Modulation of the redox status of isolated mitochondria by energy-linked substrates: quantification by high performance liquid chromatography; and "Splicing up" drug discovery, cell-based express...

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Content MODULATION OF THE REDOX STATUS OF ISOLATED MITOCHONDRIA BY
ENERGY-LINKED SUBSTRATES: QUANTIFICATION BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
AND
―SPLICING UP‖ DRUG DISCOVERY. CELL-BASED EXPRESSION AND
SCREENING OF GENETICALLY-ENCODED LIBRARIES OF BACKBONE
CYCLIZED POLYPEPTIDES

by
Harshkumar Sancheti
___________________________________________________________
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2010

Copyright 2010 Harshkumar Sancheti

ii

Dedication
This thesis is dedicated to my grandfather Rajmal Sancheti (R.I.P), my parents
Dinesh Sancheti, B.Tech., and Rajni Sancheti, MSc., my sister Prachi Parekh,
M.Com., and beloved young brother Yash Sancheti.

―Miles to go before I sleep, miles to go before I sleep‖- Robert Frost.

iii

Acknowledgements
I would like to acknowledge my sincere and heartfelt gratitude to my mentor
Enrique Cadenas, M.D, Ph.D., for his guidance, generosity and more importantly
his trust and belief in me while completing my master‘s thesis. I would also like to
thank Julio A. Camarero, Ph.D., for giving me an opportunity to work in his lab.
My appreciation for Jerome Garcia, Ph.D., and Li-Peng Yap, Ph.D. and Derick
Han, PhD. for being very supportive, willing to teach and helping me understand
the basics of mitochondrial biology. My current lab members Juliana Hwang
Ph.D., Ryan Hamilton Ph.D., Fei-Yin, Chen Li and Amit Agarwal; all have played
a part in ensuring that my master‘s thesis was a smooth ride. Also, my previous
lab members Luis Berrade, Ph.D., Getachew Woldemarian, Ph.D., Wan Wang;
all had been very helpful. I would like to thank my master‘s thesis committee
member Wei Chiang Shen, Ph.D. for his willingness to spend time in reviewing
my thesis.
Most importantly, my sincere and special thanks to Mona Verma for keeping me
optimistic and cheerful.

iv

Table of Contents
Dedication………………………………...………………………….……....ii
Acknowledgements…………………………………………….…….........iii
List of Figures…………………………………………………..................vii
Abbreviations…………………………………………….…………....……ix
Abstract..…………………………………………………….......................x
1.0 Chapter 1: Modulation of the redox status of isolated brain……….…...1
mitochondria by energy-linked substrates:
Quantification by High Performance Liquid
Chromatography
1.1 Introduction……….……………………...…………………….….....…1
1.1.1 Mitochondria structure and function…….…………….……........3
1.1.2 Tricarboxylic acid cycle.………………..………………...............4
1.1.3 Oxidative phosphorylation …...……………………………..…....6
1.1.4 Mitochondrial membrane potential……………………….….....11
1.1.5 Bioenergetics………..…………………….................................13
1.1.6 Redox status…..…….…………………………………….……...14
1.1.7 Oxidative stress……….………………..…………………….…..17
1.1.8 Nicotinamide nucleotide transhydrogenase……….………..…19
1.1.9 Mitochondria isolation considerations……..………….….....….21
1.2 Methods……………………………………….………….………….....22
1.2.1 Mitochondria isolation…………………………………….……...23
1.2.2 Mitochondrial respiration…………………..……………...…..…24

v

1.2.3 Membrane potential determination……..………..……..…...…24
1.2.4 High performance liquid chromatography….………….……....26
1.3 Results…………………………………………….……………………28
1.3.1 Shift in redox status of isolated mitochondria…….....…..……28
1.3.2 Membrane potential and pyridine dinucleotide………….……39
redox status
1.4 Discussion…………………………...……………………...…….……43
2.0 Chapter 2: Splicing up drug discovery. Cell-Based expression……....46
and screening of Genetically-Encoded Libraries of
backbone Cyclized polypeptides

2.1 Introduction…………………………..…………………...……….....46
2.2 Native Chemical Ligation………..…..………………………..…….49
2.3 Expressed Protein Ligation………………….……………………..50
2.3.1 Recombinant Polypeptide α-thioesters…….……...…………..53
2.3.2 Recombinant N-terminal Cys-containing polypeptides......….54
2.4 Biosynthesis of backbone cyclized peptides using.……................56
Expressed Protein Ligation
2.5 Biosynthesis of backbone cyclized peptides using protein…….….61
trans-splicing
2.6 Protease catalyzed protein splicing for the biosynthesis of…….…64
backbone cyclized peptides
2.7 Cell-based screening of genetically-encoded libraries of…………67
backbone cyclic polypeptides

vi

2.8 Conclusions and remarks………………..…….……...……….…...71
References…………………………………………………….…………………..74

vii

List of Figures
Figure 1: Morphology of mitochondria……………..…………..….……………..4
Figure 2: The citric acid cycle………………………………………….………….5
Figure 3: Illustrative figure of electron transfer chain…………………………10
Figure 4: The mitochondrial energy–redox axis in cell function…….……….20
Figure 5: The chemical reaction of NAD(P) in presence of CN
-
and KOH…29
Figure 6: A representative chromatogram of NAD(P)H……………..………..29
Figure 7: A representative oxygen consumption trace used to calculate…30
respiratory control ratio

Figure 8: A) NADP
+
, B) NADPH, C) NAD
+
, D) NADH, E) NADPH/NADP….34
F) NADH/NAD present in mitochodria after incubation with
complex I substrates and inhibitor
Figure 9: A representative spectrofluorometric trace of Rhodamine-123…40
used to calculate membrane potential

Figure 10: Representation of the calculated mitochondrial membrane……..41
potential using Rhodamine-123 as described in figure 9.
The values of membrane potential are calculated in terms of
ΔΨ mV. (n=3)
Figure 11: NAD(P)H/NAD(P) vs membrane potential (ΔΨ mV)……………...42
Figure 12: Backbone cyclization of polypeptides using native chemical……51
Ligation

viii

Figure 13: Biosynthesis of recombinant polypeptide α-thioesters…………..52
Figure 14: Biosynthetic approach for the in vivo production of cyclotides…60
inside live E. coli cells.
Figure 15: Production of backbone cyclized polypeptides using protein…63
trans-splicing.
Figure 16: Biosynthesis of cyclic polypeptides using intramolecular………66
sortase-mediated ligation.
Figure 17: Schematic representation of the putative mechanism of………68
protease-catalysed cyclotide cyclization.

ix

Abbreviations
G/M, glutamate/malate ; G/M + ADP, glutamate/malate + ADP; G/M + rotenone,
glutamate/malate + rotenone ; G/M + CCCP, glutmate/malate + Carbonyl cyanide
m-chlorophenyl hydrazone

x

Abstract
Chapter 1. The balance between oxidized and reduced couples, i.e., glutathione
(GSH/GSSG), pyridine dinucleotides (NADH/NAD, NADPH/NADP),
thioredoxin
reduced
/thioredoxin
oxidized,
dihydrolipoic acid/α-lipoic acid, and
lactate/pyruvate determines the cellular redox status. Oxidative stress and
altered redox status are widely considered as major components of aging and
age-related diseases. The isolation of mitochondria from organs is a widely used
tool to study mitochondrial biology. However, inherent in the long isolation
process, are alterations in mitochondrial redox status. Previous work from our
laboratory has shown that different isolation methods can alter the redox status
with respect to the glutathione pool. Substrate supplementation of isolated
mitochondria resulted in higher buffering capacity against H
2
O
2
challenges, in
part due to increased GSH levels. The aim of the present study is to extend our
previous work and monitor changes in the pyridine dinucleotide pool. Upon
substrate supplementation, changes in the pyridine dinucleotide pool were
quantified using HPLC and changes in membrane potential have been monitored
using fluorescent dye Rhodamine-123. Our data show that substrate
supplementation shifts the mitochondrial redox status towards a more reduced
state. These data are in agreement with our previous work and show changes in
redox status of pyridine dinucleotides when supplemented with mitochondrial
energy substrates.
xi

Chapter 2. The present paper reviews the use of protein splicing for the
biosynthesis of backbone cyclic polypeptides. This general method allows the in
vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA
expression techniques. Biosynthetic access to backbone cyclic peptides opens
the possibility to generate cell-based combinatorial libraries that can be screened
inside living cells for their ability to attenuate or inhibit cellular processes thus
providing a new way for finding therapeutic agent.

1
Chapter 1
Modulation of the redox status of isolated brain mitochondria by energy-linked
substrates: Quantification by High Performance Liquid Chromatography

1.1 Introduction
The isolation of mitochondria from organs is a widely used tool in studying
mitochondrial biology. Our data show that the NAD pool of isolated brain
mitochondria (by percoll gradient) is almost completely devoid of NADH, at least
up to the lowest limits of fluorescence detection; the levels of NADPH are
substantially oxidized. However, substrate supplementation by adding
glutamate/malate to isolated mitochondria or in mitochondria isolation buffer
before isolation is started can substantially shift the pyridine nucleotide pool to a
more reduced status, thus allowing for detection of greater amounts of NADPH
and NADH. These data are in agreement with our previous work and show
changes in the redox status of pyridine dinucleotides when supplemented with
mitochondrial energy-linked substrates. This study shows that mitochondrial
redox and bioenergetics are dynamically linked and subject to rapid changes.
The present method of substrate supplementation has been successful in
measuring the levels of pyridine dinucleotides present in mitochondria. However,
no current methods measure the endogenous levels of the respective pyridine
2

dinucleotides present in the mitochondria and thus, it cannot be verified if
substrate supplementation can maintain the mitochondrial endogenous redox
status of pyridine dinucleotides over the period of mitochondria isolation. Our
study shows that mitochondrial redox status can be altered by substrate
supplementation to a more reduced state, which strengthens the notion that the
actual mitochondrial redox status is changed over the period of mitochondria
isolation.
Implications of the study
1. Isolated mitochondria are used for a large number of experiments
investigating mitochondrial biology. A consideration of the changed redox status
of the isolated mitochondria can help interpret some results.
2. Examining the influence of the mitochondria redox status on the energy-
transducing capacity of mitochondria.

3

1.1.1 Mitochondria structure and function
Mitochondria are membrane enclosed organelles found conspicuously in most
eukaryotic cells. They have a sausage-like shape with variable dimensions of
approximately 3-4 µM [length] and 1 µM [diameter]. Two highly organized
membranes encircle each mitochondrion thus resulting in two distinct
compartments, a narrow intermembrane space and a much larger matrix (Fig. 1).
The size and shape of mitochondria is variable, and most significantly there is an
amazing variation in the number and structure of cristae (Scheffler, 2008).
Mitochondria are traditionally termed as the powerhouses of the cell as they are
responsible for generation of most of the ATP- coupled to respiration- required
for cellular functions (McBride et al., 2006). Mitochondria also play a role in
cellular signaling, differentiation, death, growth control and they are widely
considered to play a pivotal role in aging. Mitochondria have their own circular
DNA and reproduce independently; hence they are believed to be a case of
endosymbiosis. The number of mitochondria can vary from one to a few hundred
or thousand per cell and basically depends on the metabolic requirements of the
cell.

4

Figure 1. Morphology of mitochondria

1.1.2 Tricarboxylic acid cycle [TCA]
The TCA cycle is the 2
nd
step of respiration after glycolysis. The pyruvate
obtained from glycolysis in cytosol enters the mitochondria and is converted to
acetyl Co-A in presence of pyruvate dehydrogenase (PDH) by oxidative
decarboxylation. This is an irreversible step and, thus, pyruvate (via acetyl CoA)
is committed to the TCA cycle. The entry of acetyl Co-A yields different
intermediates and reducing equivalents such as nicotinamide adenine
dinucleotide (NADH) and flavin adenine dinucleotide (FADH
2
) (Fig. 2). The rate of
the tricarboxylic acid cycle is regulated to meet the cell‘s energy requirements.
5

Figure 2. The citric acid cycle

6

1.1.3 Oxidative phosphorylation
The first breakthrough in the understanding the concept of electron transport
chain in mitochondria was put forward by Otto Warburg through the conception of
term ‗Atmungsferment‘. Further, Mitchell‘s ‗Chemiosmotic‘ hypothesis provided
new insights into the interconversion and storage of chemical free energy in living
organisms. Finally, the structure of ATP synthase explained how a proton and
electrochemical gradient across a membrane drives the molecular rotary engine
and thus the synthesis of ATP. The reducing equivalents (i.e., NADH and FADH
2
)
obtained from the TCA cycle are utilized in the ETC. The mitochondrial electron
transfer is linked to the generation of large amount of free energy, a large fraction
of which is conserved in the form of the phosphate-bond energy of ATP in the
process called oxidative phosphorylation. NADH and FADH
2
donate electrons,
which are funneled through a series of four enzyme complexes that help maintain
a gradient for ATP generation and transports H
+
across the membrane. At the
fourth enzyme complex, the free electron and oxygen combine to generate water.
The components of the mitochondrial respiratory chain, i.e., flavoproteins, iron
sulphur centers, coenzyme Q and cytochromes are imbedded in the inner
mitochondrial membrane. All these components are arranged in four large
multimeric complexes linked by two mobile carriers and ATP synthase
The gradient generated through the transport of electrons allows the ATP
7

synthase to move and generate ATP (Figure 3 A, B, C) (Boyer et al., 1977). The
basic mechanism for energy transduction in mitochondria is generation of an
electrochemical gradient (protons, H
+
) across the mitochondrial inner membrane
during electron transport. This proton gradient is formed by pumping H
+
from the
mitochondrial matrix side of the inner membrane to the cytosolic side of the
membrane. This results in generation of an electrochemical gradient that is
coupled to the synthesis of ATP by the mitochondrial F
1
F
0
-ATPase.
Even though the outer mitochondrial membrane is highly permeable to most low
molecular weight solutes, the inner mitochondrial membrane is permeable only to
certain select moieties. It is impermeable to NAD
+
, NADH, NADP
+
, NADPH as
well to other nucleotides. However, several nucleotides such as adenosine mono
phosphate (AMP) and adenosine di phosphate (ADP), inorganic phosphate,
malate and amino acids like glutamate and aspartate, can cross the inner
mitochondrial membrane through specific membrane transport systems like
carriers, translocases, or porters (Bernardi, 1999). Cytosolic NADH is transferred
into mitochondria for oxidative metabolism and ATP production through two
NADH shuttles (Lasorsa et al., 2003), the glycerol phosphate shuttle
(MacDonald, 1981) and the malate/aspartate shuttle (LaNoue and Schoolwerth,
1979, Indiveri et al., 1987). The latter requires the concerted action of two
metabolite carriers in the mitochondrial inner membrane: the oxoglutarate/malate
carrier and the aspartate/glutamate carrier (Lanoue et al., 1974). It has been
8

shown previously shown that the transfer of reducing equivalents between
mitochondria and incubation medium can be carried out only in the presence of
both glutamate and malate (Dennis and Clark, 1977). The malate-aspartate
shuttle does not operate in the absence of either glutamate or malate (Minn and
Gayet, 1977). The malate-aspartate shuttle involves three steps of anion
exchange at the level of the inner mitochondrial membrane i.e. Glutamate-
aspartate, Glutamate-hydroxyl, Malate-α-ketoglutarate.

Inhibitors of mitochondrial respiratory chain
Rotenone is a widely used inhibitor of complex I (NADH dehydrogenase) by
shutting off the supply of electrons to the quinol (QH
2
)-cytochrome c
oxidoreductase (Chance and Hollunger, 1963, Barrientos and Moraes, 1999). As
a result, electrons derived from the NAD-linked dehydrogenases are not oxidized
in the respiratory chain. Other inhibitors of mitochondrial respiration include:
 Antimycin A: Inhibits complex III (bc
1
segment)
 Cyanide, azide, carbon monoxide: Inhibit complex IV.
The inhibitors of the various complexes along with the schematic for oxidative
phosphorylation are described in the Fig. 3A.

9

Uncouplers of respiration
Uncouplers of respiration and oxidative phosphorylation are lipophilic weak acids
that dissipate the proton gradient by transporting protons through the membranes
from the intermembrane space to the matrix. Uncouplers act by dissolving in the
membrane and functioning as carriers for H
+
, short-circuiting the normal flow of
protons through the F
1
F
0
-ATPase. Carbonyl cyanide m-chlorophenyl hydrazone
(CCCP) and 2, 4 Dinitrophenol (DNP) are the most commonly used uncouplers in
studying membrane potential and other mitochondrial experiments.

10

Figure 3 A

Figure 3 A. Illustrative figure of electron transport chain along with the respiratory
inhibitors for individual complexes. 3 B) Supracomplex view from the
intermembrane space C) A complete view of flow of electrons from the
tricarboxylic acid cycle to the electron transport chain and subsequent generation
of ATP.

Figure 3 B
11

Figure 3, continued

1.1.4 Mitochondria Membrane Potential ( Δ Ψ)
Respiration and electron transport leads to a significant membrane potential
associated with the mitochondrial inner membrane. A proton gradient exists
across the mitochondrial inner membrane as a result of proton pumping by the
enzyme complexes as explained above (Brand and Murphy, 1987, Mitchell,
1972, Tzagoloff and Myers, 1986, Lane et al., 1986) .This gradient has two
12

components: a membrane potential (protons are positively charged) and a pH
gradient (protons also determine acidity). The membrane potential gives the
energy to drive the synthesis of ATP by F
o
F
1
ATPase, which functions as a proton
turbine (Mitchell, 1979, Boyer, 1987, Boyer, 1997, Pedersen and Carafoli, 1987b,
Pedersen and Carafoli, 1987a). In most mammalian cells a membrane potential
of around -180 mV is expressed due to the electrochemical gradient.
Mitochondrial membrane potential is a very important bioenergetic parameter
and it provides a link between respiratory chain and ATP synthase. Maintenance
of mitochondrial membrane potential is essential for ATP generation, Ca
2+
uptake
and storage, and the generation and detoxification of reactive oxygen species
(ROS). ΔΨ has been shown to influence the release of NO and generation of
H
2
O
2
by mitochondria (Valdez et al., 2006, Korshunov et al., 1997). The
mitochondrial membrane potential also influences the release of cytocrome c in
apoptosis (Nicholls, 2004). Thus, ΔΨ can be considered as driving force for
maintenance of respiration and ATP generation and in turn affecting a diverse
array of functions metabolic functions and bioenergetics of mitochondria.

13

1.1.5 Bioenergetics
Each step in the electron transport chain is associated with a free energy
change, and each component undergoes a cycle of oxidation and reduction
reactions which represents a redox couple. The basis of such a redox couple can
be explained by the standard reduction potential, a property based on free
energy change. A complete reaction consists of a sum of two half reactions:-
Succinate
-
 2e
-
+ 2H
+
+ Fumarate
-
E
0
= -0.031V_________(1)
Ubiquinone + 2e
-
+ 2H
+
 ubiquinol E
0
= +0.045V_________(2)
Combining (1) and (2)
Succinate + Ubiquinone  Fumarate + ubiquinol ΔE
0
= +0.014V
The free energy change for an oxidation-reduction reaction can be calculated
from the following relation:
ΔG = -nFΔE
Where F= faraday (1 F = 96,494 calories/mol).

14

1.1.6 Redox status
The balance between oxidized and reduced couples, i.e., glutathione
(GSH/GSSG), pyridine dinucleotides (NADH/NAD, NADPH/NADP),
thioredoxin
reduced
/thioredoxin
oxidized,
dihydrolipoic acid/ α-lipoic acid, and
lactate/pyruvate determines the cellular redox status. The cellular redox status is
mainly influenced by GSH/GSSG, followed by, NADPH/NADP and NADH/NAD.
In simple terms, the redox status of cell indicates its reducing capacity or the
interconvertible oxidized and reduced couple of a specific redox couple (Schafer
and Buettner, 2001). The redox status for a particular redox couple is the
summation of the products of the reduction potential and reducing capacity of the
linked redox couples present. Reduction potential is expressed as voltage in mV
and reducing capacity can be viewed as the total charge stored (Number of
electrons available). Reducing potential can be estimated by Nernst equation as
shown mathematically below and the reducing capacity can be determined by
determining the concentration of the reduced species in the particular redox
couple.
Redox environment =


) (
1
couple n
i
Ei

× [reduced species]
i
Where E
i
= Half cell reduction potential for a particular redox pair .
[reduced species]
i
= concentration of the reduced species in that redox pair.
15

The redox state of a redox couple is defined by the half cell reduction potential
and the reducing capacity of that couple. Changes in these ratios affect the
energy status of the cell, largely determined by the ratio of [ATP] / [ADP] +
[AMP].
The pyridine dinucleotide system [NAD(P)H]: The pyridine dinucleotide system
primarily consists of NAD
+
, NADH, NADP
+
, NADPH. They are synthesized in the
cells by the well established de novo pathway, the salvage pathway and recently
suggested to be directly generated from the reduced form of nicotinamide
mononucleotide (NMNH) and ATP by the action of NMNATs (Berger et al.,
2005). Mitochondria contain a large portion of intracellular NAD
+
. The major
categories of reactions undertaken by NAD
+
and NADH are:
 Inter conversion: NAD
+
and NADH are inter converted into each other, but
the total pool of NAD
+
+ NADH remains constant.
 NAD
+
is converted to NADP
+
by the action of NAD
+
kinase.
 NAD
+
is converted to other nicotinamide-containing moieties through
enzymes like poly (ADP-ribose) polymerase-I [PARPs]
NAD
+
and NADH levels mainly affect the cellular energy metabolism, but also
play an important role in cell death (Virág and Szabó, 2002), gene expression,
aging and calcium homeostasis (Alano et al., 2004). The NAD
+
/NADH ratio can
16

also affect the mitochondrial permeability transition, while NADH levels can
interact with and inhibit voltage-dependent anion channels (VDAC). NADH/NAD
+

affects aging and oxidative damage due to its effect on cellular antioxidant
capacity (Ying, 2006). NADPH is the major source for reductive biosynthesis and
is a source of reducing equivalents for glutathione and thioredoxin systems. As
the concentration of glutathione is much higher than that of other redox systems,
it is considered to be pivotal in redox buffering in the cell. It is important to view
all redox systems as thermodynamically connected rather than isolated systems.
The oxidation of NADPH to NADP
+
yields two electrons that support reductive
biosynthesis; Conversely, NAD
+
serves as a sink for electrons. NADPH and
NAD
+
couples are maintained many orders of magnitude away from equilibrium
with each other, allowing them to fulfill their respective functions. Overall,
NAD(P)H system ratios are very critical in determining the redox state of the cell
and modulate the activity of many metabolic enzymes. These ratios are dynamic
and subject to rapid changes, thus can be viewed as metabolic sensors (Pinkas-
Sarafova et al., 2005).

17

1.1.7 Oxidative stress
The ETC is somewhat leaky, thus leads to generation of superoxide anion when
reduced components of the ETC interact with molecular oxygen; superoxide and
its dismutation product, hydrogen peroxide, are believed to contribute
significantly to the aging process and forms the central dogma of ―The Free
Radical theory of Aging‖. ROS basically comprise of a variety of diverse chemical
species including superoxide anion, hydroxyl radical and hydrogen peroxide.
Whereas, superoxide and hydroxyl radicals are short lived, hydrogen peroxide is
freely diffusible and relatively long-lived (Finkel and Holbrook, 2000). Production
of hydrogen peroxide leads to ‗Oxidative stress‘, which is an imbalance between
the cell‘s oxidants and antioxidants. Oxidants are formed as a normal product of
aerobic metabolism but are elevated under pathophysiological conditions (Sies,
1997). It needs to be understood that oxidative stress simply does not mean an
increase in oxidizing moieties or a decrease in reduced moieties. However a shift
in the equilibrium of prooxidant-antioxidant balance in favor of prooxidant is the
leading cause of oxidative stress (Sies, 1991, Sies, 1997). Hydrogen peroxide
mainly contributes to modification of cellular proteins, DNA and lipids in a way
that is deleterious for the cells and mainly inhibits protein, DNA or lipid function.
The main sources of hydrogen peroxide in the cell are electron transfer reactions
in mitochondria, xanthine oxidase, NADPH oxidase, monocytes/macrophages,
18

etc. Cells are endowed with defense mechanisms against free radicals to
minimize oxidative damage. The main cellular antioxidant mechanisms are:
 Superoxide dismutase (SOD), that catalyzes the conversion of superoxide
radical to hydrogen peroxide.
 Catalase, that converts hydrogen peroxide to water.
 Glutathione peroxidase, that converts hydrogen peroxide to water.
Catalase is localized in peroxisomes and also in heart mitochondria.
Mitochondria from other tissues do not contain catalase. Decrease in the energy
production and subsequent decline in enzymatic activities of certain key enzymes
are pre-dominant features of aging and aging related neurodegeneration
(Calabrese et al., 2006, Jones et al., 2002, Newman et al., 2007). Clinical studies
show that the earliest and most consistently seen abnormality in Alzheimer‘s
disease is depressed cerebral glucose metabolism, which results in severe
atrophy in the brain (Silverman et al., 2001, Atamna and Frey Ii, 2007). Thus,
oxidative stress or altered redox status in favor of oxidants and decreased
bioenergetic capacity (production of ATP during oxidative phosphorylation) is
considered as a major component of aging and age-related diseases (Navarro
and Boveris, 2007, Rebrin et al., 2003, Butterfield et al., 1999, Calabrese et al.,
2006, Mattson and Magnus, 2006, Boveris and Navarro, 2008).

19

1.1.8 Nicotinamide nucleotide transhydrogenase (NNT)
NNT catalyzes the reversible transfer of electrons between NAD and NADP and
equilibrates the redox levels of NAD
+
and NADP
+
. In mammals NNT is an integral
protein of the mitochondrial inner membrane responsible for the transfer of
electrons from NADPH to NAD
+
and reduction of NADP
+
to NADPH (Hoek and
Rydström, 1988). NNT is responsible for reduction of ~50% of the NADP
+
at
expense of NADH. The NNT activity is driven by membrane potential and thus
lower mitochondrial membrane potential would affect the rate of NADPH
availability. Lower NADPH would affect the ability of the glutathione redox system
to function efficiently. Overall, NNT enables the integration of an energy-redox
axis within mitochondria (Fig. 4)

20

Figure 4. The mitochondrial energy–redox axis in cell function. The mitochondrial
(a) energy axis entails the entry of glycolytic substrates into the TCA cycle and
the generation of reducing equivalents (NADH, FPH
2
) flowing through the
mitochondrial respiratory chain. The mitochondrial (b) redox axis entails redox
indicators: glutathione (GSH/GSSG), thioredoxin (Trx(-SH)/Trx-SS), glutaredoxin
(Grx), peroxiredoxins (Prx). These systems depend solely on steady flux of
NADPH. Typically viewed as independent components, the mitochondrial
metabolic state and redox status can be viewed as concerted processes, linked
primarily through inter-convertible reducing equivalents pool (i.e.
NAD(P)
+
/NAD(P)H), catalyzed by the ΔΨ
m
NNT bound to the mitochondrial inner
membrane. Perturbations in either mitochondrial metabolism or redox pathways
modulate the rate of generation of mitochondrial metabolites (e.g., H
2
O
2
) that
results in domain-specific signaling achieved through redox-mediated post-
translational modifications of cytosolic targets (e.g., GAPDH).

21

1.1.9 Mitochondria isolation considerations
Isolated rat brain mitochondria are widely used in studying the role of oxidative
stress in neurodegenerative diseases and free radical biology. Differential
centrifugation and discontinuous percoll gradient are the commonly used
methods for isolating mitochondria. Both are density-based separation methods,
relying on centrifugal force for isolation and purification of mitochondria. The
main methodical difference between differential centrifugation and discontinuous
percoll gradient is the use of percoll gradient in discontinuous percoll gradient.
However, elimination of percoll tends to make the process rigorous as it requires
a series of buffer washes. Percoll gradient methodology increases the
mitochondrial purity but prolonged percoll exposure can compromise
mitochondrial integrity, and thus utmost care must be taken to finish the isolation
process as quickly as possible.
However, inherent in differential centrifugation and discontinuous percoll gradient
isolation process, are alterations in the mitochondrial redox status. Previous work
from our lab has been focused on characterizing the changes in the
mitochondrial redox status with respect to the glutathione pool. Substrate
supplementation of isolated mitochondria resulted in higher buffering capacity
against H
2
O
2
challenges, in part due to increased GSH levels. It was found that
that the different mitochondria isolation methods, i.e., differential centrifugation
and discontinuous percoll gradient, resulted in varied effect on the redox status of
22

mitochondria, with respect to the glutathione pool. More importantly, substrate
supplementation to isolated mitochondria resulted in higher buffering capacity
against H
2
O
2
challenges, in part due to increased GSH levels, which provided the
redox buffer. Because the changes in the glutathione pool have been previously
quantified, it would be imperative to monitor the changes in the pyridine
dinucleotide pool as these two pools form the core of redox buffer systems in the
cell and maximally influence the redox status. These changes have been
characterized using percoll gradient which yields a higher purity mitochondrial
preparation.

1.2 Methods
Materials
An HPLC ZORBAX C
18
analytical column (5uM, 4.5×250mm) was used (a guard
column from agilent was always used while analyzing biological samples).
Chemicals were from Sigma Chemicals Co (St Louis, MO). An agilent 1100
series HPLC system and a Hitachi Fluorescence Spectrophotometer were used
for all analyses.

23

1.2.1 Mitochondria isolation
Adult male Wistar rats (6 months old), housed under an automated light/dark (12
h: 12 h) cycle with food and water available ad libitum were fasted overnight. All
experimental procedures were subject to the approval of the Institutional Animal
Care and Use Committee (IACUC) of the University of Southern California, Los
Angeles, CA, U.S.A. Rat brain mitochondria were isolated by percoll gradient: rat
brains were excised, chopped into fine pieces, washed and homogenized in an
isolation buffer containing 250 mM Sucrose, 20 mM HEPES and 1 mM EDTA,
1mM EGTA, protease inhibitor, plus 0.05% (w/v) bovine serum albumin, pH 7.4.
The homogenate was centrifuged at 1330×g (5 min) to remove nuclei and cell
debris and the resulting supernatant was centrifuged at 21200×g (10 min). The
pellet was re-suspended in 15% percoll and was centrifuged 21000×g for 10 min.
The resulting loose pellet was layered onto a preformed discontinuous percoll
gradient and centrifuged at 31000×g for 10 min. Mitochondrial fractions were
collected and washed twice with isolation buffer followed by washing in BSA-free
isolation buffer. Mitochondrial protein concentration was determined using Bio-
Rad protein assay dye reagent (Bio-Rad, Hercules, CA).

24

1.2.2 Mitochondrial respiration
Mitochondrial oxygen consumption was measured using a Clark-type electrode.
100 µg of isolated mitochondria were placed in the respiration chamber at 37°C
in respiratorion buffer (0.22 M Mannitol, 0.05 M sucrose, 10 mM NaH
2
PO
4
, 20
mM MOPS at pH 7.4). After initial baseline recording for 3 min, mitochondria
were energized by the addition of glutamate/malate (5 mM) as substrates (state 4
respiration) followed by addition of 150uM ADP (state 3 respiration). The rate of
oxygen consumption was calculated based on the slope of the response of
isolated mitochondria to the successive administration of substrates. The
respiratory control ratio (RCR) was determined by dividing the rate of oxygen
consumption/min for state 3 (presence of ADP) by the rate of oxygen
consumption/min for state 4 respiration (Fig.7). Only coupled mitochondria with
RCR values between 4 and 6 were used for all experiments.

1.2.3 Membrane potential determination
Mitochondrial membrane potential (ΔΨ) was determined by measuring
Rhodamine 123 (Rh-123) fluorescence (λ
exc
= 503nm; λ
em
= 527nm) with a Perkin
Elmer LS 55 spectrofluorometer at 37°C. Initially, Rhodamine-123 calibration was
done by dissolving Rhodamine-123 in ethanol and assaying
spectrophotometrically at 507 nm (ε = 101 mM
-1
cm
-1
) at different concentrations.
For mitochondrial preparations, the ethanol concentration was kept below 0.2%
25

(v/v). The fluorescence of the media (0.22 M mannitol, 0.05 M sucrose, 10 mM
NaH
2
PO
4
, 20mM MOPS) containing 0.5 μM Rh-123 was determined before
addition of mitochondria. This measurement was used as an indication of the
total dye concentration (expressed in nmol/μl). Rat brain mitochondria (0.2–0.4
mg/ml) were added to the media in the presence of 5 mM glutamate/malate, 150
µM ADP, 2 μM rotenone, 1 μM antimycin, 5 μM carbonyl cyanide 3-
chlorophenylhydrazone [CCCP]. After the addition of CCCP, the baseline was
restored and the fluorescence of the suspension was measured. The contents of
the cuvette were subsequently centrifuged to pellet the mitochondria. The Rh-
123 concentration remaining in the media ([Rh-123]
out
, in nmol/μl) was calculated
from the fluorescence values of the supernatant. The initial total amount of Rh-
123 in the cuvette ([Rh-123]
total
) and the amount remaining in the media ([Rh-
123]
out
) were used to calculate by subtraction the total amount of Rh-123 taken
up by mitochondria ([Rh-123]
mit
, expressed in nmol/mg protein). The
concentration of free Rh-123 in the matrix ([Rh-123]
in
, in nmol/μl) was calculated
using the following equation, and the binding partition coefficients at 37°C
(K
i
= 26 μl/mg, K
o
= 120 μl/mg).
[Rh-123]
mit
= K
i
[Rh-123]
in
+ K
o
[R
h
-123]
out
Mitochondrial membrane potentials (negative inside) were calculated by the
electrochemical Nernst–Guggenheim equation:
ΔΨ = 59 log ([Rh-123]
in
/[Rh-123]
out
) (Scaduto Jr and Grotyohann, 1999).
A representative trace of a spectrofluorometer has been shown in Fig. 9.
26

1.2.4 High performance liquid chromatography
The HPLC method for detection of pyridine dinucleotides (Klaidman et al., 1995)
was modified in terms of
1. Column
2. Gradient
3. pH to elute samples
4. Extraction method
The method is very sensitive and is based on formation of cyanide adducts of
NAD
+
and NADP
+
as shown in Figure 5. The mobile phase consists of 0.2 M
ammonium acetate (Buffer A) at pH 5.5 and HPLC-grade methanol (Buffer B). A
gradient program with initial conditions as 100% Buffer A and 0% Buffer B was
set. From 0-4 min, 0 to 3% B and from 4-23 min, 3-6.8 % B, followed by washing
the column with 50% A and 50% B and re-equilibrated to initial conditions for
next run. NADH and NADPH standards (1mg/ml) were prepared fresh before
injection in HPLC grade water. NADP
+
, NAD
+
(10-200 ng) standards of 1mg/ml
were prepared and stored in the freezer at -70
°
C. Prior to the run, the standards
were taken out of the freezer, thawed and diluted in an aqueous solution
containing 0.2 M KCN, 0.06 M KOH, 1 mM bathophenanthroline, allowed to react
for 5 min, the pH was adjusted (pH= 8, by adding appropriate amounts of mobile
phase) and immediately injected onto the HPLC column. Standard curves for
27

NAD
+
, NADH, NADP
+
, NADPH were prepared by adding different concentrations
between 10-200 ng of each pyridine nucleotide. This was repeated several times
and the correlation coefficient was found to be 0.99 or greater each time. The
retention times were found to be rather stable but minute fluctuations were
observed over period of time and thus standards were always injected regularly
to confirm retention time. Quantitation of pyridine nucleotides was performed by
integrating the peaks and adding the cyanide adducts as detected by the
fluorescence spectrophotometer at excitation 330 nm and emission 460 nm.
Care was taken in handling the tissue to prevent artifactual oxidation of pyridine
nucleotides. Brain homogenate and isolated mitochondria were homogenized
immediately in a homogenizing solution containing 0.06 M KOH, 0.2 M KCN and
1 mM bathophenanthroline followed by chloroform extraction. If analysis of the
sample was to be performed later, it was flash frozen in liquid nitrogen and stored
at -70°C until ready for analysis. Chloroform extraction was carried out by
centrifugation at 14000 rpm in a microcentrifuge at 4
°
C; the resulting aqueous
supernatant with soluble pyridine nucleotides was collected and extracted thrice
to remove lipids and proteins. Finally it was filtered with 0.45 µmeters positively
charged filter (Pall Life Sciences) to remove RNA and DNA in microcentrifuge at
4
°
C. The filtrate was further diluted with 0.2 M ammonium acetate and injected on
a HPLC C
18
column.

28

1.3 Results
1.3.1 Shift of redox status of isolated mitochondria
The pyridine dinucleotide pool was found to have a more oxidized redox status in
isolated mitochondria. This was reflected in the increased NADP
+
(nmol/mg of
mitochondrial protein) and comparatively decreased NADPH (nmol/mg of
mitochondrial protein) as shown in the Fig. 8A, B. Similarly, NAD
+
(nmol/mg of
mitochondrial protein) levels were increased (Fig. 8C). The NAD pool of isolated
brain mitochondria is present in almost fully oxidized form (NAD
+
) and is almost
completely devoid of NADH (Fig. 8D), at least up to the lowest limits of
fluorescence detection. This could be accounted for the oxidation of NADH to
NAD
+
over the period of mitochondria isolation (~3h) and the inability to reduce
NAD
+
back to NADH, due to the absence of substrates like glutamate/malate. It
can also be seen that the NADPH/NADP ratio, which is a good indicator of redox
status, is comparatively very low (Fig. 8E); the NADH/NAD cannot be calculated,
for NADH was not detected (Figure 8F).

29

Figure 5. The chemical reaction of NAD(P) in presence of CN
-
and KOH to form
cyanide adducts with two isomers

Figure 6. A representative chromatogram of NAD(P)H standards with a label and
elution time of respective standards specified at the top of each peak

NADP-cn1
NADPH
NAD-cn1
NADH
NADP-cn2
NAD-cn2
8.397
9.404
11.738
13.184
15.467
21.408
30

Figure 7. A representative oxygen consumption trace used to measure
respiratory control ratio (Hansatech oxygen electrode). Only coupled
mitochondria were used for all experiments with RCR ranging between 4 and 6.

Fluorescence units
G/M ADP
31

Addition of glutamate/malate
Addition of 5mM glutamate/malate to isolated mitochondria for 5 min at 25°C
resulted in a complete change in the redox status of isolated mitochondria with
respect to the pyridine dinucleotides. Addition of glutamate/malate stimulates
state 4 respiration. The amount of NADP
+
almost halved as compared to isolated
mitochondria with no substrates (Fig. 8A). On the other hand, the levels of
NADPH were increased more than 2 fold (Fig. 8A). Similarly, the levels of NAD
+

almost halved and the levels of NADH increased significantly to 0.075 nmol/mg
mitochondrial protein (from non detectable levels in isolated mitochondria not
supplemented with glutamate/malate). Thus, the mitochondrial redox status with
respect to NADH/NAD and NADPH/NADP was more reduced when mitochondria
were supplemented with glutamate/malate. The increased reduction of NAD
+

generating NADH can be accounted for by the malate-aspartate shuttle and
increase in NADPH by NNT activity. To confirm this result, mitochondria were
isolated in buffer containing 5 mM glutamate/malate. The mitochondrial isolation
procedure was kept exactly the same as previous, except for adding 5 mM
glutamate/malate in isolation buffer. After isolation, the mitochondria were
extracted and analyzed by HPLC as described before. The amount of NADH
detected was quite similar to the amount detected when isolated mitochondria
were supplemented with glutamate/malate after isolation was complete (Results
not shown).
32

Addition of glutamate/malate + ADP
Addition of glutamate/malate + ADP stimulates state 3 respiration (active
respiration), wherein the molecular oxygen and electrons combine to yield water
and ATP is generated subsequently. When isolated mitochondria are
supplemented with 5 mM glutamate/malate for 2.5 min followed by 150 µM ADP
at 25°C, it results in a further change in redox status. The levels of NADP
+
further
dropped by around 0.05 nmol/mg mitochondrial protein as compared to
mitochondria supplemented with glutamate/malate (Fig. 8A). The levels of
NADPH are increased by around 0.01nmol/mg mitochondrial protein (Fig. 8B).
NAD
+
was further decreased by 0.025nmol/mg mitochondrial protein (Fig. 8D).
However, NADH levels did not increase by addition of glutamate/malate + ADP
and showed a minor decrease of ~0.01 nmol/mg. This could be explained by the
consumption of NADH in oxidative phosphorylation stimulated by active ‗state 3‘
respiration. The NADPH/NADP and NADH/NAD ratios were further increased
and it changed the redox status to more reduced as compared to isolated
mitochondria and glutamate/malate supplemented mitochondria (Fig. 8E, F).

33

Addition of complex I inhibitor
Rotenone was used as a positive control to confirm the above results. Rotenone
is widely used as a complex I (NADH dehydrogenase) inhibitor. It shuts off the
supply of electrons to the quinol (QH
2
)-cytochrome c oxidoreductase (Chance
and Hollunger, 1963, Barrientos and Moraes, 1999). As a result, electrons
derived from the NAD-linked dehydrogenases (in the form of NADH) are not
oxidized in the respiratory chain and result in greater accumulation of NADH.
Isolated mitochondria were supplemented with 5 mM glutamate/malate for 2.5
min, followed by addition of 2 µM rotenone for 2.5 min at 25°C. It did not result in
a significant difference in the levels of NADP
+
(Fig. 8A), but as expected, the
levels of NADPH increased by ~0.02 nmol/mg of mitochondrial protein (Fig. 8B).
Similarly, the levels of NAD
+
were almost unchanged, but the levels of NADH
increased by 0.025 nmol/mg mitochondrial protein (Fig. 8C, D) as compared to
mitochondria supplemented with glutamate/malate. Both, the NADH/NAD ratio
and NADPH/NADP ratio increased more than 1-fold as compared to
mitochondria supplemented with glutamate/malate.

34

Figure 8A
Figure 8. A) NADP
+
, B) NADPH, C) NAD
+
, D) NADH, E) NADPH/NADP, F)
NADH/NAD present in mitochodria after incubation with complex I substrates.
For G/M (State 4 respiration) the incubation time was 5 min at 25°C. For
G/M+ADP (State 3 respiration) and G/M+rotenone the incubation time was 2.5
min with G/M followed by addition of ADP/rotenone for 2.5 min. The values
represented for NADP
+
, NADPH, NAD
+
, NADH are calculated in terms of
nmol/mg of mitochondrial protein (n=3).

Figure 8B

35

Figure 8, continued

Figure 8C

Figure 8D

36

Figure 8, continued

Figure 8E

Figure 8F

37

Addition of mitochondria uncoupler (CCCP)
CCCP was used as a negative control to confirm the above results. CCCP
dissipates the proton gradient by transporting protons through the membranes
from the intermembrane space to the matrix. CCCP short-circuits the normal flow
of protons through the F
1
F
0
-ATPase and thus stimulates respiration and oxidizes
the pyridine dinucleotides (Nieminen et al., 1997). On adding 3 µM CCCP for 5
min at 25°C, the levels of NADP
+
increased by 0.2 nmol/mg mitochondrial
protein and the levels of NADPH decreased by 0.015 nmol/mg mitochondrial
protein with respect to mitochondria supplemented with glutamate/malate. There
was more than one-fold increase in the levels of NADP
+
and a one-fold decrease
in the levels of NADPH with respect to mitochondria supplemented with
glutamate/malate and further inhibited by rotenone (Fig. 8A,B). As expected, the
NAD
+
levels increased by 0.075 nmol/mg of mitochondrial protein and NADH
levels decreased by 0.06 nmol/mg of mitochondrial protein with respect to
mitochondria supplemented with glutamate/malate. There was more than three-
fold increase in levels of NADP
+
and a 5-fold decrease in the levels of NADPH
with respect to mitochondria supplemented with glutamate/malate and further
inhibited by rotenone (Fig. 8C, D). The NADPH/NADP ratio almost halved, while
the NADH/NAD decreased three-fold with respect to mitochondria supplemented
with glutamate/malate. The NADPH/NADP ratio decreased around three-fold,
while NADH/NAD ratio decreased around ten-fold with respect to mitochondria
38

supplemented with glutamate/malate and further inhibited by rotenone (Fig. 8E,
F). All the results have been summarized in table 1.

NADP
+

NADPH

NAD
+

NADH

NADPH/NAD

NADH/NAD
+

(nmol/mg of mitochondrial protein)
Control
0.765 0.013 0.16 0 0.017 0
G/M
0.380 0.041 0.090 0.075 0.109 0.862
G/M + ADP
0.298 0.050 0.058 0.067 0.169 1.119
G/M +
Rotenone
0.282 0.067 0.049 0.103 0.239 2.07
G/M + CCCP
0.53 0.028 0.16 0.011 0.070 0.103

Table 1. Levels of NAD
+
, NADH, NADP
+
, NADPH (Values are represented in
nmol/mg mitochondrial protein); NADPH/ NADP
+
and NADH/ NAD
+
.

39

1.3.2 Membrane potential and pyridine dinucleotide redox status
The mitochondrial membrane potential is the driving force for mitochondrial
bioenergetics, whereas NADH/NAD and NADPH/NADP ratios indicate the
bioenergetic status in the cell. Thus, it would be essential to monitor the changes
in mitochondrial membrane potential with respect to the changes taking place in
the pyridine dinucleotide pool and a link between mitochondrial membrane
potential and NADH/NAD, NADPH/NADP is warranted. Initially the membrane
potential was determined with respect to the different substrates, respiratory
inhibitor and uncoupler. As expected, on addition of glutamate/malate (state 4
respiration was initiated) the membrane potential increased to around ΔΨ 155
mV. On stimulation of state III respiration (glutamate/malate+ADP), the
membrane potential was around ΔΨ 135 mV. Mitochondrial membrane potential
decreased on adding rotenone and further dipped as expected to around 90mV
after it was uncoupled by adding CCCP: In Fig.10A, B the net pyridine
dinucleotide redox state is plotted as a function of ΔΨ.

40

Figure 9. A representative spectrofluorometric trace of Rhodamine-123 used to
calculate membrane potential. The different time points indicate the addition of
mitochondria, substrates (G/M,ADP), respiratory inhibitor (rotenone) and
uncouplers (CCCP) as indicated in the figure. (λ
exc
= 503nm and λ
em
= 527nm)

41

Figure 10. Representation of the calculated mitochondrial membrane potential
using Rhodamine-123 as described in figure 9. The values of membrane
potential are calculated in terms of ΔΨ mV. (n=3)

42

Figure 11A.

Figure 11B.
Figure 11 A) NADH/NAD vs membrane potential (ΔΨ mV).
Figure 11B) NADPH/NADP vs membrane potential (ΔΨ mV).

43

1.4 Discussion
The mitochondrial redox status is changed during mitochondria isolation, but the
change has not been quantified. Previously, our lab has characterized this
change with respect to the glutathione pool. It is also well known that oxidative
cellular redox status leads to glutathionylation of number of important
mitochondrial proteins like complex I and complex III. Because, the reduction of
glutathione pool is mainly fueled by pyridine dinucleotides it was important to
quantify and characterize the changes in pyridine dinucleotides in isolated
mitochondria to understand the changes in cellular redox status during the
process of mitochondrial isolation. My present work has characterized the
changes in pyridine dinucleotide pool after mitochondria isolation and the effect
of energy-linked substrates in modulating this redox status. The above results
indicate that the isolated mitochondrial redox status is more oxidized. No
quantification for the endogenous amounts and ratios of pyridine dinucleotides in
mitochondria has been documented, and thus it would be difficult to quantify the
net changes taking place in isolated mitochondrial systems as compared to
endogenous mitochondria.
The following findings suggest that the redox status of isolated mitochondria is
changed drastically:

44

 No NADH could be detected in isolated mitochondria. The addition
glutamate/malate resulted in NADH detection. Endogenously, mitochondria
require NADH for a number of metabolic actions and because isolated
mitochondria lack in NADH it would be warranted to say that the NADH/NAD
redox is clearly affected in isolated mitochondrial systems.
 The NADPH/NADP is more oxidized in isolated mitochondria as compared
to mitochondria supplemented with either glutamate/malate or mitochondria
supplemented with glutamate/malate + ADP.
 Isolated mitochondrial are in state 1, wherein the membrane potential is
far from the membrane potential of actively respiring mitochondria.
 Previous data from our lab suggests a shift in the GSH/GSSG redox
status to being more oxidized in isolated mitochondria as compared to
mitochondria supplemented with energy-linked substrates. More importantly,
glutathione-protein adducts of succinyl-CoA: 3-oxoacid CoA Transferase which
plays a role in ketone body metabolism) and ATP Synthase F
1α
subunit isoform 1
were identified in isolated mitochondria. These glutathione-protein adducts were
substantially decreased in mitochondrial supplemented with glutamate/malate+
ADP.
Overall, changes taking place in isolated mitochondria suggest a change in its
redox status, which in turn could lead to a number of changes in the
45

mitochondrial proteins. Some of the most consequential effects could be
glutathionylation of important proteins and cystine bond formation in a number of
proteins. Because, isolated mitochondrial preparations are widely used in
studying mitochondrial biology, mitochondria related disorders, free radical
biology and bioenergetics this study leads to better understanding of the rapid
redox changes in isolated mitochondria.

46

Chapter 2
―Splicing up‖ drug discovery. Cell-Based Expression and Screening of
genetically-Encoded Libraries of Backbone Cyclized Polypeptides

2.1 Introduction
A significant number of natural products with wide range of pharmacological
activities are derived from cyclic polypeptides. In fact, peptide cyclization is
widely used in medicinal chemistry to improve the biochemical and biophysical
properties of peptide-based drug candidates (Hruby and Al-Obeidi, 1990, Rizo
and Gierasch, 1992). Cyclization rigidifies the polypeptide backbone structure,
thereby minimizing the entropic cost of receptor binding and also improving the
stability of the topologically constrained polypeptide. Among the different
approaches used to cyclize polypeptides, backbone or head-to-tail cyclization
remains one of the most extensively used to introduce structural constraints into
biologically active peptides.
Despite the fact that the chemical synthesis of cyclic peptides has been well
explored and a number different approaches involving solid-phase or liquid-
phase existed (Camarero and Muir, 1997, Zhang and Tam, 1997, Camarero et
al., 1998b, Shao et al., 1998, Camarero et al., 1998a), recent developments in
the fields of molecular biology and protein engineering have now made possible
the biosynthesis of cyclic peptides. This progress has been made mainly in two
47

areas, non-ribosomal peptide synthesis (Trauger et al., 2000, Kohli et al., 2002,
Walsh, 2004) and expressed protein ligation/protein trans-splicing (Camarero
and Muir, 1999a, Scott et al., 1999, Evans et al., 1999a, Camarero et al., 2001a,
Iwai et al., 2001, Abel-Santos et al., 2003). The former strategy involves the use
of genetically engineered non-ribosomal peptide synthetases and is reminiscent
of more established technologies that yield novel polyketides. The later strategy
relies on the heterologous expression of recombinant proteins fused to modified
intein protein splicing/trans-splicing units (Noren et al., 2000).
The biosynthesis of cyclic polypeptides offers many advantages over purely
synthetic methods. Using the tools of molecular biology, large combinatorial
libraries of cyclic peptides, may be generated and screened in vivo. A typical
chemical synthesis may generate 10
4
different molecules. It is not uncommon for
a recombinant library to contain as many as 10
9
members. The molecular
diversity generated by this approach is analogous to phage-display technology.
Moreover, this approach takes advantage of the enhanced pharmacological
properties of backbone-cyclized peptides as opposed to linear peptides or
disulfide-stabilized polypeptides. Also, the approach differs from phage-display
in that the backbone-cyclized polypeptides are not fused to or displayed by any
viral particle or protein, but remain on the inside of the living cell where they can
be further screened for biological activity. The complex cellular cytoplasm
48

provides the appropriate environment to address the physiological relevance of
potential leads.
Protein trans-splicing had been successfully used by Benkovic and co-workers
to generate backbone cyclized or polypeptides in vivo (Scott et al., 1999). In this
approach, the peptide to be cyclized was nested between the two split intein
fragments of the naturally occurring Ssp DnaE split intein (Wu et al., 1998)
(usually referred as N- and C-inteins) in such way that the N-terminus of the
peptide template is fused to C-intein fragment and vice versa. Protein splicing of
this chimeric protein lead to the formation of the desired cyclic peptide inside E.
coli cells. A potential limitation of this approach, however, was the requirement
for specific N- and C-extein residues at the intein junction sites (Evans et al.,
2000). These amino acids were necessary for efficient protein splicing to occur,
which restricts the sequence diversity within the sequence of the cyclic peptide.
An attractive alternative approach to the biosynthesis of circular polypeptides
was the use of an intramolecular version of Native Chemical Ligation reaction
(Dawson et al., 1994, Dawson and Kent, 2000, Tam et al., 1995). The present
paper reviews the use of these processes for the biosynthesis of circular
polypeptides (i.e. peptides and proteins) and it will discuss also the potential of
this method for the biosynthesis of cyclic polypeptide libraries inside living cells
as a complementary source for the rapid discovery of new therapeutics.

49

2.2 Native Chemical Ligation
Native Chemical Ligation (NCL) is an exquisitely specific ligation reaction that
has been extensively used for the total synthesis, semi-synthesis and
engineering of different proteins (Evans and Xu, 1999, Camarero and Muir,
1999b, Dawson and Kent, 2000, Muir, 2003). In this reaction, two fully
unprotected polypeptides, one containing a C-terminal α-thioester group and the
other a N-terminal Cys residue, react chemoselectively under neutral aqueous
conditions with the formation of a native peptide bond (Fig. 12A). The initial step
in this ligation involves the formation of a thioester-linked intermediate, which is
generated by a trans-thioesterification reaction involving the α-thioester moiety of
one fragment and the N-terminal Cys thiol group of the other fragment. This
intermediate then spontaneously rearranges to produce a peptide bond at the
ligation site. This type of thioester-based chemistry was first discovered by
Wieland in 1950‘s for the synthesis of small Cys-containing peptides (Wieland et
al., 1953, Wieland, 1988).
It is well established that when these two reactive groups, i.e. the C-terminal α-
thioester group and the N-terminal Cys residue, are located in the same synthetic
precursor, the chemical ligation proceeds in an intramolecular fashion thus
resulting in the efficient formation of a circular polypeptide (Fig. 12B). This
reaction has been successfully employed for the chemical synthesis of cyclic
50

peptides and small protein domains (Camarero and Muir, 1997, Shao et al.,
1998, Camarero et al., 1998a, Camarero et al., 1998b).

2.3 Expressed Protein Ligation
The discovery of protein splicing and advances in protein engineering have made
also possible the introduction of the C-terminal α-thioester group and N-terminal
Cys residue into recombinant proteins. These important developments made
possible the use of NCL between synthetic and/or recombinant fragments. This
technology, called Expressed Protein Ligation (EPL), allows access to a
multitude of chemically engineered recombinant proteins including biosynthetic
circular polypeptides (Muir, 2003).
51

Figure 12. Backbone cyclization of polypeptides using native chemical Ligation.
A. Principle of Native Chemical Ligation (NCL). B. Intramolecular NLC leads to
the formation of a backbone cyclized polypeptide.

52

Figure 13. Biosynthesis of recombinant polypeptide α-thioesters. A. Scheme
representing the proposed canonical mechanism for protein splicing mediated by
a Cys-intein. B. Expression and purification of recombinant polypeptide thioesters
using a modified intein fusion protein. In the modified intein (represented with an
asterisk) the last Asn residue of the intein has been mutated to Ala to prevent C-
terminal cleavage and splicing. This mutation allows trapping a thioester
intermediate that can be cleaved with a thiol to provide the corresponding
thioester function.

53

2.3.1 Recombinant Polypeptide α-thioesters
Recombinant protein α-thioesters can be obtained by using engineered inteins
(Muir et al., 1998, Severinov and Muir, 1998, Evans et al., 1998, Camarero and
Muir, 1999b). Inteins are self-processing domains which mediate the naturally
occurring process called protein splicing (Xu and Perler, 1996) (Fig. 13). Protein
splicing is a cellular processing event that occurs post-translationally at the
polypeptide level. In this multi-step process an internal polypeptide fragment,
called intein, is self-excised from a precursor protein and in the process ligates
the flanking protein sequences (N- and C-exteins) to give a different protein. The
current understanding of the mechanism is summarized in Figure 13A and
involves the formation of thioester/ester intermediates (Xu and Perler, 1996). The
first step in the splicing process involves an N→S or N→O acyl shift in which the
N-extein is transferred to the thiol/alcohol group of the first residue of the intein.
After the initial N→(S/O) acyl shift, a trans-esterification step occurs in which the
N-extein is transferred to the side-chain of a second conserved Cys, Ser or Thr
residue, this time located at the junction between the intein and the C-extein. The
amide bond at this junction is then broken as a result of succinimide formation
involving a conserved Asn residue within the intein. In the final step of the
process, a peptide bond is formed between the N-extein and C-extein following
an (S/O)→N acyl shift (similar to the last step of Native Chemical Ligation, see
54

Fig. 12A). Mutation of the conserved Asn residue within the intein to Ala blocks
the splicing process in midstream thus resulting in the formation of an α-thioester
linkage between N-extein and the intein (Xu and Perler, 1996) (Fig. 13B). This
thioester bond can be cleaved using an appropriate thiol through a trans-
thioesterfication step to give the corresponding recombinant polypeptide α -
thioester. The IMPACT expression system, commercially available from New
England Biolabs (Chong et al., 1997, Chong et al., 1998), allows the generation
of recombinant α -thioester proteins by making use of such modified inteins in
conjunction with a chitin binding domain (CBD) for easy purification by affinity
chromatography (see Fig. 13B).

2.3.2 Recombinant N-terminal Cys-containing polypeptides
The introduction of N-terminal Cys residues into expressed proteins can be
readily accomplished by cleaving (by proteolysis or auto-proteolysis) the
appropriate fusion proteins. The simplest way to generate a recombinant
polypeptide containing a N-terminal Cys residue is to introduce a Cys
downstream to the initiating Met residue. Once the translation step is completed,
the endogeneous methionyl aminopeptidases (MAP) removes the Met residue,
thereby generating in vivo a N-terminal Cys residue (Hirel et al., 1989, Dwyer et
al., 2000, Iwai and Pluckthum, 1999, Camarero et al., 2001a, Cotton et al., 1999).
55

Other approaches involve the use of exogenous proteases. Verdine and co-
workers added a Factor Xa recognition sequence immediately in front of the N-
terminal Cys residue of the protein of interest (Erlandson et al., 1996). After
purification, the fusion protein was treated with the protease Factor Xa which
generated the corresponding N-terminal Cys protein. Tolbert and Wong had also
showed that the cysteine protease from tobacco etch virus (TEV) can also be
used for the same purpose (Tolbert and Wong, 2002). This protease is highly
specific and it can be overexpressed in E. coli. Other proteases that cleave at the
C-terminal side of their recognition site, like enterokinase and ubiquitin C-terminal
hydrolase, could be also used for the generation of N-terminal Cys residues.
Protein splicing can also be engineered to produce recombinant N-terminal Cys-
containing polypeptides. Several inteins have been already mutated in such a
way that cleavage at the C-terminal splice junction (i.e. between the intein and
the C-extein, see Fig. 13B) can be accomplished in a pH- and temperature-
dependent fashion (Evans et al., 1999b, Southworth et al., 1999, Mathys et al.,
1999).
Proteins with N-terminal Cys can be also obtained by the convenient modification
of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Liu et al
(Liu et al., 2008) successfully generated the Csk and Abl tyrosine kinase
domains with N-terminal Cys using this method.
56

More recently, Hauser et al had used the N-terminal pelB leader sequence to
direct newly synthesized fusion proteins to the E. coli periplasmic space where
the corresponding endogenous leader peptidases (Dalbey et al., 1997, Paetzel et
al., 2002) can generate the desired N-terminal cysteine-containing protein
fragment (Hauser and Ryan, 2007).

2.4 Biosynthesis of backbone cyclized peptides using Expressed Protein
Ligation
The approach employed for the biosynthesis of backbone cyclized polypeptides
using EPL is depicted in Figure 14. The target polypeptide to be cyclized was
fused at the N-terminus with a peptide leading sequence immediately followed by
a Cys residue, and at the C-terminus with an engineered intein. The N-terminal
leading sequence can be cleaved in vitro or in vivo by a proteolytic or self-
proteolytic event thereby generating the required N-terminal Cys residue. This
Cys residue then reacts in an intramolecular fashion with the α-thioester
generated by the engineered intein at the C-terminus thus providing a
recombinantly generated backbone cyclized polypeptide. This approach had
been used for the in vitro and in vivo biosynthesis of different backbone cyclized
polypeptides.
57

The demonstration of this biosynthetic cyclization strategy was first reported in
vitro by Camarero and Muir in 1999 using the N-terminal SH3 domain of the c-
Crk protein as model protein (Camarero and Muir, 1999a). In this work, the SH3
domain was fused to a modified VMA intein at the C-terminus and to the
MIEGRC motif (which contains a Factor Xa proteolysis site) at the N-terminus.
After expression in E. coli and purification, the intein fusion protein was treated
with Factor Xa protease. This proteolytic step afforded a N-terminal Cys-
containing SH3-intein fusion protein which spontaneously reacted in an
intramolecular fashion to yield the corresponding cyclized SH3 domain. The
cyclization process was extremely clean and fast, and the resulting cyclic SH3
protein domain was fully active (Camarero et al., 2001b). Intriguingly, this
intramolecular process did not require the presence of a thiol cofactor [absolutely
necessary to facilitate intermolecular ligation reactions (Camarero and Muir,
1999b)]. This interesting result was explained on basis of the close proximity of
both reacting groups in the folded state of the SH3 domain (the N- and C-termini
of the natively folded SH3 are located within 6Å), which was able to increase the
local concentration of both reacting groups. This effect has been already reported
in the cyclization of different small protein domains (Camarero et al., 1998b,
Camarero et al., 1998a).
Iwai and Pluckthum had also reported the biosynthesis of a cyclized version of
the β-lactamase protein using a similar approach (Iwai and Pluckthum, 1999). In
58

their case, the N-terminal Cys residue was generated in vivo by removal of the
initiating Met residue by an endogenous Met amino peptidase. After purification
of the N-terminal Cys-containing intein fusion protein at pH 8.0, the cyclization
was triggered by addition of a thiol cofactor at pH 5.0. The resulting cyclized
protein was found to be more stable against irreversible denaturation upon
heating than the linear form.
Based on the high efficiency observed during the in vitro cyclization of the SH3
domain (Camarero and Muir, 1999a, Camarero et al., 2001b), Camarero and
Muir used a similar approach to test the possibility of carrying out the cyclization
of the SH3 domain inside living cells. For this purpose, the Factor Xa recognition
leading sequence in the SH3-VMA intein fusion protein was replaced by a Met
residue. During the expression of the resulting fusion protein in E. coli cells, the
Met residue was efficiently removed by an endogenous Met aminopeptidase
(Hirel et al., 1989). This in vivo proteolytic event unmasked the N-terminal Cys
residue which then reacted in an intramolecular fashion with the α-thioester
group induced by the C-terminal engineered VMA intein (Camarero et al.,
2001a). Analysis by SDS-PAGE showed that most of the SH3-intein fusion
protein (>90%) was cleaved in vivo. Remarkably, when the entire soluble cell
fraction was analyzed by reverse-phase HPLC, the expected cyclic SH3 protein
and the cleaved intein were found to be the major components in the mixture. It
is worth noting that no linear SH3 domain was found in the cellular mixture,
59

suggesting that in vivo hydrolysis of the α-thioester linkage present in the
precursor protein was minimal. This work demonstrated the first example of a
polypeptide chemical ligation reaction performed in the complex cytoplasmic
environment of a living cell, and represents an important milestone in current
efforts to generate and screen libraries of cyclic polypeptides inside living cells.
More recently, we have applied the same approach for the biosynthesis of
cyclotides inside living bacterial cells (Fig. 14) (Camarero et al., 2007). Cyclotides
are small globular microproteins with a unique head-to-tail cyclized backbone,
which is stabilized by three disulfide bonds (Craik et al., 1999). The number and
positions of cysteine residues are conserved throughout the family, forming the
cyclic cystine-knot motif (CCK) (Craik et al., 1999) that acts as a highly stable
and versatile scaffold on which hyper-variable loops are arranged. This CCK
framework gives the cyclotides exceptional resistance to thermal and chemical
denaturation and enzymatic degradation. Moreover, several cyclotides had been
found able to cross eukaryotic cell membranes (Greenwood et al., 2007). All
these unique properties make them ideal candidates for the development of
peptide-based drugs (Craik et al., 2002). Our group has recently developed and
successfully used a bio-mimetic approach for the biosynthesis of several folded
cyclotides inside cells by making use of intramolecular EPL in combination with
modified protein splicing units (Kimura et al., 2006, Camarero et al., 2007) (Fig.
14). Our important finding makes possible the generation of large libraries of
60

cyclotides (≈10
9
) for high throughput cell-based screening and selection of
specific sequences able to recognize particular biomolecular targets (Camarero
et al., 2007, Kimura et al., 2006).

Figure 14. Biosynthetic approach for the in vivo production of cyclotides inside
live E. coli cells. Backbone cyclization of the linear cyclotide precursor is
mediated by a modified protein splicing unit or intein. The cyclized product then
folds spontaneously in the bacterial cytoplasm.

61

2.5 Biosynthesis of backbone cyclized peptides using protein trans-
splicing
An alternative approach to EPL for the cell-based biosynthesis and screening of
backbone cyclized polypeptides in vivo is the use of protein trans-splicing (Fig.
15). This approach was first reported by Benkovic and co-workers and makes
use of Ssp DnaE split intein. Protein trans-splicing is a naturally occurring post-
translational modification similar to protein splicing with the difference that the
intein self-processing domain is split in two fragments (called N-intein and C-
intein, respectively, Fig. 15A) (Saleh and Perler, 2006, Xu and Evans, 2005).
These two intein fragments are inactive individually, however, they can bind each
other with high specificity under appropriate conditions to form a functional
protein splicing domain (Fig. 15A). By rearranging the order of the elements of
the intein (i.e. N-intein and C-intein, see Fig. 15) the result of the splicing
produces a backbone cyclized polypeptide (Scott et al., 1999, Naumann et al.,
2005). This methodology, also denominated SICLOPPS (split intein circular
ligation of proteins and peptides) has been used to generate several natural
cyclic peptides (Abel-Santos et al., 2003) as well as large genetically-encoded
libraries of small cyclic peptides (Tavassoli and Benkovic, 2007).
It should be noted, however, that these systems require the presence of specific
amino acid residues at both intein-extein junctions for efficient protein splicing to
occur (Scott et al., 1999, Scott et al., 2001, Abel-Santos et al., 2003). In contrast
62

to protein trans-splicing, the only absolute sequence requirements for native
chemical ligation is the presence of a N-terminal cysteine. Model studies had
also shown that all 20 natural amino acids located at the C-terminus of a
polypeptide α-thioester can support ligation (Hackeng et al., 1999). Moreover, the
engineered inteins used to generate recombinant polypeptide α-thioesters are
compatible with most amino acids upstream of the cleavage site (Noren et al.,
2000). Thus, our native chemical ligation approach may be quite general with
respect to the sequence of the linear peptide precursor and is potentially a
powerful tool to generate a diverse array of backbone cyclized polypeptides in
vivo.

63

Figure 15. Production of backbone cyclized polypeptides using protein trans-
splicing. A. Scheme representing the proposed canonical mechanism for protein
trans-splicing mediated by a split Cys-intein. B. Cyclization of polypeptides using
protein trans-splicing. To facilitate cyclization, the N-intein and C-intein moieties
are fused the C- and N-terminus of the polypeptide to be cyclized, respectively

64

2.6 Protease catalyzed protein splicing for the biosynthesis of backbone
cyclized peptides
Protease catalyzed protein splicing, also known as transpeptidation, was
employed in prokaryotes to attach proteins to peptidoglycan in the cell-wall
envelope (Marraffini et al., 2006). For example, sortases are transpeptidase
enzymes found in most Gram-positive bacteria that are specialized in this task.
Among several isomorphs and homologues discovered so far, the
Staphyloccocus aureus Sortase A (SrtA) (Mazmanian et al., 1999) had been
widely employed for protein engineering (Mao et al., 2004, Tsukiji and
Nagamune, 2009). SrtA recognizes substrates that contain an LPXTG sequence
and catalyzes the cleavage of the amide bond between the Thr and Gly by
means of an active-site cysteine (Cys184) residue (Fig. 16A). This process
generates a covalent acyl-enzyme intermediate. The activated carboxyl group of
the Thr residue then undergoes nucleophilic attack by an amino group of
oligoglycine substrates (in S. aureus, a pentaglycine Gly
5
cross-bridge on branch
lipid II precursor) producing ligated products. In the absence of oligoglycine
nucelophiles, the acyl-intermediate is hydrolyzed by a water molecule.
An intramolecular version of the sortase-mediated ligation can be used for the
generation of backbone cyclized polypeptides (Fig. 16B). Bouder and co-workers
reported the SrtA-mediated cyclization of EGFP containing the (Gly)
3
and LPTEG
65

peptides at the N- and C-terminus, respectively (Parthasarathy et al., 2007).
SML-based protein cyclization had also been reported by other groups for the
cyclization of dyhydrolate reductase (DFHR) and peckstrin homology (PH)
domain (Tsukiji and Nagamune, 2009). Protease-catalized protein splicing have
been also recently found in animals and plants (Saska and Craik, 2008). For
example, recent studies have shown that the biosynthesis of cyclotides in plants
(see above) involves an asparaginyl endopeptidase (AEP) catalyzed
transpeptidation event (Gillon et al., 2008). Cyclotides are naturally processed
from precursor proteins that have both N- and C-terminal pro-regions. Mutation of
a highly conserved Asn residue at the C terminus of the cyclotide domain
eliminates cyclization, as does deletion of the C-terminal pro-region following this
residue (Gillon et al., 2008). Together, these findings indicate that an
endopeptidase with specificity for asparagine residues is likely to be involved in
the cyclization process (Saska et al., 2007). The protease that cleaves the N-
terminal pro-region, however, has yet to be identified. The identification of all the
proteins required for the cyclization of cyclotides could provide an alternative
method to intramolecular EPL for the biosynthesis of genetically-encoded
libraries of cyclotides inside living cells. However, it should be emphasized the
simplicity of the EPL-based biomimetic approach, which only uses one single
self-processing protein that can easily expressed in any type of cell for rapid
screening of genetically-encoded libraries.
66

Figure 16. Biosynthesis of cyclic polypeptides using intramolecular sortase-
mediated ligation. A. Principle of sortase-mediated ligation. Sortase A first
recognizes an LPXTG sequence within polypeptide 1 and cleaves the amide
bond between the Thr and the Gly with an active-site Cys184, generating a
covalent acyl-enzyme intermediate. The thioester intermediate is then attacked
by an amino group of the oligo Gly-containing polypeptide 2, which allows the
ligation of the two polypeptides by a native peptide bond. B. Polypeptide
cyclization of a dual tagged polypeptide by an intra-molecular transpeptidation
reaction.
67

2.7 Cell-based screening of genetically-encoded libraries of backbone
cyclic polypeptides
The ability to create cyclic polypeptides in vivo opens up the possibility of
generating large libraries of cyclic polypeptides. Using the tools of molecular
biology, genetically encoded libraries of cyclic polypeptides containing billions of
members can be readily generated. This tremendous molecular diversity forms
the basis for selection strategies that model natural evolutionary processes. Also,
since the cyclic polypeptides are generated inside living cells, these libraries can
be directly screened for their ability to attenuate or inhibit cellular processes.
In contrast to phage display, where the screening takes place in vitro, screening
that takes place in the cytoplasm offers the advantages conferred by a native
physiological environment where diverse biochemical events may be examined.
In addition, problems resulting from the presence of a fusion tag (in this case the
viral particle), in a phenomenon known as template effect, may be circumvented.

68

Figure 17. Schematic representation of the putative mechanism of protease-
catalysed cyclotide cyclization. Cyclotides are a large family of plant defence
proteins characterized by a cystine knot and cyclic backbone. Their prototypic
linear precursor protein (top) comprises an endoplasmic reticulum (ER)-targeting
sequence (dark blue), a pro-region (purple), an N-terminal repeat region (Ntr;
green), a cyclotide domain (light grey) and a C-terminal tail (red). It features also
a conserved asparagine (yellow) at the C-terminal cleavage point of the cyclotide
domain. The precursor is processed in the ER and vacuole: disulfide bonds are
formed (yellow) and a range of unidentified proteases (brown) trim the precursor.
In the final stage the active-site cysteine of an AEP (yellow) displaces the C-
terminal tail to form an enzyme-acyl intermediate (boxed). This intermediate is
then attacked by the cyclotide N-terminal glycine to form the mature cyclic
peptide

69

Backbone cyclized polypeptides are relatively more stable and more resistant to
cellular catabolism than linear polypeptides or disulfide-based cyclic
polypeptides. Naturally occurring cyclic peptides often exhibit diverse therapeutic
activities ranging from immunosuppression to antimicrobial activity. The stability
of backbone cyclized polypeptides that display certain pharmacological
properties suggests that they may be suitable scaffolds on which to graft the
molecular diversity of an intracellular library (Craik et al., 2002, Scott et al.,
2001).
A number of advances in vivo library generation and screening have recently
been made. Scott and Benkovic used protein trans-splicing to generate the cyclic
peptide Pseudostellarin F, an eight amino acid circular peptide with tyrosinase
inhibitory activity (Scott et al., 1999). The in vivo biosynthesized Pseudostellarin
F was fully active and successfully screened in vivo for its tyrosinase inhibitory
activity. More recently, cyclic peptide libraries based on the Pseudostellarin F
scaffold demonstrated the structural requirements for this system. Apparently,
several amino acids positions near the intein-extein junction are critical for
expression and cyclization and the authors estimated that 70% of their library
produced cyclic products.
Payan and coworkers also used a similar protein trans-splicing approach to
generate random cyclic peptides in the cytoplasm of human cells using a
retroviral expression vector (Kinsella et al., 2002). Screening of the library for
70

modulation of the IL-4 signaling pathway, led to the identification of several cyclic
peptides that selectively inhibit the ɛ promoter activity. The library was based
upon a five amino acid coding strategy, and the potential complexity of the library
was about 160,000 members at the amino acids level. Of the 565 clones tested,
twenty-three hits were identified. These small circular peptides are potential
therapeutic agents against allergy and asthma and may serve in the future as
leads for the development of more potent compounds. These results
demonstrate an efficient functional screen for cyclic peptides in vivo in
mammalian cells.
More recently, Cheng et al (Cheng et al., 2007) have also used protein trans-
splicing to select backbone cyclized peptides able to inhibit the bacterial ClpXP
protease from genetically encoded of octapeptides, where only five residues
were randomized. Screening of potential inhibitors was performed in E. coli cells
using fluorescence activated cell sorting in combination with a genetically
fluorescent reporter. The selected inhibitors had little shared sequence similarity
and were able to interfere with unexpected steps in the ClpXP mechanism in
vitro. One of the selected cyclic peptides showed antibacterial activity against
Caulobacter crescentus, a model organism that requires ClpXP activity for
viability.
Tavassoli et al (Tavassoli et al., 2008) have also recently used protein trans-
splicing for the screening and selection of genetically-encoded libraries of
71

cyclized peptides able to interfere with the Gag-TSG101 interaction, which is
required for the release of HIV particles from virus-infected cells. In this case the
peptide library (composed of ≈ 10
6
different octamers) was screened in E. coli
cells using a reverse bacterial two-hybrid system (RTHS) (Tavassoli and
Benkovic, 2005, Horswill et al., 2004). This bacterial RTHS system uses chimeric
repressor fusion proteins and promoter sequences to link the disruption of
targeted fusion protein heterodimers to the expression of several reported genes
that allows the selection of peptide inhibitors. This approach yielded several
cyclic octapeptides that when linked to the cell penetrating peptide (CPP) TAT
were able to inhibit the production of virus-like particles in human cells with a
reported IC
50
of 7 µM.

2.8 Conclusions and Remarks
The ability to biosynthesize backbone cyclic peptides using EPL, protein trans-
splicing or SPL has important implications for drug-development efforts. The
capability to screen for biochemical events in an environment as complex as the
cell‘s interior will result in valuable and unique information about potential leads
identified by this method. Indeed, peptide-based libraries have been already
shown to be effective in producing drug candidates in bacterial as well as
72

mammalian systems (Scott et al., 1999, Kinsella et al., 2002, Cheng et al., 2007,
Tavassoli et al., 2008).
In summary, we have reviewed several approaches as well as recent
developments on the use of different types of protein splicing for the biosynthesis
of circular polypeptides. Protein trans-splicing has revealed itself as a powerful
tool for the biosynthesis of circular polypeptides that include small peptides and
large proteins (Scott et al., 1999, Evans et al., 2000, Iwai et al., 2001, Abel-
Santos et al., 2003, Tavassoli and Benkovic, 2007). It also has been shown that
this approach can be used for the generation of large libraries of circular
polypeptide inside living cells where they can be directly screened for biological
activity (Abel-Santos et al., 2003, Kinsella et al., 2002, Cheng et al., 2007,
Tavassoli et al., 2008). However, it has been shown that specific residues of the
native N-extein and C-extein are required for efficient protein trans-splicing to
occur with the naturally split DnaE intein (Evans et al., 2000). It is conceivable,
therefore, that this could restrict or bias the sequence diversity in the
corresponding circular peptide libraries generated by this method. Similar
sequence requirements are also found in Sortase-mediated intramolecular
ligations, where 9 extra residues are required for the Sortase-catalyzed
cyclization to take place.
Intramolecular EPL, on the other hand, had also been used successfully for the
73

in vitro and in vivo biosynthesis of cyclic polypeptides (Camarero and Muir,
1999a, Iwai and Pluckthum, 1999, Evans et al., 1999a, Camarero et al., 2001b,
Camarero et al., 2001a, Kimura et al., 2006, Camarero et al., 2007). In contrast
with the protein trans-splicing approach, EPL is compatible with most of the
amino acids at the cyclization site (Hackeng et al., 1999, Noren et al., 2000),
making this approach general with respect of the sequence of the linear
polypeptide sequence.
Cyclic peptides represent an exciting new tool for deciphering complex biological
processes. As the genomics era unfolds, there will be a continuing demand for
innovative strategies to address difficult biological questions, and the cyclic
peptides offer biologists access to highly diverse molecular libraries for genetic
experiments, approaches previously restricted to synthetic chemists. Moreover,
methods are being developed for the application of cyclic peptides in a variety of
experimental endeavors, providing a toolbox that should benefit evolving
genomics-based approaches. In the coming years, scientists will continue
unraveling the overwhelming amount of genomic data encoding complex
biochemical pathways and protein networks, and cyclic peptides are an attractive
complementary tool that can aid this challenging task.

74

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Abstract (if available)

Abstract Chapter 1. The balance between oxidized and reduced couples, i.e., glutathione (GSH/GSSG), pyridine dinucleotides (NADH/NAD, NADPH/NADP), thioredoxinreduced/thioredoxinoxidized, dihydrolipoic acid/alpha lipoic acid, and lactate/pyruvate determines the cellular redox status. Oxidative stress and altered redox status are widely considered as major components of aging and age-related diseases. The isolation of mitochondria from organs is a widely used tool to study mitochondrial biology. However, inherent in the long isolation process, are alterations in mitochondrial redox status. Previous work from our laboratory has shown that different isolation methods can alter the redox status with respect to the glutathione pool. Substrate supplementation of isolated mitochondria resulted in higher buffering capacity against H2O2 challenges, in part due to increased GSH levels. The aim of the present study is to extend our previous work and monitor changes in the pyridine dinucleotide pool. Upon substrate supplementation, changes in the pyridine dinucleotide pool were quantified using HPLC and changes in membrane potential have been monitored using fluorescent dye Rhodamine-123. Our data show that substrate supplementation shifts the mitochondrial redox status towards a more reduced state. These data are in agreement with our previous work and show changes in redox status of pyridine dinucleotides when supplemented with mitochondrial energy substrates.

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Creator Sancheti, Harshkumar (author)

Core Title Modulation of the redox status of isolated mitochondria by energy-linked substrates: quantification by high performance liquid chromatography; and "Splicing up" drug discovery, cell-based express...

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 05/10/2010

Defense Date 03/26/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cyclotides,drug discovery,high performance liquid chromatography,membrane potential,mitochondria,OAI-PMH Harvest,protein splicing,pyridine dinucleotide,redox status

Language English

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Advisor Cadenas, Enrique (committee chair), Camarero, Julio A. (committee member), Shen, Wei-Chiang (committee member)

Creator Email hsanchet@gmail.com,hsanchet@usc.edu

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