the ATP synthase c-subunit leak channel (ACLC)
becomes relatively more closed in a synaptic stimulus-dependent manner during synapse
maturation. The mechanism is related to rapid movement of the anti-apoptotic protein Bcl-xL
to mitochondria (Li et al.,2019)followed by assembly of newly synthesized F-ATP synthase
F1 components with free c-subunit rings, the latter of which are already present in the
mitochondrial inner membrane [128]. This new synthesis adds newly assembled ATP
synthase to that already existing. If this increased ATP synthase assembly fails to occur at a
critical time point, synapses do not develop normally. The function of the free c-subunits
earlier in development may be to provide a persistent inner membrane leak that supports an
aerobic glycolytic metabolic phenotype. Aerobic glycolytic metabolism allows for a high rate
of protein synthesis(Licznerski et al.,2020)and perhaps lipid biosynthesis during the
developmental period. The study suggests that the high rate of protein synthesis associated
with the glycolytic metabolism normally decreases at the time of synapse maturation,
regulated by relative ACLC closure (Grifftihs et al.,2020)

As mentioned earlier, Ca2+ release is only one of numerous signals that may be associated
with t-mPT. These include transient bursting of ROS and ATP generation, alkalinization of
matrix pH, and occurrence of fluxes of other ions like Na+. These t-mPT mediated localized
signals could have profound effects on cell regulation including development, differentiation,
maturation and aging, and localized organelle-organelle crosstalk signaling. The t-mPTP
openings in intact cells have been detected through small molecule or protein-based
fluorescence probes and mitochondrial ion channel recordings (Mishra et al.,2019). Although
living cell ion channel recordings are ideal to detect rapid response kinetics, they fail when
attempting to visualize pan-cellular mitochondrial events, and imaging techniques are not yet
sensitive enough to record rapid and smaller mPTP flickers. Future development of new
probes coupled with advanced imaging techniques may be able to address the question of
whether t-mPT occurs only in a very small subset of mitochondria or if it is a more universal
event for the mitochondria (Agarwal et al.,2020).

20
21
1.5.3 THE ROLE OF MMPTP IN INNATE IMMUNITY

Another important role for inner membrane permeabilization by mPTP is related to

mitochondrial DNA release during innate immunity. Certain extracellular signals known as
pathogen or danger associated molecular patterns interact with toll-like receptors (TLRs) on
macrophages (Jones and Divakaruni,2020). Signaling downstream of TLRs helps to
inactivate extracellular pathogens by formation of an intracellular complex called the
inflammasome (Yu et al.,2020). The inflammasome is often further activated by newly
synthesized mitochondrial DNA,some of which is exposed to oxidative stress. Oxidized
mitochondrial DNA is released through mPTP into the cytosol and can further propagate the
inflammatory signal to neighboring cells through mitochondrial DNA exocytosis (De maria
et al.,2019). However, mitochondrial DNA release may not be entirely dependent on mPTP
since other studies have shown that activation of Bax/Bak can lead to mitochondrial
herniation and DNA release. Notably, this method of mitochonrial DNA release was
indepdent of CypD as cells lacking CypD still underwent Bax/Bak-dependent mitochondrial
DNA release. Given these data, it is clear there is a relationship between MOMP, mPTP
sensitization, and inner membrane integrity that remains to be explored (Riley et al.,2018).

1.5.4 THE ROLE OF MMPTP IN ISCHEMIA/REPERFUSION INJURY

Reperfusion of the heart (and other tissues) following an extended period of ischemia can
exacerbate the damage caused by ischemia itself and is known as ischemia reperfusion injury
(IRI)(Halestrap and Richardson,2015). It has long been known that ischemia is associated
with elevated intracellular [Ca2+] and phosphate and that reperfusion causes the production
of ROS. These are exactly the conditions that would be expected to cause mPTP opening and
this was subsequently shown to be the case in both isolated heart cells and the perfused heart.
Furthermore both pharmacological and genetic (Baines et al.,2005)attenuation of CypD
activity was shown to protect from IRI, and it is now widely accepted that opening of the
mPTP during reperfusion plays a key role in IRI in a range of tissues. Indeed, inhibition of
mPTP opening can be induced by a variety of pharmacological, preconditioning and post-
conditioning protocols that lead to cardioprotection in models of IRI(Bauer and
Murphy,2020). However, CsA itself has not proved to be an effective cardioprotective agent
in some animal models of IRI or in large scale clinical trials(Ramachandra et al.,2020). This
probably reflects the ability of the mPTP to open independently of CypD if the trigger is

22
sufficiently large. Furthermore, if F1 is lost during prolonged cellular toxicity, as was shown
recently, CsA and other reagents that bind within the F1 might no longer inhibit the
channel(Mnatsakanyan et al.,2022).

It seems highly probable that the observed rise in intracellular [Ca2+] during ischemia plays a
key role in priming mitochondria for mPTP opening during reperfusion when re-energisation
leads to uptake of the accumulated Ca2+ into the mitochondria(Griffiths,2009). In support of
this view, pharmacological or genetic inhibition of mitochondrial uniporter activity does
reduce IRI as does over expression of the Na+/Ca2+ exchanger that mediates Ca2+ efflux
from the mitochondria (Luongo et al.,2017). The other widely accepted trigger for mPTP
opening during reperfusion is increased ROS production. This has long been recognized to
accompany reperfusion, although targeting ROS as a cardioprotective strategy has proved
disappointing. In ischemic cell Ca2+ overload, the mitochondrial inner membrane is the first
location of death channel (mPTP) activation before the outer membrane Bcl-2 machinery
(Rasola and Bernadi,2017) which activates MMPTP. The dysregulated Ca2+ accumulation
that occurs in the necrotic core leads to opening of MMPTP, but mPTP can also be activated
in the surround of the core or penumbra. mPTP opening leads to mitochondrial
depolarization, inhibition of ATP synthesis and finally Bax activation, as the outer membrane
gets involved. This then results in the release of pro-death factors into the cytosol including
cytochrome c (Hetz et al.,2005). During ischemic or toxic cell death, anti-apoptotic proteins
such as Bcl-2 and Bcl-xL can be proteolytically cleaved by caspases and calpain to form pro-
death molecules. The N-terminal cleaved version of Bcl-xL has been implicated in neuronal
ischemic cell death and in activation of Bax(Park and Jonas,2017). The effect of cleaved Bcl-
xL on inner mitochondrial membrane depolarization is inhibited by Bcl-xL inhibitors and by
CsA, suggesting its direct action on mPTP. ATP synthase c-subunit knock down also protects
against mitochondrial inner membrane depolarization during glutamate toxicity, suggesting
that the mechanism involves initial activation of an mPTP-associated channel (Bonora et
al.,2013;Park et al.,2017)

23
Figure 1.5 Possible mechanisms involved in the opening of the mPTP in ischemia
reperfusion injury and its prevention by ischemic preconditioning. This diagram summarizes
key factors that have been proposed to initiate and amplify mPTP opening during reperfusion
of the heart after a period of prolonged ischemia.

Source:

24
1.5.5 THE ROLE OF MMPTP IN AGING

Triggers for the mPT in cells in culture in vitro or ex vivo, which include oxidative stress,
energy depletion, and high cytoplasmic Ca2+ concentrations are well described. However,
these triggers are largely unachievable in vivo except in extreme circumstances such as
ischemia or reperfusion injury. Thus, major contributors to the mPT in vivo in aging remain
less clear and have been extrapolated by logical extension, for example, by common
inhibition of both the decline in mitochondrial membrane potential in aging tissues and of the
mPT in cells in vitro by the CypD inhibitor CsA(Crompton,2004). A growing series of
compelling data indicate that the mPTP has a greater propensity to open in aging (Rottenberg
and Hoek,2017). Firstly, mitochondrial membrane potential and efficiency of oxidative
phosphorylation decline with age, across multiple tissues and in many model organisms
(Green et al.,2011). While this could implicate factors such as accumulated damage to the
respiratory chain, evidence indicates that increased mPT may play a causal role in the decline
in mitochondrial membrane potential and decline in ATP generation with age. Secondly, a
major stimulus for the mPT, increases in oxidative stress and associated damage of cellular
structures, is a central hallmark of the aging process (Lopez et al.,2013). Evidence in model
organisms indicates that primary exposure to mitochondrial ROS may prompt a hormetic
response, priming stress-defense pathways to protect against further oxidative insults (Van
Raamsdonk and Hekimi,2009). As organisms age, however, oxidative insults prompt cellular
damage that declining defense mechanisms cannot ward off, driving mitochondrial and
organismal decline. Thirdly, and perhaps most compellingly, indirect measurements of mPTP
opening in isolated mitochondria or permeabilized cells indicate that a signature common to
multiple tissues in aging is enhanced mPTP activation. It should be stressed that these
measurements were made by exposing cells or mitochondria to non-physiologic stimuli
known to induce the mPT such as high concentrations of Ca2+ and inorganic phosphate
(Paradies et al.,2013). Less perturbational methods for assessing mPT in aging require
exposing cells or mitochondria to Ca2+ sensitive fluorophores, non-endogenous metabolites,
or examination of structural changes in mitochondria such as swelling. However, even the
latter has been examined to a greater extent upon stimulation with nonphysiologic levels of
Ca2+. It should also be noted that changes in mitochondrial architecture and swelling are
likely to have multiple causal factors during aging, with mPT representing but one (Arrazola
and Inestrosa,2015).

25
1.6 MITOCHONDRIAL ATPase

Mitochondrial ATP production is the main energy source for intracellular

metabolic pathways (Schapira 2006). The human mitochondrial (mt) ATP synthase, or
complex V is the 5th multi subunit oxidative phosphorylation (OXPHOS) complex. It
synthesizes ATP from ADP in the mitochondrial matrix using the energy provided by the
proton electrochemical gradient (Zeviani and Di Donato 2004). ATP is synthesized from its
precursor, ADP, by ATP synthases. This enzyme is found in the cristae and the inner
membrane of mitochondrial. ATP synthesis is the most widespread chemical reaction inside
the biological world. ATP synthase is the very last enzyme in oxidative phosphorylation
pathway that makes use of electrochemical energy to power ATP synthesis (Ahmad and
Cox,2014).
ATP synthase is one of the most ubiquitous and plentiful protein on earth, accountable
for the reversible catalysis of ATP to ADP and Pi. This is also one of the most conserved
proteins in Bacteria, Plants and Mammals with more than 60% of the amino-acid residues of
the catalytic β-subunit resisting evolution (Feniouk,2012). The mitochondrial ATP synthase
is a multi-subunit protein complex having an approximate molecular weight of 550 kDa. The
human mitochondrial ATP synthase or F1/ F0 ATPase or complex V is the fifth component
of oxidative phosphorylation chain. This enzyme is the smallest known biological nanomotor
and plays a crucial role in ATP generation (Jonckheere et al., 2012).

1.7 STRUCTURE OF ATPase

The ATP synthase, also called Complex V, has two major subunits designated F0 and F1.
The F0 part, bound to inner mitochondrial membrane is involved in proton translocation,
whereas the F1 part found in the mitochondrial matrix is the water soluble catalytic domain.
F1 is the first factor recognized and isolated from bovine heart mitochondria and is involved
in oxidative phosphorylation. It was named so from the term ‘Fraction 1’. F0 was named so
as it is a factor that conferred oligomycin sensitivity to soluble F1 (Wittig and
Schagger,2008). The structure of enzyme ATP synthase mimics an assembly of two motors
with a shared common rotor shaft and stabilized by a peripheral stator stalk. The F1 part of
ATP synthase is made up of 8 subunits, 3α, 3β, γ, δ and ε, where the γ, δ and ε subunits add
up to the central stalk (or the rotor shaft) and an alternate arrangement of 3α and 3β form a
hexameric ring with a central cavity. The γ subunit inserted in the central cavity protrudes out

26
to meet ε which binds on its side and together they bind the F0. Two of the Fo subunits,
subunit a and subunit A6L are encoded by the mtDNA ATP6 and ATP8 genes, respectively.
The detailed structure of most of the bovine mitochondrial complex V subunits has been
resolved by X-ray crystallography by John Walker and his group (Rees et al.,2009).

1.7.1 THE F1 ATPase

The mitochondrial F1 ATPase has the subunit composition α,β,γ,δ,ε. The molecular
weight of the subunits are 55 kDa, 51 kDa, 30 kDa, 15 kDa, and 5.7 kDa for the mature
bovine α, β, γ, δ, and ε subunits respectively. The X-ray crystal structure of the bovine F1-
ATPase provided the first look at the structure/function relationship of the subunits in the F1
ATPase (Devenish et al.,2008)and subsequent structures, some with bound inhibitors, have
given greater understanding into the molecular mechanism of ATP synthesis (Rees et
al.,2012).
The α-and β-subunits share only about 20% identity but they form nearly identical folds
and the α-carbon atom positions differ only by about 2.8 Å rmsd (root mean square deviation)
with the greatest spatial divergence in the last 40 residues. The nucleotide binding sites are at
the interfaces between the α- and β-subunits with the catalytic site formed primarily by the β-
subunit and the non-catalytic site formed primarily by the α-subunit. In accordance, there are
6 nucleotide-binding sites in the α3β3γ assembly with 3 being catalytic binding sites and 3
being non-catalytic binding sites. Support for the residues involved in the binding and
catalysis deduced from the crystal structure was obtained by extensive mutagenesis of
primarily the E. coli enzyme, prior to and after the structure determination (Devenish et
al.,2008).
The P-loop (residues 156–163) is integral to the catalytic site with β-subunit residues
Gly161/Lys162/Thr163 being nearly invariable with only Ser163 being a functional variant.
For ATP hydrolysis, β-Glu188 has been concluded to act as a catalytic base that activates a
water molecule in a reaction mechanism where in the transition state, the γ-phosphate
assumes a trigonal-bipyramidal arrangement of the oxygen atoms. ATP hydrolysis is the
reverse of ATP synthesis and microscopic reversibility is widely accepted for this reaction
(Walker and Dickson,2006)

27
Figure 1.6 Schematic subunit composition of ATP synthase. Subunits γ and ε shape a
primary stalk that non-covalently interacts with the membrane bound, hydrophobic c-ring.
The proteolipidic oligomer c rotates in opposition to the peripheral stator that is made from
ab2 and δ. The stator restricts rotation of the α3β3 hexamer, which provides six nucleotide
binding sites (each located at αβ-subunit interface) for reversible ATP synthesis. Ions are
translocated across channels at the a–c ring interface, generating a torque on the central stalk
γε. The γ-subunit which forms α-helical coiled-coil structure is embedded in the F1-hexamer.
The torque of γ causes alternating conformational adjustments in the β-subunits, leading to
differences in nucleotide binding affinities (open, tight and free), which eventually bring
about ATP synthesis or hydrolysis. Mitochondrial ATP synthase possess the bacterial core set
of subunits, but contain additional subunits in the F0 domain (subunits DAPIT and 6.8
kDaProteolipid) and in the stator (subunits d and F6).
Source: Wiley online library

28
1.7.2 THE F0 FACTOR

Factor Fo is also commonly referred to as F0 (F zero). Efraim Racker named factor Fo for
the factor that conferred sensitivity of the F1 ATPase to the antibiotic, oligomycin.
Oligomycin is now known to bind to subunit-c of Fo and prevent proton flow. Recently, the
structure of oligomycin bound to yeast subunit c has been determined by X-ray
crystallography at 1.9 Å resolution providing molecular details into oligomycin binding and
an understanding of how oligomycin blocks proton translocation (Meier et al.,2005). Based
on the structural and genetic data, it is postulated that oligomycin binds on the face of the c-
ring at the region comprised of the proton half channel, formed in part by subunit-a
(Symersky et al.,2012).
The Fo sector is defined by the number of subunits in the biochemical preparation, all of
which have the common characteristic of being membrane proteins or tightly associated with
the membrane proteins. The Fo portion of the ATP synthase comprises the proton turbine as
well as the base of the stator, while the rotor is composed of γδε (Lee et al.,2010). The core of
Fo is composed of an oligomer of the c-subunits, which contain an essential carboxylate from
the side chain of either a glutamate or aspartate residue. The side chain carboxyl acts as the
proton donor and acceptor in proton translocation pathway. Two half-channels (or pathways)
are postulated to exist and are thought to be formed by subunits-a, which provide access to
the essential carboxylate on subunit-c and to the aqueous phase of the matrix and
intermembrane spaces of the mitochondrion (Dong and Fillingame,2010). Other subunits,
such as A6L, may either modulate or provide structural support for the Fo portion of the
enzyme. A recent model of the bovine ATP synthase using cryo-electron microscopy was
able to show the arrangement of subunits a, b, c, e, f, g, and A6L, within the membrane
domain (Baker et al.,2012).
The number of c-subunits varies between species with known stoichiometries ranging
from 8–15 (Watt et al.,2010). BovineFo is composed of a c8-ring while yeast Fo is composed
of a c10-ring (Symerskey et al.,2012). The c-ring rotates within the membrane in steps
determined by the number of c-subunits in the c-ring. As such,movement of 8 protons are
used to rotate the bovine cring 360° driving the synthesis of 3 ATPs. This analysis provides
insight into the thermodynamic considerations for ATP synthesis and provides theoretical
values for ATP/ H+ ratios and therefore, P/O ratios (Watt et al.,2010).

29
30
1.7.3 THE STATOR

The stator is also referred to as the peripheral or extrinsic stalk. The stator functions to
hold F1 fixed to allow rotation of the rotor within the core of F1. The stator provides a
structural support and is not involved directly in the catalytic reaction. The stator is composed
of the oligomycin sensitive conferring protein (OSCP, subunit 5), subunit b, subunit d, and F6
and the X-ray structure has been solved for the peripheral stalk of bovine enzyme (Rees et
al., 2009). Subunit b is anchored to the membrane while OSCP is locked to the top of F1.
(The naming of subunit 5 as OSCP is unfortunate since OSCP does not participate in forming
the oligomycin-binding site.) OSCP was so named because oligomycin prevents proton flow
through Fo and inhibits ATP hydrolysis only if F1 is functionally attached and coupled to
proton movement (Campanella et al.,2009).
Breaking the stator uncouples ATP hydrolysis from proton translocation because the F1
core can spin instead of the rotor. The primary structure of the stator proteins shows low
conservation between bovine and yeast, as evidenced by yeast subunit h and bovine F6,
which have just 14.5% sequence identity. However, the function is conserved because
expression of a cDNA encoding bovine F6 complements the deletion mutation of the gene
encoding subunit h in yeast (Cross and Muller,2004).

1.8 FUNCTIONS OF ATPase

1.8.1 ATP synthesis by ATPase

ATP synthase is sometimes described as Complex V of the electron transport chain

(Jonkheere et al., 2012). The FO component of ATP synthase acts as an ion channel that
provides for a proton flux back into the mitochondrial matrix. It is composed of a, b and c
subunits. Protons in the inter-membrane space of mitochondria first enter the ATP synthase
complex through an a subunit channel. Then protons move to the c subunits (Garrett and
Grisham 2012). The number of c subunits determines how many protons are required to make
the FO turn one full revolution. For example, in humans, there are 8 c subunits, thus 8
protons are required (Fillingame et al., 2003). After c subunits, protons finally enter the
matrix through a subunit channel that opens into the mitochondrial matrix. This reflux
releases free energy produced during the generation of the oxidized forms of the electron
carriers (NAD+ and Q) with energy provided by O2. The free energy is used to drive ATP

31
synthesis, catalyzed by the F1 component of the complex (Berg et al.,2002). Coupling with
oxidative phosphorylation is a key step for ATP production.

1.9 MEDICINAL PLANTS

1.9.1 PLUMBAGO ZEYLANICIA

The genus Plumbago belonging to the family Plumbaginaceae, comprises 10 genera and 280
species (Mukherjee et al.,2007). Three main species included in genus Plumbago namely,
Plumbago indica L., Plumbago auriculata L. and Plumbago zeylinica L. Among these
species, Plumbago zeylanica L. is more popular due to its therapeutic properties. Plumbago
zeylanica L. usually referred to as Ceylon leadwort, doctor bush and wild leadwort, is one of
the well-known herbal plant. It also named as chitramula and chitrak in Ayurveda. Chitrak is
a perennial herb cultivated in shady places in the garden for its brilliant inflorescence
(Jijhotiya et al.,2018). It is widely distributed throughout India and Sri Lanka. The present
compilation aimed to highlight the phytochemicals present in Plumbago zeylanica L. and
provide a comprehensive data regarding its pharmacological potentials.

TAXONOMY

Plumbago zeylanica L., is an abundantly branched perennial herb with alternate leaves
(Fig.1.7). It an annual plant. The plant grows up to height of 3–4ft. Leaves are thick, fleshy,
sessile, oval and lance-elliptic in shape. Flowers of this plant are 10–25cm long and arranged
in terminal and axillary elongated spikes.

32
Figure 1.7 Showing the root of Plumbago zeylanica

Source: Ladoke Akintola University of Technology, Ogbomoso

Kingdom Plantae
Subkingdom Tracheobionta
Class Magnoliopsida
Subclass Caryophyllidae
Superdivision Spermatophyte
Division Magnoliophyta
Order Caryophylllales
Family Plumbaginaceae
Genus Plumbago
Species Plumbago zeylanica

33
PHYTOCONSTITUENTS OF PLUMBAGO ZEYLANICA

Different researchers report phytochemicals from different parts of the plant. Large range of
therapeutic activities possessed by this plant are due to presence of this valuable constituent.
Number of secondary metabolites such as naphthoquinones flavonoids, alkaloids, glycosides,
saponins, steroids, tannins, triterpenoids, coumarins, carbohydrates, phenolic compounds,
fixed oils, fats and proteins reported to be present in Plumbago zeylanica(Vishnukanta and
Rana,2010). Major naphthoquinones present in the plant are plumbagin, chitranone, 3-
biplumbagin, chloroplumbagin, elliptone, coumarins includes seselin, 5-methoxy seselin,
xanthyletin, suberosin. Other phytoconstituents present in the plant includes plumbagic acid,
β sitosterol, 2-dimethyl-5hydroxy-6-acetylchromene, saponaretin, and isoaffinetin(Arpita and
Navneeta,2017). Several other naphthoquinones, difuranonaphthoquinones
binaphthoquinones, coumarins, di-phenyl sulfone, carboxylic acids and esters, monoterpenes,
tri-terpenoids, amino acids, anthraquinones, steroids, steroid glucosides, sugars and other
compounds are also reported to be present(Datta and Mishra,2014). The roots of the plant
contains plumbagin, and other constituents such as 3-chloroplumbagin, binaphthoquinone
named as 3′,6 ′-biplumbagin, 3,3′-biplumbagin and four other pigments reported as
isozeylanone, zeylanone, elliptone, and droserone(Chauhan,2014).

PHARMACOLOGICAL AND THERAPEUTIC ACTIVITIES

Plumbago zeylanica is a pharmaceutically important plant. It exhibits broad range of

pharmacological activities, which includes antibacterial, antifungal, antiinflammatory,
antidiabetic, anticancer, antioxidant, hepatoprotective, cytotoxic and wound healing. The
reported pharmacological activities of various parts of Plumbago zeylanica L. are detailed
below:

ANTIMICROBIAL ACTIVITY

Shweta and Dubey studied antimicrobial properties of the leaves extracts of the plant against
some known drugs. The in-vitro antimicrobial activity and the minimum inhibitory
concentration (MIC) of the crude extract and the standard antibiotics were studied. Maximum
inhibition was reported with leaves extracts as compared to the standard antibiotics(Shweta
and Dubey,2015). In another study, Singh and colleagues investigated methanolic extracts of
the stem and the leaves against six bacterial species and nine fungal species for antimicrobial
studies. Both the extracts showed antimicrobial activity in a dose-dependent manner.

34
Moreover, the antimicrobial activities assayed from the zones of inhibition. Leaves extract
indicated maximum antimicrobial activity against both Staphylococcus aureus and Fusarium
oxysporum whereas the stem extract was noted to be more antimicrobial against the
Pseudomonas aeruginosa and the Penicillium expansum species. Study suggests that the
methanolic extract of Plumbago zeylanica L. stem possess significant antibacterial
activity(Singh et al.,2017). In another study, Ogunleye and coworkers carried an
investigation to evaluate the antibacterial activity of the ethanolic extract of Plumbago
zeylanica L. root bark against seven bacteria extracted from two dumpsites within the city of
Akure. Study revealed, antibacterial activity of the extract enhances with increasing
concentration(Ogunleye and Akinneye,2019). In a recent experiment Jain et al., investigated
Plumbago zeylanica L. for its antifungal activity. Antifungal potential was studied against
four pathogenic fungal species Fusarium oxysporum, Rhizoctonia solanii, Alternaria sp. and
Sclerotium rolfsii. Study suggested excellent inhibitory activities against Alternaria spp.
whereas least against S. rolfsii at 62.5μg/ml(Jain et al.,2020).

ANTIOBESITY

Kotecha and Rao investigated anti-obesity activity of Plumbago zeylanica L. A clinical study
was conducted on obese patients taken from I.P.G.T & R. Hospital at Jamnagar, Gujarat.
During the investigation, an intervention of Plumbago zeylanica L. and haridra powder within
the dose of 500mg and 1g (4 times a day) respectively administered in a capsule form to the
patients for 45 days with restricted diet schedule of low calorie diet. Proposed intervention of
Plumbago zeylanica L. and haridra powder showed potential reduction in the weight of the
patient as compared to the haridra alone(Kotecha and Rao,2007).

HEPATOPROTECTIVE ACTIVITY

Kanchana and Sadiq, reported hepatoprotective activity of petroleum ether extract of

Plumbago zeylanica L. roots against paracetamol induced liver damage. Various biochemical
parameters were studied to evaluate the hepatoprotective activity. Elevated levels of markers
in the animals treated with paracetamol confirmed the severe hepatic damage by paracetamol.
Following the administration of extract, significant reduction was noted in the serum markers
indicating the effect of the extract in restoring the normal functional ability of the
hepatocytes(Kanchana and Sadiq,2007).

35
 CHAPTER TWO

MATERIALS AND METHOD

2.1 Materials and Reagents

Chemicals and reagents used include distilled water, CuSO 4.5H2O, NaOH,
Ammonium hydroxide, phosphate buffered saline (PBS), Trypan blue 0.4 percent (Gibco,
New York, USA), and other analytical grade chemicals and reagents. Filter paper, conical
flask, beakers, reagent bottles, homogenizer flasks, spectrophotometer, cuvette, magnetic
stirrer, stopwatch, stopwatch, dissection set and so on.

2.2 Plant Material

P. zeylanica root will be bought from Akoda, Ede, Osun State. This will be identified and
authenticated in the Department of Plant Biology, University of Ilorin, Ilorin, voucher
number will be obtained.

2.2.1 Preparation of the crude aqueous extract of Plumbagozeylanica

1. The preparation of Plumbagozeylanica roots will be done according to the method
described by Agbaje and Adeniran, 2009 and modified.
2. Briefly, the fresh roots of Plumbagozeylenica will be washed with distilled water,
drained and air-dried for 1hr.
3. A known weight of chopped bits (500g) of the air-dried Plumbagozeylanica will be
macerated in 5000ml distilled water for 24h.
4. The macerated mixture will be filtered using muslin clothe.

2.2.2 Partitioning of Aqueous P. zeylanica Crude Fraction with Organic Solvents

The crude aqueous extract of P. zeylanica will be mixed with various organic solvents (n-
hexane, ethylacetate, butanol, and water) for 1 hr and separated using a separating funnel to
obtain the HF, EF, BF, and AF fractions of the crude aqueous extract.
The separated organic portions will be concentrated using rotary evaporator at 50°C and
weighed to calculate the percentage yield. The obtain fractions of Plumbagozeylanica are
then orally administered to normal albino rats for the assessment of their effects on rat
MMPTP.

36
2.3 Experimental Design
Twenty male Wistar rats weighing between 100 g and 150 g were used for the experiment.
2.4 Protein Estimation
Protein estimation was estimated by use of (Lowry et al., 1951) that makes use of
serum derived from cows called Bovine serum albumin.

2.4.1 Principle

Proteins with aromatic amino acids have tyrosine and tryptophan present which produce a
blue-black colour complex, reading the maximum absorbance of 750nm wave length when
reacted with sodium tungstate molybdate and phosphate (FolinCiocalteau). The intensity of
the colour depends on the amount of these aromatic amino-acids present and will thus vary
for different proteins. Bovine serum albumin was preferred because of the availability, due to
low costs and even due to the purity of BSA. This reaction is dependent on following: pH at
range of 9 to 10.5, incubation time for about 30mins and sensitivity, about 10 ug/ml will give
an accurate result.3.3.2 Standard Protein Solution
1mg/ml BSA solution was prepared by dissolving 5 mg of BSA in 5 ml of distilled water.

Reagent A

2% Na2CO3 in 0.1M solution: 2 g of Na 2CO3 and 0.4 g NaOH pellets were dissolved in 50 ml
distilled water and then made to 100 ml of distilled water in standard volumetric flask. The
reagents were stored at room temperature.

Reagent B
2% Na-K Tartarate solution: 2 g of Sodium potassium Tartarate Na-KC 406 where dissolved in
50 ml of distilled water and made up to 100 ml of distilled water in standard volumetric flask.
The reagent was stored at room temperature.

Reagent C

1% of Copper Sulphate Solution: 1 g of Hydrated Copper Sulphate (CuSO 4.5H2O) was

dissolved in 50 ml of distilled water and made up to 100 ml in a standard volumetric flask.
The reagent is stored at room temperature.

37
Reagent D

Alkaline Copper Solution: This was prepared fresh by mixing 50 ml of reagent A with 0.5 ml
of reagents B and 0.5 ml of reagent C. The Tartarate solution was added first to prevent
solution from getting cloudy

Reagent E
FlolinCiocalteau Reagents Solution: This is the color reagent and it contains
phosphomoylbdictungstics complex and bromine water. It is commercially available in 2 N
but it is diluted with distilled water to 1 N before use. The reagent was kept in a black
container because it is photolytic.

2.4.3 Procedure
Varying volumes of the prepared standard BSA solution was used as shown in
protocol. Each test tube was filled with 1ml distilled water and 3ml of reagent D was added to
the protein samples, mixed and allowed to stand for 10 minutes at room temperature. 0.3mls
of reagent E was added very quickly and the mixture vigorously shaken immediately. After
3mins, the absorbance were read at 750 nm using UV Spectrophotometer. The resulting
values obtained were used to plot the standard protein curve.

Table 2.1: Protocol for Protein Estimation

Test tubes 1 2 3 4 5 6

Standard BSA (µL) - 100 200 300 400 500

Distilled water 1000 900 800 700 600 500

Reagent D 3.0 3.0 3.0 3.0 3.0 3.0

Reagent E 0.3 0.3 0.3 0.3 0.3 0.3

38
39
2.5 Determination of Mitochondrial Protein

10 µL of the mitochondria was dissolved in 990 of distilled water in duplicate test

tubes.3.0 ml of reagent D was added to the test tubes and allowed to stand for 10 minutes.
After this, 0.3 ml of Folin C was added to all the test tubes and allowed to stand for 30
minutes after which readings were taken at 750 nm wavelength on a UV Spectrophotometer
(Lowry et al., 1951).

2.6 Assay for Mitochondrial Atpase Activity In Rat Liver Mitochondria

2.6.1 Principle
According to Bassir's method (1963). The idea behind it is that when molybdic acid
reacts with inorganic phosphate, it produces a yellow color that reduced to produce a blue
molecule. Ascorbic acid can be used as the reducing agent, and the intensity of colour that
results is directly correlated to the amount of inorganic acid present.

2.6.2 Procedure for Isolation of liver mitochondrial from male wistar rats
All apparatus needed were iced (i.e., conical flask, ependdof tube, homogenizing
flask, beaker, measuring cylinder and reagent bottle containing 0.25M Sucrose). The
centrifuge containing the centrifuge tube was precooled at 2000 rpm at 4 ⁰C. The animal to be
sacrificed was isolated (fasted) for 24 hours. The fasted animal was swirled round in order to
disorientate it and was immediately sacrificed by cervical dislocation. The animal was
dissected and the liver located below the diaphragm was immediately excised and dapped on
a filter paper to drain the blood on it and was weighed. The weighed liver was transferred into
a beaker placed on ice where it was minced with a pair of scissors, the minced liver was
diluted with calculated volume of buffer (0.25M Sucrose) in a Teflon-glass cup
homogenizing flask and was homogenized. This whole process was carried out on ice (4 ⁰C)
to preserve the integrity of the mitochondria. The homogenate was transferred into the
centrifuge tubes and was placed in the rotor inside the centrifuge machine. The homogenate
was sedimented twice at 2500 rpm for 5 mins to remove nuclear fractions and cell debris. The
supernatant obtained was centrifuge at 13000 rpm for 10 mins in order to get the
mitochondria fraction. The sedimented mitochondria pellet obtained after the supernatant has
been discarded was washed with buffer (0.25M Sucrose) and centrifuged at 12000 rpm for 10

40
mins thrice. The pure mitochondrial fraction obtained was used to prepare an aliquot, using
200 µl of buffer (0.25M Sucrose). The aliquot was preserved on ice for further assay.

2.6.3 Procedure for determination of Adenosine Triphosphate Enzyme (ATPase) activity

Determination of Adenosine Triphosphate Activity was determined by modification
of the method of Lardy and Wellman (1953).

For the ATPase activity, eleven test tubes with duplicates were used in all. The test
tubes were labeled as follows: the first test tube as the blank, the second as mitochondria
only, the 3rd as ATP only, the 4th as mitochondria and ATP, while the 5th to 7th test tubes were
labeled as varying volume of the extract (40 mg/100g body weight (bw), 50 mg/100g bw and
60 mg/100g bw respectively), the 8th test tube as zero-time. 200 µl of 25Mm sucrose was
pipette into all test tubes using a micro pipette. 200µl of KCl was also put in all the test tubes;
1300 µl of Tris was put in all the tubes. Varying volumes of the extract 40mg, 50mg and
60mg, were put in 5th, 6th, and 7th test tubes respectively. Calculated volume of distilled water
was put in all the test tubes. 40 µl of ATP in all test tubes except the 1 st and 2nd test tubes. 1ml
of 10% sodium dodecyl sulfate (SDS) was put in the zero-time test tube after the addition of
ATP in order to stop the reaction in the tube. The test tubes were transferred into a water Bath
of 30⁰C temperature and were shaken vigorously for 30 minutes for incubation. While in the
water bath, 20 µl of mitochondria was put in all test tubes except the 1 st and 3rd test tubes at
an interval of 30 secs. After the 30 mins, SDS was added to the test tubes except for the zero
time test tube. To end the reaction 4ml of distilled water was added to each of the test tubes to
dilute. 1 ml was picked from each test tube and pipette into the duplicate set of test tubes
respectively. 1 ml of ammonium molybdate was added into all the new set of test tubes. L-
ascorbate was added into the solution in the new set of test tubes at 30 seconds interval for 30
minutes. After the 30 mins, the spectrophotometer was read at 660 nm.

2.7 Determination of Inorganic phosphate

2.7.1 Principle
This was performed according to the method described by Bassir (1963). The
principle behind the method is based on the fact that molybdic acid in the presence of
inorganic phosphate forms a yellow colouration complex which is reduced to give a blue
coloured compound. Ascorbate can be used as a reducing agent and the intensity of the colour
thus formed as directly proportional to the concentration of inorganic phosphate.

41
2.7.2 Procedure
1 ml of distilled water was picked from each test tube and pipetted into the duplicate set of
test tubes respectively. 1 ml of ammonium molybdate was added into all the new set of test
tubes. L-ascorbate was added into the solution in the new set of test tubes at 30 seconds
interval for 30 minutes, after the 30 minute, the spectrophotometer was read at 660 nm.

Table 2.2: Protocol for Mitochondrial ATPase Activity

Test-tubes Sucrose KCL Tris H2O UCP ATP Mitochondrial
(µL) (µL) (µL) (µL) (µL) (µL) (µL) (µL)
Blank 200 200 1300 300 - - -
Mit. Only 200 200 1300 280 - - 20
ATP only 200 200 1300 260 - 40 20
Mit. + ATP 200 200 1300 240 - 40 20
UCP 200 200 1300 190 - 40 20
Zero time 200 200 1300 240 - 40 20

2.8 Assays for Mitochondria Swelling.

300 μMCaCl2 per mg of mitochondria protein was used to induce MPTP opening
according to a modification of the lapidus and sokolove (1993).

2.8.1 Procedure
The low ionic strength mitochondria was isolated using a method described by
Johnson and Lardy (1967). The animals not fed overnight were sacrificed by “cervical
dislocation” and dissected quickly. The liver was rapidly excised, trimmed to remove excess
tissue and washed in buffer A. Thereafter the liver was weighed, chopped and suspended in
buffer A to make a 10% suspension of tissue in buffer. Immediately the liver was
homogenized on ice using a glass of Teflon potter homogenizer. The homogenate was
sediment twice at 2500 rpm for 5 mins to remove the nuclear fractions and cellular debris,
supernatant obtained was centrifuged at 1300rpm for 10 minutes and mitochondria fraction
obtained was washed three times at 12000 rpm for 10 mins with buffer B. The mitochondria
was immediately dispensed into 1ml Eppendorf tubes and aliquots and used fresh.
Mitochondria obtained from intubated animals were pre-incubated in the presence of

42
rotenone in swelling buffer for about 3 minutes at 30 0C prior to addition of 250mM Sodium
Succinate which was added 30 seconds later and swelling rate was quantified at 540nM based
on the procedure described by Lapidus and Sokolove (1993).For standard induction of
MMPTP of untreated mitochondria; isolated Mitochondria were pre-incubated in the
presence of rotenone in swelling buffer for about 3 minutes at 30 0C prior to the addition of
CaCl2 (triggering agent). 30 seconds later 250 mM sodium succinate was added and swelling
rate was quantified at 540 nm based on the procedure described by Lapidus and Sokolove
(1993). For assay without triggering agent, addition of CaCl 2 was omitted; spermine was
added immediately after the addition of rotenone, just before the addition of mitochondria
and the mixture was incubated for 3 minutes in a water bath at 30 0C with thorough mixing for
spermine.

2.8.2 Reagents
Preparation of 4.9 mM Spermine
0.05 g of spermine was dissolved in a little water and then made up to 50 ml in 50 ml
standard volumetric flask.
Preparation of 2.5 mM Rotenone
0.05 g of Rotenone was dissolved in a little quantity of 95% Ethanol and then made up to 50
ml with Ethanol in a 50 ml volumetric flask

Preparation of 250 mM Sodium Succinate

0.68 g of sodium succinate was dissolved first in a little quantity of distilled water and then
made up to 10 ml marked volumetric flask.

Preparation of 18 mM CaCl2
0.1 g of CaCl2 was dissolved in a little quantity of distilled water and then made up to 50 ml
volumetric flask.

Buffer A (Isolation buffer)

210 mMMannitol, 70 mM Sucrose, 5 mM HEPES, 1M KOH and 1 mM EGTA (pH =7.4)
Preparation: 0.60g of HEPES, 19.16g of Mannitol, 12.0g of sucrose and 0.19g of EGTA were
dissolved in 200ml of distilled water, standardized with 1M KOH (Ph = 7.4) and then made
up to 500ml

43
Buffer B (Washing buffer)
210 mMMannitol, 70 mM Sucrose, 5 mM HEPES, 1M KOH and 1 mM EGTA (pH =7.4)
Preparation: 0.30 g of HEPES, 9.55 g of Mannitol, 6.0 g of sucrose and 1.25 g of BSA were
dissolved in 100 ml of distilled water, standardized with 1M KOH (Ph = 7.4) and then made
up to 500 ml

Buffer C (Suspension buffer)

210 mMMannitol, 70 mM Sucrose, 5 mM HEPES, 1M KOH and 1 mM EGTA (pH =7.4)
Preparation: 0.30 g of HEPES, 9.55 g of Mannitol, 6.0 g of sucrose were dissolved in 100 ml
of distilled water, standardized with 1M KOH (Ph = 7.4) and then made up to 250 ml.

Buffer D (swelling buffer)

210 mMMannitol, 70 mM Sucrose, 5 mM HEPES, 1M KOH and 1 mM EGTA (pH =7.4)
Preparation: 0.35 g of HEPES, 9.55 g of Mannitol, 6.58 g of sucrose were dissolved in 100
ml of distilled water, standardized with 1M KOH (pH = 7.4) and then made up to 250 ml. All
were stored at 4°C.

44
45
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