This course will focus on developing integrative skills through directed reading and analysis of the current primary literature to enable the student to develop the capstone project as the overall final exam for the specialization in systems biology.
Lecture 2B - Introduction to Research Article 1- Part 2
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Del curso dictado por Icahn School of Medicine at Mount Sinai
Integrated Analysis in Systems Biology
47 calificaciones
Icahn School of Medicine at Mount Sinai
47 calificaciones
Curso 5 de 6 en SpecializationSystems Biology and Biotechnology
De la lección
General Features of Systems Biology Studies
Conoce a los instructores
Susana Neves, PhDAssistant Professor
Department of Pharmacology and Systems Therapeutics, Systems Biology Center New York (SBCNY)
So we talked a little bit about the protein DNA arrays that they used to
identity transcription factors there were activated by HIVAN.
The authors also determined which genes were differentially express in
Tg26 mice versus wild-type using micro-arrays.
And they identify 434 genes that were up regulated,
and 72 genes that were down regulated.
Then using promoter analysis, the list of up regulated
genes was analyzed using transfac matrices and
chip enrichment analysis to identify any putative
transcription factors that could in theory regulate these genes.
From the TRANSFAC analysis nine transcription factors were identified,
while the, the chip enrichment analysis identifies 62 transcription factors.
That could account for the differential gene expression observed.
The data from the protein DNA arrays identified
35 transcription factors that were regulated by HIV.
So, the top five transcription factors from each pro,
promoter enrichment analysis was selected.
So five from the TRANSFAC, and five from the CHIA analysis.
Along with any overlapping transcription factor that appeared
in the protein DNA array.
From the TRANSFAC analysis,
three transcription factors overlap with those identified in the protein DNA array.
And those are shown in yellow.
Whereas the gealist, had five transcription factors that appeared
in the, also appeared in the protein DNA array.
From these 18 transcription factors of Protein Protein interaction network
using Gs to networks.
And finally this kinase enrichment analysis, KEA, was used to
identify any upstream kinases that could target the transcription factors.
And thus could be punitive distal regulators.
The gene expression changes induced by HIVAN.
This pipeline resulted in a list
of kinases that could regulate the original expression changes in HIVAN.
And one of the kinases was HIP Kinase 2, which is a novel regulator for HIVAN.
The rest of the figures and the results deal with the extensive
experimental validation of the role of HIP Kinase 2 and
the kidney fibrosis induced by HIV.
>> So the computational analysis predicted that HIP Kinase-2 was
a regulator of the gene expression induced by HIV.
So the question was whether HIV or HIVAN could actually regulate HIPK2.
Given the format of this online course,
it is not possible to go through every panel and the figures.
I will cover some of the selected panels in detail
to provide you with the framework to evaluate the rest of the figures.
The problem set at the end of this lecture,
will require you to review all the panels, not just the ones that I will cover.
So one of the key experiments that was done is
to determine whether HIV can change the expression or
activity or both of HIP kinase-2.
And the way was done through doing amino staining to look at
whether the infection with HIV could enhance expression in the glomerulus and
the tubules, and as you can see by the red staining that is enhanced
in the Tg26 mice, it does seem to be increased.
This is further confirmed using amino blotting, where the tissue is
ground up and the lysates then run on a gel.
And as you can see, the HIP Kinase band seems to be more intense in
the Tg26 independent of whether in the glomerulus or the cortex.
The cortex contains most, most of the tubules.
As a normalization they also blot for
the actin and as you can see that doesn't change much.
Then they decided to determine whether this increase in HIP 2
kinase was due to transcriptional regulation.
And they looked at the mRNA and as you can see from the mRNA data,
there doesn't seem to be an effect suggesting that this occurs
post-translational to support the increase in levels.
They also saw an increase in kinase activity, total kinase activity.
So given how there was no affect at the mRNA level,
the authors thought that maybe the effect was a protein stability level.
They did an experiment where they treated cells
with G, MG132 which is a protiozone inhibitor and
what they notice is that there was a time dependent accumulation of HIP2 kinase
suggesting that normally HIP2 kinase is degraded via the.
And within a few hours,
it could reach levels that were comparable to those induced by HIV infection.
It was already known from the literature that SIAH
was a regulator of HIP kinase degradation.
And so, the authors wanted to determine whether SIAH,
an upstream regulator of HIP kinase.
And so what they did is that they actually looked at the expression levels
of SIAH compared to HIP Kinase 2.
As expected in Tg26 mice,
where the levels of HIPK2 are enhanced, the levels of SIAH seem to be reduced.
The opposite is, is seems to be the case in the wild-type mouse
where the Siah level seems to be much more prominent and
the hip kinase levels seem to be decreased.
In panel 3B shows that there is
a negative correlation between SIAH levels and HIP kinase 2.
>> In panel 3F, there is
an experiment that actually demonstrate that the relationship between SIAH and
HIPK appears to be causal.
>> The authors directly modulated the expression of SIAH either by using siRNA,
small interfering RNA, or by overexpressing it using a plasmid DNA.
And as you can see, that decreasing the levels of SIAH
was sufficient to increase the levels of HIP kinase 2.
And the opposite was observed.
When you overexpress SIAH,
you could see a drastic reduction In HIP Kinase-2.
To determine how HIV may be actually modulating SIAH levels
the authors treated, kidney cells with a N
acetyl cysteine, which is an oxygen scavenger.
So he reduces reactive oxygen species.
And what they found is that, as expected in the cells that were exposed to HIV,
you saw a decrease in the expression of SIAH.
And a concurrent increase in HIPK2 expression.
But in the cells that were pretreated with these oxidant scavenger,
the HIV infection was not able to decrease.
Other levels of SIAH and thus the levels of HIP kinase remain low.
One of the cellular events induced by HIVAN is the rapid
apoptosis of the renal tubulal epithelial cells.
And since HIP Kinase 2 was
known to be involved in apoptotic events in other systems,
the authors wanted to determine whether HIV regulation
of HIPK2 could be mediating this apoptosis event.
So what they decided to do is look at CAS base 3 activity.
CAS base 3 is one of the main mediators of apoptosis.
And so, as we can see in figure 4b,
HIV infection increases significantly the activity of kaspace3.
If you overexpress a muton of HIP Kinase 2 that lacks Kinase activity,
or you treat the cells with an siRNA to decrease the levels of Kinase 2.
You reduce this increase in Caspase 3 activity.
Suggesting that HIP Kinese 2 may be mediating this apoptotic events.
So far the authors have made a very compelling case for
the role of HIPK2, and HIV apoptosis and
also in the role of the epithelial to mesichimal transition.
But we haven't really seen so, any NBVO data.
And this is where Figure 6 comes in, where we actually see
what is the effect of reducing the levels of HIP Kinase
2 in [INAUDIBLE] or effective with HIV.
And one of the, the key panels here
is looking at the level of albionin and
creatine ratio in the urine of these mice.
And they have measured the amount of protein
in their urine as a measure of kidney function.
And we have four groups.
The wall type, the knockout, no to little expression of HIP kinase,
the Tg26 and also the,
the Tg26 that are also low expressors of HIP kinase too.
And, as expected, the Tg26 mice exhibit
very prominent proteinuria, or protein in their urine and
this decrease in function appears to be
significantly reduced in animals that have low expression
of HIPK2, suggesting that reducing the activity of
HIPK2 can have a protective effect in HIVAN.
>> And having low levels of HIPK2 can
protect against proteinuria, suggesting an enhanced filtration and kidney function.
So, we're going to conclude this lecture with a couple of take home messages.
One of the reasons why we chose this research paper is
because of the methodology used.
A research paper where they were able to analyze
high-throughput data and gain mechanistic insight.
One of the key aspects of this paper is that they had a very nice disease model,
the Tg26 mice that were expressing HIV in the kidney.
They also have done extensive
experimental validation of the role of HIP Kinase 2 at
the cellular level and all the way to the whole animal level.
So there has been extensive validation of the predictions.
And finally they have also targeted HIP Kinase 2, and
shown that it can counteract some of the kidney fibrosis observed.
Suggesting that their, they,
the HIP kinase 2 could be a therapeutic target for a kidney fibrosis.
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