Characterization of p16 Mutants within Cancer

Kobe Ly

Rubin Lab, UCSC

Introduction

Role of p16

Within the Rubin lab, we study the cell cycle and the proteins that regulate it. For this project, we are looking at a tumor suppressor protein called p16, which regulates the G1-S phase checkpoint within the cell cycle by binding to CDK4/6 and inhibiting the complex formation of CDK4/6-Cyclin D. When CDK4/6-Cyclin D forms, the complex with phosphorylate a protein called retinoblastoma, Rb, which will release off a protein called, E2F, and the cell cycle will continue. p16 plays a major role in the inhibition of the formation of the CDK4/6-Cyclin D complex. If p16 does not work, Rb will be phosphorylated, E2F will be free to act, and the checkpoint will be passed.

Lollipop Diagram

This lollipop diagram is a representation of p16 (CDKN2A) missense mutations found in cancer. Light green lollipops represent the variant of unknown significance of p16 mutations. Dark green lollipops represent proposed drivers in p16 missense mutations reported in cancer.

Hypothesis

We hypothesize that mutations within p16 results in structural destabilization that leads to loss of CDK4/6 binding
We are testing this by determining the structures of these complexes through X-Ray crystallography
We hypothesize that small molecules can bind to mutant p16 and thermodynamically stabilize the protein, allowing p16 to bind and properly inhibit CDK4/6
Our goal is to use a compound to restabilize p16 and restore tumor suppressor function, preventing cell cycle mis-regulation.

Crystal Structure Diagram

Pymol Structure

While we currently do not have the full crystal data structure (currently a work in progress), I went into Pymol and created an example co-complex crystal structure to replicate a possible result that we would want to see. Looking at the complex on the right, the protein in blue is CDK6, the protein in pink is p16, and the protein in red is NB09 nanobody. The nanobody is here to act as a stabilizer for p16. When p16 was attempted to be purified and crystallized before, it was extremely unstable and always precipitated. The nanobody helps stabilize the p16 and make it a little happier. For this specific structure, I made a site-specific mutagenesis, turning the methionine at the 53 position of p16 into an isoleucine (M53I).

Method/Workflow

Culturing Cells

Purification / Concentration

FPLC / SEC

Setting up Plates

SEC Fractions

This is an example of what a protein gel looks like after our protein is put into the FPLC. The FPLC separates the protein in our solution by size and deposits it in 500uL fractions, with larger material exiting earlier and smaller material latter. This is a gel of our NB09 nanobody, which has a molecular weight of 12kD. When purifying our p16, we should see a similar gel, but the molecular weight should be closer to 17kD and it would be released in earlier fractions.

Crystals

Future Directions

Optimizing ADP Glo Assay to determine loss of function of multiple mutations
Obtaining X-ray crystallography data for multiple mutations
Possible development of small molecule that can stabilize p16 molecule

Acknowledgements

Carina Villegas for being an amazing and supportive mentor
Dr. Seth Rubin and the Rubin lab for their guidance and support
The Koret Scholars Program
The STEM Diversity Program
NIH Grant R35 GM145255
NSF Grant S-001061

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