Student Research at Duke

So because Wednesday was July 4th, this week has been slower than usual but nonetheless much was accomplished. First, on Friday Jack and I presented an article about TGF-B signalling in prostate cancer stromal cells for our lab’s biweekly journal club meeting. It was interesting to learn about what approaches other labs are doing to study TGF-B. We did another Western blot to detect if our ”protein X” fragments were successfully expressed. However, since we still didn’t detect any signal for the fragments of interest, we immunoprecipitated the fragments with an antibody before doing the Western to reduce any nonspontaneous proteins binding to the membrane. As far as the mutagenesis experiment is going, we have transformed and minipreped the truncation mutants (1-193, 1-200) and the point mutant (V175A) and sent those for sequencing. On Friday, we found out that luckily one of samples of ”protein X” 140-215 had the correct sequence so we will be able to maxiprep the bacteria and be ready to use it for future experiments.

This week we have been busy presenting our chalk talks to our fellow students and this afternoon Dr. Willard will be lecturing about genomics. I enjoyed listening to everyone’s talks and found it particularly interesting when a familiar technique, cell line, etc. was mentioned, reminding me just how much I have learned these past four weeks. On Monday I followed Tam and did my first maxiprep, a technique to grow large quantities of bacteria with your cloned vectors and later transfected those in Cos-7 cells. Tuesday I ran a gel to check our miniprep products from the previous week so that Wednesday we were able to send the DNA (”protein X” fragments 1-139 and 140-215) for sequencing. We also started our mutagenesis experiments and are busy PCRing, digesting and transforming these past two days. Unfortunately one of our gels had holes in it so I realized too late that our samples were leaking out so we have to redo those digestions. Also we’re doing a Western blot for the nonmutated “protein X” fragments (that we sent for sequencing). Anywho after locopops at the seminar today, Julie Stevenson and I are off to her house for the weekend. Look out for an update next week then!

Here’s an overview of my project. If you want to learn more, come to hear my chalk talk next week!

Protein-protein interaction between ”Protein X” and ALK-1

Big Picture:
Transforming growth factor beta (TGFβ) is a regulator of multiple cellular processes including proliferation, differentiation, and cell survival. Specific cell surface receptors bind to the TGFβ ligand and initiate an intracellular signaling cascade through the phosphorylation of transcription factors, the Smads.                                                                                                                        Here is a picture of the canonical TGF-beta signaling pathway.  (Blobe, GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med. 2000 May 4;342(18):1350-8.)

One specific TGFβ receptor, ALK-1, expressed specifically on endothelial cells is essential for angiogenesis. A mutation of ALK-1 is proposed to result in the disease hereditary hemorrhagic telangiectasia type 2 (HHT-2). HHT is an autosomal dominant disease characterized by vascular dysplasia. ALK-1 has been shown to interact with several intracellular binding partners. One such partner has been identified as the regulatory subunit of “protein X”. Studying how ALK-1 interacts with these other proteins might allow us to better understand the ALK-1 signaling pathway and propose a way to target the pathway and treat such a devastating human disease.

Research Questions:
1) Do “protein X” and ALK-1 interact?
Yes, according to yeast two hybrid screen performed previously and confirmed by GST pull-down and co-immunoprecipitation experiments                                                                          

2a) Where is the binding site for ALK-1 on “protein X”?
-Hypothesis: somewhere near the C-terminus (similar to other serine/threonine kinases) -What we’re working on now (Week of June 4/11/17): Molecular Cloning of Truncation Mutants of “Protein X”—creating plasmids that contain fragments of “protein X” to identify the location of binding site