The (transgenic mice) for the initiation of LPS

The outer membrane of Gram-negative bacteria consists of LPS which
contributes to its structure and protects the membrane from biological attacks
in many forms. LPS makes a strong response from animal immune systems. 1

In this study we look at animal
models (transgenic mice) for the initiation of LPS. The main
aim is to identify which signalling pathways are activated in response to
treating the cells with LPS. To do this, we use the heavy to light (H/L) ratios
for the detected phosphopeptides to determine upregulated peptides.

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The results from keg analysis shows at least ten enriched pathways.
The proteins Mapk1 and Akt1 are present in all signalling pathways as there is
an increase in catalytic activity following treatment with LPS. Sqstm1 and
Eif4b only have a single activation in the mTOR and osteoclast differentiation
signalling pathways respectively (indicated by lower H/L ratio compared to
Mapk1 and Akt1). Cdk2 has two phosphorylation sites which are activated in
progesterone mediated and prostate cancer pathways. NF-kB can be activated in
several signalling pathways as they overlap and is also the only protein to
show cross linkages between Lys-Gly upon amino acid modification.


Lipopolysaccharides are large molecules which contribute to the
structural integrity of bacteria by protecting the membrane from biochemical
attacks. LPS also increases the negative charge of the cell membrane and helps
stabilize the overall membrane structure. 2

Therefore, lipopolysaccharides (LPS) are
a key component of the outer membrane of Gram-negative bacteria. It is
localized in the outer layer of the membrane, in non-capsulated strains,
exposed on the cell surface. LPS is the potent activator of monocytes and
macrophages which triggers many proinflammatory cytokines such as tumour
necrosis factor alpha and interleukin-1 (TNF-? and IL-1 respectfully). This is promoted by the LPS active
receptor on macrophages, CD14 and the serum protein (known as LPS-binding
protein) gives LPS a higher affinity to bind to CD14. 3

In this
study we look at animal models (transgenic mice) for the initiation of LPS where
overexpression of CD14 causes hypersensitivity and conversely, mice with
smaller levels of CD14 don’t respond to a lower dosage LPS stimulation. 4

To analyse the protein/ peptide complement of a cell, tissue or
organism, we use proteomics. This works by fractionating peptide mixtures,
using mass spectrometry to identify individual proteins/ peptides, followed by
analysing the mass spec data with bioinformatics. The use of bioinformatics
allows us to process raw mass spectral data into protein data by analysing the
peptide mapping/ mass spec results and determining the protein or peptide
sequence that most closely matches the experimental data.5

Post translational modifications (mainly phosphorylation’s) for
residues (Serine, Threonine and Tyrosine) control signalling cascades
intracellularly. Its significance means we need to be able to distinguish its
phosphoproteomes in the mass spectrometer. 6

This is done through quantitative methods where isotopes are
labelled with 13 C, 15 N, 2 H, or 18 O which allows us to see a difference in
mass between peptides with the same amino acid sequence and modi?cation state.

Integration of stable isotopes are achieved by labelling the
entire proteome or by in vitro reactions with stable isotope-containing
chemicals. In this study, we focus only on the metabolic labelling strategy called
Stable Isotope Labelling by Amino acid in Cell culture (SILAC).

SILAC is a widely used approach in mass spec phospho/- proteomics with
an accurate quantitative strategy when using cell culture systems to study the
aspects of signalling. The practicality of SILAC come from its ability to encode
cellular proteomes through standard metabolic processes and its ability to incorporate
non-radioactive, heavy stable isotopes enriched for amino acids and newly
synthesized proteins. 7

In terms of this study (and most
cases), labelling is based on the use of heavy Arg and Lys amino acids
containing the stable 13 C and 15 N isotopes for mouse macrophage cell line
(RAW264), grown as two identical cultures. The speci?c mass difference between
the heavy labelled and unlabelled amino acids used during cell growth allows
the phosphoproteomes to be distinguished by mass spectrometer. 8

The main aim is to identify which signalling pathways are
activated in response to treating the cells with LPS. To do this, we use the heavy
to light (H/L) ratios for the detected phosphopeptides to determine upregulated



To conduct this experiment, the acquired DNA had to be transfected
into cells and then grown in culture due to the mutation we implement. The mouse
macrophage cell line (RAW264) is one of several methods used to generate cell
lines. It is derived from a retrovirus (Abelson murine leukaemia virus) used to
induce transformation of mouse white blood cells. RAW264 is useful in this
study as the cell line has a high efficiency for DNA transfection and is
sensitive to RNA interference.

The cell line was grown as two identical cultures and the heavy
culture was treated with lipopolysaccharide (LPS) for 30 minutes. One of the
cultures was labelled with heavy arginine and lysine using the SILAC method 9
Metodieva et al (2013). For protein extraction, trypsin digestion and
phosphopeptide enhancement, an equal number of cells from LPS treated and light
cell cultures were mixed. Phosphopetide samples obtained from this were further
analysed by nano-scale LCMS/MS on an LTQ/Orbitrap Velos system and the
generated data were then analysed using MaxQuant. 10

The cell lines are grown in media deficient of an essential amino
acid but enhanced with a non-radioactive, isotopically labelled form of that
amino acid. This experiment uses deuterated leucine (Leu-d3) which is
incorporated after five doublings in the cell lines so that the proteins can be
studied. Protein populations from the experimental and control samples (Leu-d0
and Leu-d3) are mixed directly following collection, and mass spectrometric
identification can be conducted as all peptides containing leucine are
incorporated by all normal leucine (Leu-d0) or all Leu-d3.11

 MS data is analysed by the MaxQuant
search engine. MaxQuant searches are performed using a reverse database to
calculate the false discovery rate (FDR). This is a method of theorising errors in the null hypothesis testing methods
are conducting multiple comparisons. To examine SILAC ratios, for
unmodified peptides and phosphopeptides, the quantitative analysis performed by
MaxQuant was further evaluated by statistical tests using Microsoft Excel.

The ratio data when presented was skewed so Log transformations of
the H/L ratios was conducted to make the data distribution more normal like. By
using log2, the results where easier to interpret. A unit of difference in the
log2 scale means a two-fold difference in the ratio scale. The formula =LOG (X,
2) is typed into excel where X is the H/L ratio to be transformed. To calculate
robust Z scores, positive and negative log2 values the median and two
percentiles where used. Finally, raw p values are calculated, and significant
peptides selected. 12


Table 1: A
table to show the peptide sequences contained in the specific genes.

The data in table 1 is linked to the enrichment analysis
identified in the pathway sequences. Uniprot accession ID lets us view the
amino acid sequences and peptides. By doing this we can identify the specific
phosphorylation sites. The modifications column shows how many peptides are
altered and the type that occurs; the main form being phosphorylation with a few
oxidation reactions which leads to activation of the pathways. The column showing
the H/L ratios allow us to detect the possible phospho peptides which in turn
allow us to determine upregulated peptides. These H/L ratios are significantly
larger than expected which goes against the null hypothesis which states that
there is no change in response to the treatment with LPS. The ratio data is
skewed but log-transforming lets us interpret the ratio.

Figure 1: The Kegg analysis shown in figure 1 indicates the
proteins that are activated via phosphorylation on the MAPK signalling pathway.
These proteins are present in in several signalling pathways such as VEGF, Fc
epsilon RI signalling pathway, toll like receptor signalling pathway, Chagas
disease (American trypanosomiasis) and the prostate cancer signalling pathway. However,
because the MAPK pathway shows these proteins (ERK, p38 and ATK) and the nuclear
factor kappa activated B cells (NFkB) which is crucial in DNA transcription and
cytokine production. These will be specified in the discussion.


Figure 2: Figure 2 is the keg analysis of the mTOR
signalling pathway. It shows the Mapk1 and Akt1 proteins which are present in
all ten of the enriched pathways. In addition, the eukaryotic translation
initiation factor 4B (EIF4B gene) is also present. This protein is not present
in any other signalling pathway but is still relevant as its phosphorylation
must increase catalytic activity.



Figure 3: The data shows that 5 different proteins are
activated in this signalling pathway. It contains the same proteins (NFkB, ERK
and Akt) present in the other enriched pathways. The p38 protein is another formula
for the MAP kinase. Figure 3 also shows an additional protein (p62) which is
not present in any other pathway. This is the sequestosome 1 protein (also
known as the ubiquitin-binding protein) which is essential in cellular processes
such as transport and catabolism. Although this contains a phosphorylation site
(as shown in the table) it occurs in more than one site (see discussion).



Figure 4: Figure 4 presents multiple activated pathways
compared to the other enriched signalling pathways. However, these are all
different forms of the genes Mapk1 and Akt1. It also contains the cyclin dependent
kinase 2 protein (CdK2) which is only present in 3 other enriched pathways. Prostate
cancer signalling show 3 androgen receptors which are responsible for cellular processes such
as cell growth and apoptosis (cell death).


From the table of calculated data, we can see that there is
an abnormally large number of genes in the list mapping on to specific pathways
compared to the number of genes mapping on pathways in the entire genome. The H/L
ratios can be deduced as the amount of activation which occurs in the pathway
when its treated with LPS. For example, the MAPk1 protein has its phospho peptide
activity increased by a factor of 15.6 in response to the treatment. Table 1
shows that 2 phosphorylation’s occur in in the peptide sequence however, this is
derived from the keg sequence analysis meaning its only the expected result. To
prove this hypothesis, we must look at the uniprot data of mouse Mapk1 which
gives us the full peptide sequence encoding the gene. This will manually validate
information inferred from a combination of experimental and computational
evidence. The peptide sequence is between 171 and 189 with amino acid modifications
occurring at residue 183 and 185. This is the activation of phospho threonine
and phospho tyrosine which is phosphorylated by MAP2K1/ MEK1 and MAP2K2/MEK2 on
Thr-183 and Tyr-185 respectively in response to external stimuli e.g. insulin. This
phosphorylation causes dramatic conformational changes, which enable full
activation and interaction of MAPK1/ERK2 with its substrates. It is known that
all eukaryotic cells possess multiple MAPK pathways which manage mitosis
regulation, apoptosis, and cell survival. Therefore, LPS strongly activates it
in all the signalling pathways and evidence for this is shown in the enrichment

MapK14 is another MapK derivative as its activated in most
of the enrichment pathways. The table shows a high H/L ratio (12.5) when treated
with LPS but more importantly, that an oxidation reaction can occur on one of
its two phosphorylation sites. Oxidation and phosphorylation on Thr-180 can
occur to activate/ inhibit signalling pathways and Tyr-182. MAPK14 can
represent p38 MAPKs which have an important role in the cascades of cellular
responses caused by extracellular stimuli such as proinflammatory cytokines leading
to the activation of transcription factors. Kegg analysis shows this as p38 is
activated in Osteoclast differentiation, Progesterone mediated oocyte
maturation, MAPK signalling, VEGF, Fc epsilon RI signalling, Toll like receptor
signalling and Chagas disease pathways.

The Akt1 proteins follow in a similar pattern as Mapk1 as it
is activated in all pathways. The modified amino acid residue occurs at phospho
serine 124 and 126 which happens to be the same residues altered in human cells
for upstream transcription activation. The peptide sequence of cyclin dependent
kinase 1 shows a fairly low H/L ratio (6.2) compared to Mapk1- 14 and Akt1. This
is because the protein is only activated in three enriched pathways
(progesterone mediated, prostate cancer and pathways in cancer). From the
uniprot data we can see the peptide sequence occurs between 10 and 20 in the
whole chain and although table 1 specifies two phosphorylation sites, there are
three possible amino acid modifications. Treatment with LPS would activate the specific
pathways using phospho threonine at 14 whereas phosphorylation at Tyrosine 15 can
inhibit cell growth thereby slightly reducing kinase activity.

Nuclear Factor Kappa B is the transcription factor present
in most cell types and is the endpoint of a series of signal transduction
events that are initiated by stimuli related to many biological processes such
as differentiation, cell growth, tumorigenesis and apoptosis. Nevertheless, it
shows a lower H/L ratio as there is only one phosphorylation site for
activation in the peptide sequence. The uniprot data presents this as a cross
link between Lys-Gly at peptide 855. NF-?B belongs to the category of rapid acting
primary transcription factors which allows NF-?B to be a first responder to
harmful cellular stimuli. From this we can assume activation by LPS has a more important
practical application. Sequestosome 1 is only activated in osteoclast
differentiation as a p62 protein. Phosphorylation occurs on Threonine 269 and