Molecule Building - Carolyn Fisher used small molecule mimics to study isomer-specific signaling events involved in APP processing and signal transduction. She tested these small molecules on H4 neuroglioma cells and monitors changes in APP proteolytic products via Western Blots. She had also used ESI+ LCMS to monitor cellular uptake of these small molecules, Interestingly, a specific small molecule shows great promise for its effect on decreasing amyloidogenic processing within cells. Stay tuned for her upcoming 2015 paper documenting these results! (This project is open for new students to join)
PIN1 - Monique Rogals has been working with Pin1, which is a peptidyl-prolyl isomerase, meaning that it speeds interconversion of the xxx-Proline peptidyl bond (the omega torsion angle) between cis (0degrees) and trans (180degrees). It specifically targets phosphorylated Threonine and phosphoSerine, which is unique among human peptidyl prolyl isomerase. This allows potential regulation of the pool of potential Pin1 substrates by either addition or removal of a phosphate. The significance of this function cellullarly is varied. The most common is in protein folding, where trans peptidyl bonds are usually required to properly fold the protein. pT/pS-proline experiences a relatively high percentage of cis bonds naturally in solution, so isomerases can come along and speed interconversion back to trans bonds, commonly allowing folding. It can also alter the ability of proteins to bind with other proteins, which is largely what we in this lab care about. Monique is trying to determine if IRAK1 (Interleukin-1 receptor activated kinases 1) can interact bivalently with Pin1 and what the effects of the interactions will be on the cell environment, as well as determining if any other proteins can interact with Pin1 in a similar manner. She uses NMR titration experiments to quantify the strength of the interaction by measuring the binding affinity between the two proteins.
More PIN1 - Jeahoo Kwon is working with Pin1 as well, by using NMR to determine dissociation constants KD for phosphorylated IRAK-M (pIRAK-M) and Pin1 and the phosphomimetic mutation IRAK-M S110E and Pin1 using the 1H-15N HSQC experiment. He has also measured the Pin1 catalyzed isomerization of IRAK-M S110E and the isomerization of wild type of pIRAK-M using the ROESY NMR experiment.
VASP - Andrea Acevedo is working with VASP, an actin polymerization regulatory protein, within the domain of EVH1, and its relationship with the Zyxin, a cytoskeletal protein. She has done NMR titrations of zyxin into the EVH1 domain of VASP. The relationship of these two proteins suggests a functionality in stress fiber repair within the cell as Zyxin recruits VASP as a stress response mechanism. Andrea has analyzed the spectroscopy from the titrations together with previous experimental data from a past lab member to calculate the binding affinity of the Zyxin to the EVH1 domain, as well as assigning residue identity to peaks shown from the NMR spectroscopy of EVH1 Y39E, a mutant used to better understand the interaction models between the proteins. She is currently solving the structure of a Chimera protein between EVH1 and Zyxin by NMR spectroscopy.
Tori is working with Andrea to study if the model predicted from the analysis of the NMR spectroscopy is reflected in a more physiological conditions.
Andrea is also starting a new excited project to study the auxin-responsive negative circuit in rice, working from a molecular perspective up to a phenotypical perspective.
IRAK - Akito Nicol is working along side Jaehoo Kwon with IRAK1 in its N-terminal death domain. IRAK1 is one of many proteins involved in the complex Toll-like receptor (TLR) and Interleukin-1 receptor (IL-1R) pathways, which are used in the innate recognition of pathogen-associated molecules. Upon activation of TLR/IL-1R pathways, IRAK1 and IRAK4 are recruited by MyD88, which leads to the phosphorylation (and eventual autophosphorylation) of IRAK1. This activates the kinase activity of IRAK1. IRAK1 and IRAK4 then dissociate from the MyD88-TLR complex, and associate with TRAF6, continuing the cascade. Akito is attempting to obtain a form of IRAK1DD that is soluble as a monomer via point mutations based on a homology model between IRAK1 and IRAK-M. This would allow for structural and functional analysis of IRAK1 via NMR spectroscopy. In the long term, it is believed that this information will open new avenues for the development of drugs to treat asthma.