results

In human hepatic cells (HepG2), PTP1B expression was silenced by using small interfering RNA (siRNA), in order to be compared with control hepatocytes. They were treated with Thapsigargin (10 μL from stock  1μΜ into 2 ml  media, experiment 1) and Tunicamycin (10 μL from stock 5 mg/mL into 2 ml  media, experiment 2) in order to cause them ER stress . They were cultured in DMEM media (10% Fetal Bovine Syrom, 1% Pen/Strep). They were collected after 0.5h, 1h, 1.5h, 2h, 4h, 6h and 8h, and they were lysed. By using Western Blotting technique the following results were extracted.

Results and Outcomes

Experiment 1: ER stress caused by Thapsigargin on human HepG2 cells +/- PTP1B

Figure 1: the first experiment was conducted for n=2 but due to implications to western blotting it was analysed for n=1. This figure shows that there is an increase in PTP1B levels with ER stress in control cells (CTL siRNA) after the 6^th hour. PTP1B siRNA seems to prevent this increase.

Phosphorylation of eIF2-alpha peaked ~2h of ER stress induction and then reduced to normal levels by 8h. BIP expression levels increased more slowly, peaking at 8h. No significant differences were seen with PTP1B siRNA in this experiment.

Experiment 2: ER stress caused by Tunicamycin on human HepG2 cells +/- PTP1B

Figure 2: in order to support the results of experiment 1 the same procedure was followed, but this time the design was changed for practicing reasons. Each sample was loaded in duplicates. This figure shows that PTP1B siRNA decreases PTP1B levels inconsistently. Also it was concluded that Tunicamycin did not work as well as Thapsigargin in terms of causing ER stress in this experiment.

 Unfortunately, there was not enough time to repeat the experiments, and due to difficulties with western blotting I was not able to present significant results.  However, some conclusions can be extracted. ER stress agents thapsigargin and tunicamycin cause phosphorylation of eIF2-alpha and increase expression of Bip in a time dependant manner. SiRNA technology was inconsistent in knocking down PTP1B levels in HepG2 cells. In order to improve this outcome of this short-time project in the future we should repeat the experiments, change the concentrations of primary and secondary antibodies and finally investigate the role of PTP1B in association with the other ER stress factors, e.g. eIF2a, ATF4, BIP, GADD34.

Thanks to British Biochemical Society and Dr Mody, I had the opportunity to practice my techniques and to apply my knowledge acquired from the taught lectures, as well as to learn new methods and to get involved with the latest technology. Now I feel much more confident in the laboratory. I learned how to extract, analyse and interpret  data, how to efficiently cooperate within a laboratory team, and how to overcome the everyday’s difficulties and frustrations that research reserves, motivating myself to work harder. Until having this opportunity, research was only one of my many career aspirations. Now, after finishing this studentship, I feel that my future belongs to research!

Thank you very much

Petros

1st Week

Hi!!

To be honest I didn’t expect such a work load. I thought that the first days would have been spent on boring introductions and a lot of DON’Ts, but no….

After meeting the lab group I wore my lab-coat and straight to the lab. The fact is that I had already been in the same lab last summer voluntary spending a month on familiarizing myself with the “lab” concept and gaining my first experiences on field. So there was no excuse for time wasting. However rehearsing was necessary so I did a lot of pipetting   and practicing. Also safety issues were discussed and of course I met our mice!!!! I had no clue until now on how working with animals and I have to admit that I am really impressed on how people here treat them, how respectful they are and how strict the rules are. Anyhow the first contact was really something special for me. I could never imagine myself holding a mouse, but now I feel that I am doing that for ages……

Let’s have a look on the project now.

The project I am involved with is the delineation of the role of protein tyrosine phosphatase 1B (PTP1B) in endoplasmic reticulum stress in liver and adipose tissue.

Endoplasmic reticulum (ER) is an organelle responsible for many functions such as protein folding, lipid biosynthesis, calcium storage and release. Many physiological or chemical changes in ER lead to ER stress which can contribute to insulin resistance. Prevention of ER stabilization leads to cell apoptosis and cell death. A homeostatic response to this ER stress is the activation of unfolded protein response pathway (UPR).  UPR works in 3 ways: 1) activates the transcription factor ATF6 that up-regulates ER chaperones to prevent the protein aggregation. 2) Inhibits protein synthesis through the PERK kinase activation and 3) disposition of accumulated unfolded proteins through proteosomal degradation (IRE1).

We are particularly interested on PERK (protein kinase RNA – like ER kinase). PERK decreases translation by phosphorylating eukaryotic translation initiation factor (eIF2)-α which regulates the mRNA mechanism, resulting cell cycle arrest which attenuates global protein translation through inhibition of the eIF2B complex, leading to a rapid reduction in the abundance of folding proteins in the ER. eIF2α  phosphorylation stimulates Activating Transcription Factor 4 (ATF4) production, which activates the transcription of  CHOP and GADD34, that help in the dephosphorylation of eIF2α, through the activation of the Protein Serine/Threonine Phosphotase (PP1), allowing the resumption of protein synthesis. 

    

Simplifying, obesity is a factor that causes ER stress. ER stress causes cease of protein synthesis. This inhibition is retracted by Protein Phosphatase-1 bounded with GADD34.

As I mentioned before, ER-stress is associated with insulin resistance. PTP1B plays an essential role in metabolism by decreasing insulin and leptin signaling because is a negative regulator of metabolism.The phosphate group from tyrosine phosphorylated proteins is removed by Protein Tyrosine Phosphotases (PTPs). The prototype of them is the PTP1B. Therefore PTP1B seems to play an important role in obesity and diabetes. The aim of this project is to investigate how PTP1B regulates the ER-stress response.

It is not simple at all!!!!

I spent my first week learning about cultures. How important it is to follow the rules, especially when you are working under the hood, I learned to hate contamination, and to use the equipment propertly. However the theory is not enough. You have to get involved. So I got my own cells to run my project! I started with HepG2 (hepatic) cells, and I learned how to change the media, and afterwards how to collect them and split them in new 6 wall plates and flasks. The next step was to cause them pharmocological ER stress for different periods of time and then to extract the protein for further analysis. Then we used BCA protein assay  (colorimetric assay) determine the concentration of the protein. Next step : Western Blotting. Next week I hope that I will be able to represent you with some first results.

Until now everything is exciting. Every hour I am learning something new. The secret: keep taking detailed notes, asking questions and reading!

Before finishing my first post I want to thank British Biochemical Society for giving me this studentship, and for giving me the chance to work in an active lab.