Student Research at Duke

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

The last couple of days have been the busiest by far with days lasting from 9am to 6:30pm and coming in on the weekend Well that’s research for you! Though I can’t complain since most of the time is down-time when I’m waiting for proteins to bind, DNA to replicate, etc. Jack and I have been trying to construct and clone truncation mutants of the regulatory subunit of “protein X” which we believe interacts with our protein of interest, ALK-1. I’ll talk more about the details in my next post). Using a computer simulation program, PDraw, we planned out primers with specific restriction sites that we could use for PCR. While we waited for our primers to arrive, we went ahead and started a restriction digest with a previous aliquot of “protein X”, running a ligation over night, and transforming DH5-alpha bacteria with the “protein X” and vector constructs the next day. Additionally to show me how he had previously confirmed the existence of an interaction between ”protein X” and ALK-1, we started a Western Blot that confirmed the protein-protein interaction between the two molecules through a pull-down assay.                                                                                                                                                                                                                            As soon as the primers arrived two days later, we dived into conducting some PCR, restriction digests, gel purification, ligation, transformation…But wait we still had to purify the DNA from the DH5-alpha bacteria through a miniprep and run it on a gel to check if it worked alright…Oh and how could I forget, we needed to add the antibodies to our Western blot…essentially by Thursday, we were doing three experiments at once! While it was confusing switching between experiments (I have twelve pages of notes for Thursday and Friday alone!) it saved us lots of time–by the time we finished the first experiment and found it yield unsuccessful results, we could look on the bright side that we hadn’t wasted 4 days just doing that one experiment but still had two others in progress. Unfortunately out of the three experiments only one worked and so this week we are repeating our cloning but slightly tweaking our strategies to troubleshoot. And if we’re lucky, Jack says we might get to start a site-directed mutagenesis where we will get to make mutated constructs of our molecules of interest. I’m going to write a proposal of my research project and post it soon so stay tuned.

Yesterday I “interviewed” Dr. Blobe to find out how he got hooked on research. He has enjoyed science as long as he can remember and was proud to share with me the original copy of his first scientific paper, “The Effects of Diet Rate on Mice”…from his sixth grade science class! Although the material might be considered elementary, a glimpse of the paper showed the beginnings of a true “lab rat” as Dr. Blobe likes to refer to himself now. He continued to do research during high school and had his first research experience in a lab the summer after his freshman year at Notre Dame University. He recalls spending countless hours for six or even seven days a week (!) doing “manual labor” such as purifying ribonuleases, growing bacteria, and doing gel electrophoreses that today with the help of fancy smancy machines can be completed much more efficiently.                                                                                                                                                                                                                   After graduating with a chemistry major, Dr. Blobe went on to attend the MD/PhD program at Duke University. After completing the program, he went back to the lab for 2 years as a postdoc, got a research position while studying Protein Kinase C (PKC) and then his own lab at Duke. He recalled that the first few years were tough because in order to keep your lab you have to raise significant monetary support by writing many grants. Dr. Blobe chooses to employ all levels of researchers in his lab from undergraduates to medical students to post-docs “to provide them the opportunity and building blocks to figure out if they want to do research as a career”.                                                                                                                                                                                                                           A typical day for Dr. Blobe nowadays is as follows. He arrives at his office at 8 am and spends the day answering emails, writing grants/papers, reviewing papers, attending meetings/seminars, giving lectures to medical students/graduate students… On Tuesdays, he goes to the clinic to treat patients with colon and pancreatic cancer. At the end of the work day, he goes home, spends time with his kids, climbs on the Stairmaster while reading papers, and later spends about an hour more writing grants.  Dr. Blobe chose to conduct research particularly in cancer because he wants ”to make an impact in the future on the lives of patients with significant [medical] problems”. Finally when asked if he would like to share any last words, his first words were, “It’s the best job on earth!” He enjoys the ”constantly stimulating, changing, and growing environment and the opportunity to interact with bright, talented people.” Dr. Blobe-as both a researcher and mentor-hopes to make an impact on both the lives of patients as well as students interested in pursuing cancer research.                                                   

Welcome to my blog! As apart of the Howard Hughes Research Fellows Program I will be blogging about my summer experience and hope to share with you what I am learning. Dr. Blobe’s lab focuses on the transforming growth factor-beta (TGF-B) signal transduction pathway and and its dichotomous role in both promoting and inhibiting tumorigenesis. I will be working with Jack (a Duke surgery resident currently doing research) to find if a particular protein-let’s call it “protein X” for our purposes- interacts with the TGF-B receptor, Alk-1, and then characterize its effect on receptor signaling.                                                                                                                                                                                                                                                                          I have just finished my first week at the lab. On Monday, Tuesday, and Wednesday I observed Jack as he taught me how to do a cell culture and transfection. On Thursday and Friday he had to attend a conference. As a resident, he is a likely target for pharmaceutical companies so his entire trip-transportation, hotel, and meals-has been provided for by a company. I guess that’s one of the perks of the medical profession   So instead on Thursday I watched Liz (a fifth year graduate student) perform the final step-”detection”- of a Western Blot, i.e. incubation with a primary and secondary antibody and developing pictures of the gel. Unfortunately the results weren’t as good as Liz hoped. If I had worked nearly five days preparing and conducting such an experiment only to get so-so results I would expect to surely feel disappointed. Yet Liz didn’t appear to be too disappointed. As I have learned in the lab this past week, one shouldn’t expect to get great results his or her very first try (or even the third or fourth tries!) It takes time (and much tweaking) to get results that can even considered for publication. And though I was only in the lab for 2 hours today, I definitely had the most fun experience today. Because Jack is away, he bestowed on me the responsibility to take care of his cells so I got to do my very first cell culture!                                         Here’s  some pictures of the Blobe lab:                                               Jack at his bench pipetting for a transfection.                                       Liz and Ryan deep in conversation on the project they’re working on.                                And me at my very own desk with lots of reading to do.Thanks for reading my blog. Talk to you soon.