Lipidomics based analysis in magnaporthe oryzae

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LIPIDOMICS-BASED ANALYSIS IN MAGNAPORTHE ORYZAE"
Table of Contents
Title Page……………………………………………………………… … I
Declaration….……………………….………………………………… … II
Acknowledgement………………………….………………………… … III
Publication List ……………………………………………………… … IV

SUMMARY" "4"
List of Tables" "5"
List of Figures" "6"
List of Symbols" "8"
CHAPTER 1: INTRODUCTION" "11"
1.1 Challenges in Food Supply" "11"
1.2 Life Cycle of M. oryzae and Rice Blast Disease" "12"
1.3 Important Signaling Pathways of M. oryzae" "15"
1.4 Lipids and Their Metabolism" " 17"
1.4.1"Phospholipids" "17"
1.4.2"Neutral"Lipids" "24"
1.4.3"Sphingolipids" "28"
1.5 Recent Advances of M. oryzae Research" "29"
1.6 Proposed Model for Turgor Pressure Production" "31"
1.7"In#silico"Inh ib ito r"Design" "34"
1.8 Aims of the Project" "36"
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CHAPTER 2: MATERIALS AND METHODS" "38"
2.1 Growth Medium" "39"
2.2 The Fungal Strain, Growth Condition and Appressorium Formation" "39"
2.3 Lipid Body/Droplet Staining by BODIPY" "39"

2.4 Live Cell Fluorescence Microscopy" "40"
2.5 Lipid Extraction" "40"
2.6 Lipid Profiling and Quantification by HPLC/MS" "41"
2.7 Analysis of Phosphatidylinositol Phosphates" "43"
2.8 Inhibitor Design by Bioinformatic Approaches" "44"
2.8.1 Homologous Modeling of Tps1’s Structure" "44"
2.8.2 Screening of MLSMR and Docking Results Analysis" "44"
2.8.3 Lead Optimization" "45"
2.8.4 Molecular Dynamics Simulation by Gromacs" "45"
CHAPTER 3: GENERAL LIPIDOMIC ANALYSIS" "48"
3.1 Lipid Body Staining" "48"
3.2"Profiling"of"the"Lipidome" "54"
3.3 Semi-quantitative Analysis of the Lipidome" "60"
3.3.1"Quantification"of"Phospholipids" "61"
3.3.2"Quantification"of"Ceramidehexosides" "64"
3.3.3"Quantification"of"TAGs"and"DAGs" "68"
3.3.4"Quantification"of"Phosphoinositides" "71"
3.4"Conclusion" "73"
CHAPTER 4: BETA OXIDATION AND PATHOGENICITY" "74"
4.1 Phospholipids and TAGs in Mutants and WT" "75"
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4.2 Conclusion" "79"
CHAPTER 5: IN SILICO INHIBITOR DESIGN" "80"
5.1 Structure Modeling by Modeller" "83"
5.2 Screening of MLSMR for Inhibitors" "86"
5.3 Lead Optimization" "90"
5.4 Molecular Dynamics by Gromacs" "92"
5.5 Conclusion" "94"
CHAPTER 6: DISCUSSION" "95"

6.1 Lipid Body Staining" "95"
6.2 Profiling and Quantification of the Lipidome" "96"
6.3 Validation of the Mechanism for Turgor Pressure Production" "99"
6.4 Beta-oxidation: Mitochondria vs. Peroxisomes" "100"
6.5 Lead 25 for Blast Disease Control" "102"
CONCLUSION" "103"
REFERENCES" "105"
APPENDICES" "123"






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SUMMARY
Magnaporthe oryzae (M. oryzae) is the causal agent of the rice blast disease.
Triacylglycerides (TAGs) were one of the major sources used to generate
turgor pressure as a means for M. oryzae to penetrate into host’s leaf. Lipids
therefore play a very important role in the pathogenesis. However, there is up
to date no lipidomics study of M. oryzae available. As Part I of project, our
research for the first time analyzed the lipidome of M. oryzae and quantified
the lipid species across different time points along the pathological cycle. The
lipidomics study as a platform was further used to analyze two beta oxidation
pathway mutants and proposed possible explanation for their nonpathogenicity.
Our data had also shown interesting information and was suggestive of a
possible mechanism for turgor production.
Previous studies already discovered that trehalose synthase (Tps1) was not
only responsible for the production of trehalose and utilization of nitrogen

source, but also the regulation of several NADPH-dependent transcriptional
corepressors, namely Nmr1, Nmr2, and Nmr3, which can each bind NADP.
Therefore, as for Part II of the project, the structure of Tps1 was modeled for
screening of possible inhibitors in silico against a database of 400k compounds,
and molecular dynamics studies were also done for some of the best hits.
Advice was then given for future inhibitor design in the context of rice blast
control.
To summarize, this project had: 1) profiled the lipidome of M. oryzae; 2)
identified key lipid species for turgor generation of M. oryzae; 3) employed
the lipidomics approach as the platform to study some nonpathogenic mutants;
4) proposed a possible mechanism for turgor production; 5) screened chemical
databases for possible inhibitors of a key enzyme (Tps1) involved in
pathogenesis.
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List of Tables



Page



Supplementary
Table 1
Lipid species identified and their (accurate)
masses
124-128

Supplementary
Table 2
Top 45 compounds with the lowest-energy
binding confirmations
136










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List of Figures


Page
Figure 1
The pathogenesis cycle of M. oryzae
13
Figure 2
A proposed model for turgor generation in

appressoria
33
Figure 3
A brief illustration of the experimental
procedures
38
Figure 4
The staining the lipid droplets of M. oryzae
50-53
Figure 5
The elution profiles of different lipid classes
& tabulation of lipid species identified
56-59
Figure 6
Quantification of phospholipids
62-64
Figure 7
Analysis on CMHs
66-67
Figure 8
Quantification of TAGs and DAGs
70
Figure 9
Quantification of PI-3,4-P2 and PI-4,5-P2
72
Figure 10
TAG analysis on WT, ΔEch1 and ΔFox2
77
Figure 11
Proposed function of trehalose metabolism

82
Figure 12
The modeled structure of Tps1
84
Figure 13
Structures of Tps1, 1gz5 and 2wtx aligned
85
Figure 14
The validation of Vina’s performance
88
Figure 15
The binding confirmation of the best 3
compounds when docked to Tps1
89
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Figure 16
The structure, binding confirmation and
computed LogP of Lead 25
91
Figure 17
MD study of Lead 25
93
Supplementary
figure 1
TAG:DAG:PLs ratios over different time
points of the pathogenesis of M. oryzae
129-132
Supplementary
figure 2

Total PLs in WT, ΔEch1 and ΔFox2 in
conidia and appressoria
133
Supplementary
figure 3
Multiple sequence alignment of Tps1 and
2wtx
134
Supplementary
Figure 4
Multiple sequence alignment of truncated
Tps1,2wtx, 1gz5 and 1uqt
135
Supplementary
Figure 5
The binding confirmation of the best 45
compounds when docked into Tps1
137-148
Supplementary
Figure 6
The 2D structures of the best 45 compounds
149-152
Supplementary
Figure 7
The 2D structures of the 26 modified
compounds based on Compound 24789937
153-154

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List of Symbols
AA
arachidonic acid
cAMP
cyclic 3', 5' adenosine monophosphate
CM
complete medium
DAG
diacylglycerol
DHAP
dihydroxyactone phosphate
DMSO
dimethyl sulfoxide
󳵜C
degree Celcius
C. albicans
Candida albicans
DNA
deoxyribonucleic acid
g
grams
g
relative centrifugal force
g/L
grams per litre
GFP
green fluorescent protein
h

hour
HPLC
high performance liquid chromatography
IP(1,4,5)P3
inositol-1,4,5-trisphosphate
kb
kilobase
LB
lysogeny broth
µg
microgram
9"
"
µL
microlitre
mg
milligram
min
minute
mL
millilitre
mm
millimetre
mM
millimolar
M
molar
MRM
multiple reaction monitoring
MS

mass spectrometry
MAPK
mitogen-activated protein kinase
MM
minimal medium
NADPH
nicotinamide adenine dinucleotide phosphate
ng
nanogram
PCR
polymerase chain reaction
PI-3-P
phosphatidylinositol 3-phosphate
PI-3,4-P2
phosphatidylinositol 3,4-bisphosphate
PI-3,4,5-P3
phosphatidylinositol 3,4,5-trisphosphate
PI-4,5-P2
phosphatidylinositol 4,5-bisphosphate
PKA
protein kinase A
rpm
resolutions per minute
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S. ferax
Saprolegniaferax
U. maydis
Ustilago maydis
TAG

triacylglyeride
%
percentage
% w/v
percentage weight by volume
% v/v
percentage volume by volume












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CHAPTER 1: INTRODUCTION
1.1 Challenges in Food Supply
It might be fair to say that the world today has never been able to solve
the problem of the security of food supply, despite of the advancement of
technology. Possible causes would be climate change, increased demand for
biofuels, structural shifts in food and agricultural systems, trans-boundary
movement of disease and widespread land degradation (Thompson, Cohen et
al. 2012, Thornton 2012). Taking China as an example, the rapid urbanization
has a big and long term impact on both the public health (Van de Poel,

O'donnell et al. 2012)and its own environment (Zhu 2012), while the loss of
farming lands would be one of the direct consequences. One more example is
the conversion of corn to ethanol in the United States since 2005,which was
considered a major cause of global food price increases during that time
(Albino, Bertrand et al. 2012).
Meanwhile, crops diseases could further contribute to the insecurity of
food supply. One example could be seen in the case of Phytophthora infestans
(P. infestans), the Oomycete agent of potato late blight and the primary cause
of the great Irish famine of the nineteenth century. The disease caused
significant yield losses in Ireland due to the wetness of the climate while there
was a large proportion of the population who almost totally depended on
potato as their food supply (Large 1940); eventually 1.5 million out of 8
million population died of starvation and another 1.5 million emigrated, when
about a quarter of the emigrants died in transit (Klinkowski 1970). A recent
case however would be the wheat stem rust, as it could infect 90% of the
wheat strains and cause a global threat to the wheat production and threaten
the food supply of billions (Singh, Hodson et al. 2011).
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How would the issue of food shortage be effectively handled? To answer the
question, many ideas and suggestions could be given in relation to policy
making and resource management; but as for scientists, research on the crops
and the possible pathogens in the context of the crop diseases management
should be apriority.

1.2 Life Cycle of M. oryzae and Rice Blast Disease
Magnaporthe oryzae (M. oryzae) represents a serious threat to global rice
production due to its role as a causal agent of the rice blast disease, the most
severe disease affecting cultivated rice, causing 10%-30% loss of rice
production (Talbot 2003).

M. oryzae reproduces both sexually (Saleh, Xu et al. 2012) and asexually.
Asexual spores are involved in rice blast disease; sexual reproduction, on the
other hand, gives rise to perithecium, the fruiting body that carries numerous
eight sporedasci under proper conditions (Valent, Farrall et al. 1991).
Figure 1 (taken from Wilson and Talbot 2009) illustrates the life cycle of
M. oryzae. Rice blast infection is initiated when conidia attach to the
hydrophobic surface of a rice leaf by producing an adhesive material at the
apex of the conidium (Hamer, Howard et al. 1988). The conidia could
germinate even only in the presence of water and form germ tubes within 2
hours (Talbot 2003). The germ tubes continue to extend for about 15-30 µm
before swelling at their tips. The tips would become flattened against the leaf
surface, and differentiated into specialized dome-shaped structures called the
appressoria (Bourett and Howard 1990). Some possible conditions for
appressorium

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Figure 1. Table taken from Wilson and Talbot, 2009. A conidium when having landed on
the rice leaf or hydrophobic surface would germinate, form an appressorium, penetrate
through the leaf surface and start the intracellular growth. Lastly, conidiation would occur
before the cycle restarts.




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differentiation are leaf surface topography and the absence of exogenous
nutrients (Dean 1997). When the surface is hydrophilic, some chemicals could
be used to still induce appressorium differentiation, taking cutin and lipid
monomers for example (Gilbert, Johnson et al. 1996). The cell wall of an
appressorium is rich in chitin, and a layer of melanin is deposited underneath
the cell wall of the appressorium (Bourett and Howard, 1990). The melanin
layer enables M. oryzae to withstand the physical force produced by the
appressorium when penetrating the plant cuticle (de Jong, McCormack et al.
1997).
A conidium contains three nuclei in all and one in each cell. During
appressorium differentiation, nuclear division takes place in the germ tube:
about 4 to 6 h after inoculation, one nucleus migrates to the germ tube, and
this nucleus then differentiates into two daughter nuclei by a single mitotic
division (Veneault-Fourrey, Barooah et al. 2006). One of the two daughter
nuclei would then move into the appressorium while the other returns to the
conidium. After 12 to 15 h, the three nuclei in the conidium begin to degrade
and only the nucleus in the appressorium remains (Veneault-Fourrey, Barooah
et al. 2006). The nutrient storage is also mobilized from the original conidium
into the appressorium as soon as mitotic division takes place. After all these, a
specialized septum would be developed and separate the appressorium and the
collapsing conidium. Autophagic cell death of the conidium would then follow
and the appressorium is left intact on the plant leaf (Veneault-Fourrey,
Barooah et al. 2006, Kershaw and Talbot 2009).
After the leaf cuticle is ruptured, M. oryzae starts to form a penetration
peg that swells into a primary infection hyphae; the infection hyphae in turn
differentiates into a series of branched and bulbous invasive hyphae (Talbot
2003). After the initial epidermal cell is colonized, invasive hyphae begins to
move into adjacent cells and infect host plant tissue (Valent, Farrall et al. 1991,
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Talbot, Ebbole et al. 1993). After 4 days, disease lesions appear on the surface
of the leaf and M. oryzae releases very large numbers of conidia into the
atmosphere and restarts the whole process of pathogenesis (Wilson and Talbot
2009).
There are 2 major symptoms on rice plants by the infection of M. oryzae,
namely a leaf spot disease with large ellipsoid lesions on the surface of rice
leaves (Talbot 1995) and also neck blast panicle blast symptoms in older rice
plants (Wilson and Talbot 2009). The key to its pathogenesis, as what has been
mentioned earlier on, is the formation of the appressorium, a specialized cell
that serves to facilitate the process of invasion and penetration into plant
tissues during infection by generating substantial amount of turgor pressure
and physical force. Glycerol was reported as the most abundant solute in the
appressoria (Talbot 2003) and therefore represents the main osmolite
responsible for generating the high turgor pressure (Thines, Weber et al. 2000).
The high turgor pressure, which could be up to 8 mPa (Howard, Ferrari et al.
1991), enables the penetration peg to mechanically penetrate through the leaf
surface of the host and thus allows the delivery of the fungal materials into the
host cell (Howard, Ferrari et al. 1991). As appressorium formation does not
involve an exogenous supply of nutrients, the source of glycerol should be
found inherently within the conidia, which consists of substantial amounts of
lipids, glycogen and other storage products (Talbot 2003).

1.3 Important Signaling Pathways of M. oryzae
Three signaling pathways have been shown to play an important role in
plant infection by M. oryzae: the cyclic 3', 5' adenosine monophosphate
(cAMP) signaling pathway (Lee and Dean 1993, Xu and Hamer 1996,
Umemura, Ogawa et al. 2000), the Pmk1 mitogen-activated protein kinase
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(MAPK) signaling pathway (Xu and Hamer 1996, Bruno, Tenjo et al. 2004)
and the Mps1 MAPK signaling pathway (Xu, Staiger et al. 1998).
M. oryzae uses the cAMP signaling pathway for surface recognition and
triggering of appressorium formation (Lee and Dean 1993, Umemura, Ogawa
et al. 2000). Appressorium formation was restored in the presence of
exogenous cAMP for a Δmac1 mutant (Choi and Dean 1997). However, there
are evidences that cAMP signaling pathway has broad and divergent impact on
growth and pathogenesis (Adachi and Hamer 1998).
In M. Oryzae, there are three MAPK pathways characterized: the Pmk1
MAPK pathway which is involved in appressorium formation and penetration,
the Mps1 MAPK pathway which is involved in conidiation and penetration,
and the Osm1 MAPK pathway required for osmoregulation; however, only the
first two genes are affecting the pathogenicity (Xu and Hamer 1996, Xu,
Staiger et al. 1998, Dixon, Xu et al. 1999). It was also proposed that PMK1
acts downstream of a cAMP signal for appressorium formation since pmk1
mutants develop abnormal germ tubes in the presence of cAMP on hydrophilic
surfaces (Xu and Hamer 1996). Apart from M. Oryzae, PMK1 homologues in
appressorium-forming fungi, including M. oryzae, Colletotrichum lagenarium
(C. lagenarium), Cochliobolus heterostrophus (C. heterostrophus), and
Pyrenophorateres, were found to be essential for appressorium formation and
plant infection (Lev, Sharon et al. 1999, Takano, Kikuchi et al. 2000,
Ruiz-Roldán, Maier et al. 2001). For other fungi like Fusarium oxysporum (F.
oxysporum) and Botryti scinerea (B. cinerea), PMK1 homologues are still
required for their pathogenicity (Zheng, Campbell et al. 2000, Di Pietro, Garcí
a‐Maceira et al. 2004). It might be fair to say that for many fungi the PMK1
pathway is conserved for appressorium formation and other plant infection
processes. However, although PMK1 homologues have been identified in
several fungi, it is still an on-going research on the activation of PMK1 and its
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downstream effectors, and one example could be the work on MST12 (Park,
Xue et al. 2002). Meanwhile, distortion of the MPS1 MAPK pathway resulted
in the failure of the remodeling of the appressorium wall and the consequential
inability of appressoria to penetrate plant cell surfaces (Xu, Staiger et al. 1998).
MPS1 homolog in the maize pathogen C. heterostrophus was also shown to be
involved in melanin synthesis (Eliahu, Igbaria et al. 2007). In the case of C.
lagenarium, knocking out MPS1 would even prevent the fungus from forming
appressoria (Kojima, Kikuchi et al. 2002).

1.4 Lipids and Their Metabolism
Lipids are key components of cell membranes and actively involved in a
range of biological functions: i.e. energy storage, as precursors for hormone
synthesis and also signaling (Sul and Wang 1998, Bozza, Melo et al. 2007).
Lipid biosynthesis occurs in the cytoplasm and utilizes acetyl-CoA as the
precursor through a process known as lipogenesis (Kersten 2001). Because of
the shared source of acetyl-CoA, lipid metabolism is tightly connected to
carbohydrate metabolism. It is even believed that proper lipid metabolism is a
key molecular integrator of energy homeostasis, membrane structure and
dynamics, and signaling (Wenk 2010).

1.4.1$Phospholipids$
Phospholipids are major components of cellular membranes that
participate in a range of cellular processes. Their structures exhibit a high
degree of het- erogeneity: saturated fatty acyl groups are predominated in the
sn-1 position, whereas unsaturated fatty acids are commonly found at the sn-2
position. Fatty acid remodeling is believed to have contributed to the
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incorporation of unsaturated fatty acids at the sn-2 position. Our understanding
of the phospholipids’ roles and functions has been broadened by recent

discoveries as well. In the following sessions, they are briefly discussed based
on the classes.

1.4.1.1 Phosphatidic acids (PAs)
PA is the simplest diacyl-glycerophospholipid and occurs only in small
amounts (often less than a few mol%) in biological membranes but yet is
crucial for cell survival, because it is a key intermediate in the biosynthesis of
phospholipids and is involved in many signaling events.
The synthesis is by two major de novo biosynthetic pathways that utilize
either glycerol 3-phosphate (G-3-P) or dihydroxyacetone phosphate (DHAP)
as precursors (Carman and Zeimetz 1996). In the case of G-3-P, it is acylated
by G-3-P acyl- transferase at the sn-1 position to produce lysophosphatidic
acid (LPA). DHAP is acylated at the sn-1 position by DHAP acyl- transferase
to produce 1-acyl-DHAP, which is reduced by 1-acyl-DHAP reductase to form
LPA. At this stage, LPA is further acylated by LPA acyltransferase in the sn-2
position to yield PA. Phospholipids, including phosphatidylserine (PS),
phosphatidylethanolamine (PE), and phosphatidylcholine (PC), are
synthesized from PA through the cytidine diphosphate diacylglycerol pathway.
Different organelles (i.e., the ER, mitochondria, and in addition peroxisomes
of mammalian cells, lipid particles of yeast and chloroplasts of plants) could
be involved in the biosynthesis PA, and the redundancy of the biosynthetic
systems could be that different pools of PA may serve as precursors for the
synthesis of complex phospholipids and/or triacylglycerols (TAG) in different
organelles, though the regulatory mechanisms for channeling PA to form the
various species of acylglycerolipids under specific physiological conditions
are still under research (Athenstaedt and Daum 1999).
PA and LPA are known to be involved in membrane fission and fusion
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events (Weigert, Silletta et al. 1999), and a conversion of LPA into PA may

induce negative spontaneous monolayer curvature and membrane bending
(Kooijman, Chupin et al. 2003). In the light of that, PA has been found to
facilitate the coalescence of contacting LDs in forming "supersized" lipid
droplets in yeast (Fei, Shui et al. 2011). Meanwhile, an important determinant
of the biological functions of PA is its anionic headgroup (Kooijman and
Burger 2009), which is related to many PA-protein interactions. It was also
found to be involved in mTOR signal transduction and protein synthesis, being
also a direct link between mTOR and mitogens (Fang, Vilella-Bach et al.
2001). PA on the endoplasmic reticulum directly binds to the soluble
transcriptional repressor Opi1p and makes it inactive outside the nucleus; upon
the rapid consumption of PA by the addition of the lipid precursor inositol,
Opi1p is released from the endoplasmic reticulum for its nuclear translocation
and repression of target genes (Loewen, Gaspar et al. 2004); such interaction
between PA and Opi1p is known to be pH dependent and linked membrane
biogenesis with nutrient availability as well (Young, Shin et al. 2010).

1.4.1.2 PCs
PC is a major component of the cellular membrane, and its role could not
be replaced by PE (Wu, Ye et al. 2010).
As for the synthesis, both de novo biosynthesis (the Kennedy Pathway)
and remodeling processes (the Lands cycle) take place: the saturated fatty
acids found frequently at the sn-1 position of PC are believed to be derived
from de novo biosynthesis, whereas the unsaturated fatty acids, usually found
at the sn-2 position in PC, are esterified mainly through the remodeling
process (MacDonald and Sprecher 1991).
PC could be converted into PA, phosphocholine and DAG, which are in
turn functioning as signaling molecules. One example is seen in the case of the
hydrolysis of PC by PC-PLC: the resulting phosphocholine and DAG in the
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case of being stimulated by cytokines, growth factors, mitogens, and calcium
ions have been implicated in intracellular signal transduction involved in the
regulation of cell metabolism, growth, differentiation and induction of
apoptosis (Szumilo and Rahden-Staron 2008). PC-specific phospholipase C
has inhibitory effects on the pathways responsible for constitutive epithelial
ovarian cancer cell stimulation and cell proliferation (Spadaro, Ramoni et al.
2008), and that the elevated phosphocholine pool detected in epithelial ovarian
cancer cells primarily results from upregulation/activation of ChoK and
PC-phospholipase C involved in PC byosinthesis and degradation, respectively
(Iorio, Ricci et al. 2010).

1.4.1.3 PEs
In yeast, PE is synthesized by multiple pathways located in different
subcellular compartments (i.e., ER, Golgi and mitochondria) whose PE
products are functionally different，and one example is seen in the case of
LysoPE that supports growth and replaces the mitochondrial pool of PE much
more efficiently than and independently of PE derived from the Kennedy
pathway (Riekhof and Voelker 2006).
The mitochondrial inner membrane contains two non-bilayer‐forming
phospholipids, PE and cardiolipin, which affect the stability of respiratory
chain supercomplexes differently (Böttinger, Horvath et al. 2012).
Mitochondrial PE deficiency impairs formation and/or membrane integration
of respiratory supercomplexes (Tasseva, Bai et al. 2013). Recently, cardiolipin
and mitochondrial PE are required to maintain tubular mitochondrial
morphology and have overlapping functions in mitochondrial fusion (Joshi,
Thompson et al. 2012). PE has other important roles besides being a
membrane component, and very often it is associated with other proteins. Psd2,
a PS decarboxylase, is responsible for the synthesis of vacuolar membrane PE;
the loss of the enzyme causes a specific reduction of the vacuolar membrane
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PE but not the total PE levels and the subsequent loss of normal activity of a
vacuolar ATP-binding cassette transporter protein called Ycf1: the mutant
yeast strain is sensitive to cadmium (Gulshan, Shahi et al. 2010). PE is
involved in autophagy: the formation of Apg8-phosphatidylethanolamine has
an essential role in membrane dynamics during autophagy (Ichimura, Kirisako
et al. 2000). PE plays an important role in the regulation of APP proteolysis
and thus Aβ generation too (Nesic, Guix et al. 2012).

1.4.1.4 PSs
Being a metabolically related metabolite of PE, PS is present in
membranes of all eukaryotic and prokaryotic cells. Depending on the type of
organisms, different pathways would be used for synthesizing PS: as what is
reviewed, in prokaryotes and the yeast, all PS is synthesized by a PS synthase
that uses CDP-diacylglycerol and L-serine, while in mammalian cells a
calcium-dependent base-exchange reactions in which the polar head-group
(choline or ethanolamine) of a pre-existing phospholipid (PC or PE,
respectively) is exchanged for L-serine is used; one more thing to note of is
that mammalian mitochondria do not make PS whereas bacteria do (Vance and
Tasseva 2013).
PS is not symmetrically distributed across the two leaflets of the
membrane bilayer and studies on the erythrocyte membrane indicate that more
than 96% of PS resides on the inner leaflet of the bilayer (Zachowski 1993).
However, during the blood-clotting cascade, the trans-bilayer asymmetry of
PS in the plasma membrane of activated platelets is markedly altered so that
PS becomes exposed on the cell surface and the clotting factors would be
recruited to the surface of platelets (Williamson, Bevers et al. 1995, Majumder,
Quinn-Allen et al. 2008).
Due to the anionic nature of PS, the positively-charged motifs of some
key signaling proteins, such as the tyrosine kinase Src, as well as the Ras and

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Rho family of GTPases will bind to PS for their membrane targeting and
activation (Sigal, Zhou et al. 1994, Finkielstein, Overduin et al. 2006,
Lemmon 2008, Yeung, Heit et al. 2009). The catalytic activity of several key
signaling proteins such as such as synaptotagmin, dynamin-1 (Yeung, Heit et
al. 2009) through the interaction between the C2 domains and PS. PS could
also interact with proteins containing PH domains, such as
3-phosphoinositide- dependent kinase-1 (Lucas and Cho 2011) and Akt
(Huang, Akbar et al. 2011).

1.4.1.5 Phosphatidylinositols (PIs) & Phosphoinositides (PIPs)
PI can be phosphorylated to form phosphatidylinositol phosphate (PI-4-P
or PIP), phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol
trisphosphate (PIP3).
Sec14p is a major yeast PI transfer protein (PITP), regulating an essential
interface between lipid metabolism and protein transport from Golgi
membranes to the cell surface (Xie, Fang et al. 1998) and even involved in the
execution of developmentally regulated polarized membrane trafficking
pathway (Vincent, Chua et al. 2005).
PI could be metabolized into various inositol phosphates like mono- to
polyphosphorylated inositols (i.e., PIP1, PIP2, PIP3…). The various PIPs play
crucial roles in diverse cellular functions, such as cell growth, apoptosis, cell
migration, endocytosis, and cell differentiation. PI(4,5)P(2) is enriched in
HIV-1, the depletion of which causes reduced HIV-1 budding (Chan, Uchil et
al. 2008). The budding yeast Saccharomyces cerevisiae uses PI(3,5)P2 as its
chromatin architecture-modulating agent (Han and Emr 2011). At the same
time, it is known that association with PI(3,4,5)P3 at the membrane facilitates
phosphorylation and activation of Akt by PDK1 (Lawlor and Alessi 2001):
subsequently, a host of other proteins could be phosphorylated and affect

many cellular events. Furthermore, it was demonstrated in hematopoietic cells
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that functionally distinct PI(3,4,5)P3 pools exist (Bohnacker, Marone et al.
2009). Recently, a specific interaction between a RhoGAP domain of Rgd1p
and phosphoinositides was discovered, which was used by phosphoinositides
to specifically stimulate the RhoGAP activity of Rgd1p on Rho4p (Schlame
and Otten 1991, Odaert, Prouzet-Mauleon et al. 2011).

1.4.1.6 Other phospholipids
Being generally regarded as being a mitochondrion-specific phospholipid
and is particularly enriched in mitochondrial inner membranes (Krebs, Hauser
et al. 1979), cardiolipin (CL) is a unique phospholipid with four acyl chains
and an unusual composition of molecular acyl species (Schlame, Beyer et al.
1991). In humans, defective CL acyl-chain remodeling is associated with a
severe genetic disorder, the Barth syndrome (Schlame, Towbin et al. 2002).
Analysis of relative phospholipid contents of lipoproteins showed that CL and
phosphatidylethanolamine are selectively enriched in high density lipoprotein
(Deguchi, Fernández et al. 2000).
Synthesis of CL takes place at the inner mitochondrial membrane by the
transfer of the phosphatidyl group of cytidinediphosphate-diacylglycerol
(CDP-DAG) to PG, catalyzed by CL synthase (CLS); this conversion is so
effective that only contain trace amounts of PG is found in mitochondrial
membranes (Daum 1985). Tam41 is also required for such synthesis (Kutik,
Rissler et al. 2008). Mitochondrial fusion was found to do with the loss of CL
and mitochondrial PE, which further leads to reduced levels of small and large
isoforms of the fusion protein Mgm1p (Joshi, Thompson et al. 2012). In yeast,
lacking both CL and PE is synthetically lethal (Gohil, Thompson et al. 2005).
Recently, the yeast protein Taz1p was shown to function as a transacylase,
catalyzing the reacylation of monolysocardiolipin to mature cardiolipin (Xu,

Malhotra et al. 2006)."We now identified the protein encoded by reading frame
YGR110W as a mitochondrial phospholipase, which deacylates de novo
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synthesized CL (Beranek, Rechberger et al. 2009).
Ceramide 1-phosphate (Cer-1-P) is the product of ceramide kinase and its
product, and mediates calcium ionophore- and interleukin-1 β -induced
arachidonic acid release, due to the activation of a species of PLA2 (Pettus,
Bielawska et al. 2003). Ceramide can also be broken down by ceramidases to
sphingosine, which in turn is phosphorylated by sphingosine kinases to
generate sphingosine 1- phosphate (S1P) (Spiegel and Milstien 2003).
Activation of the S1P1 receptors stimulates downstream signals important for
cell locomotion and lymphocyte recirculation and tissue homing (Hobson,
Rosenfeldt et al. 2001, ROSENFELDT, HOBSON et al. 2001, Matloubian, Lo
et al. 2004). Sphingomyelin (SM) is an abundant constituent of cellular
membranes in a wide range of organisms. Its high packing density and affinity
for sterols help provide a rigid barrier to the extracellular environment and
play a role in the formation of lipid rafts, specialised areas in cellular
membranes with important functions in signal transduction and membrane
trafficking (Simons and Toomre 2000, Holthuis, Pomorski et al. 2001)." SM
synthesis is mediated by a SM synthase, which transfers the phosphorylcholine
moiety from PC onto the primary hydroxyl of ceramide, thus generating SM
and diacylglycerol (DAG) (Ullman and Radin 1974, Voelker and Kennedy
1982, Marggraf and Kanfer 1984).
Above, phospholipids are playing broad structural and signaling roles, and
to identify the identities and study the functions would be of fundamental and
tremendous significance for any lipidomics study.

1.4.2$Neutral$Lipids$
Neutral lipids normally include TAGs (as well as the closely related DAGs

and MAGs), steryl esters (SEs) and wax esters (WEs). They lack charged
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groups and cannot integrate into bilayer membranes in substantial amounts.
The neutral lipids stored in lipid droplets are often used as nutrients and
energy storage after enzymatic hydrolyzation; further more, the hydrolyzation
products like sterols, DAG and fatty acids can also serve as building blocks for
membrane formation, synthesis of steroid hormones and even energy purposes.
Some of the examples are briefly discussed here.

1.4.2.1 DAGs
DAG has a glycerol backbone attached with two acyl chains and is often a
product of TAG hydrolysis. However, de novo synthesis of DAG from glucose
hydrolysis via dihydroxyacetone phosphate and glycerol 3-phosphate is possible
(Rossi, Grzeskowiak et al. 1991). Hydrolysis of PI(4,5)P2 by phospholipase C (PLC)
yields DAG as well. Meanwhile, DAG can be formed through the breaking down of
phosphatidylcholine by a phospholipase C-mediated mechanism (Besterman, Duronio
et al. 1986). In mice, some acyl CoA:monoacylglycerol acyltransferase (MGAT)
activity is found in its acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) as
well, and this DGAT1 exhibits additional acyltransferase activities like wax
monoester and wax diester synthases, and acyl CoA:retinol acyltransferase (ARAT) .
DAG works together with calcium to activate protein kinase C, which goes on to
phosphorylate other molecules, leading to altered cellular activity. DAG is also
involved in the fusion and fission of membranes (Bankaitis 2002, Baron and Malhotra
2002). In diabetes, DAG plays a role in the glucose-induced activation of glomerular
PKC and the subsequent glomerular hypertrophy (Craven, Davidson et al. 1990).
Further, DAG could be converted to PA and exert impacts through that.

1.4.2.2 TAGs
TAG is the most common lipid-based energy reserve in nature. The main

pathway for synthesis of TAG is believed to involve three sequential
acyl-transfers from acyl-CoA to a glycerol backbone (Bell and Coleman 1980,