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Access Type

WSU Access

Date of Award

January 2018

Degree Type


Degree Name



Biochemistry and Molecular Biology

First Advisor

Zhe Yang


SMYD proteins are a family of proteins known to possess a methyltransferase activity and has been shown to play important roles in cancer and immunology. Currently there are limited studies about SMYD4 linking it to muscle development and breast cancer. Our lab previously showed that SMYD4 interacts with hsp90 tail. Hsp90 is an important protein in the chaperon complex that is responsible for degrading between 30-40% of proteins through delivering them to the lysosomes. My study aimed to further understand SMYD4 by attempting to proof its’ localization via immunofluorescence microscopy inside the lysosomes of U2OS cells. Our results showed SMYD4 to not be localized inside the lysosomes of U2OS cells.

The Nogo-B receptor is known for being essential for angiogenesis where its knockdown results in embryonic lethality due to defects in vascular formation. A recent study also shows NgBR to be involved in the binding and activation of Ras to the plasma membrane. Currently the NgBR crystal structure has not been yet solved but previous studies suggested it to be a type I transmembrane protein. However, BLAST alignment and SAX analysis performed previously by our lab members suggests NgBR to be a well folded cytosolic protein resembling the structure of cis-IPTase UPPS protein. We chose to further confirm our previous data through the purification of NgBR (79-293) and using circular dichroism which indeed showed a high percentage similarity between the secondary structure of NgBR and UPPs. Additionally, we were able to purify a new NgBR (55-293) protein that includes additional amino acids from NgBR extracellular domain.

The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. SAX analysis performed using NgBR protein that includes the extracellular and transmembrane domains which contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs).

Methods such as NMR spectroscopy, electron microscopy, and X-crystallography are currently used to determine protein structure and its interactions with other proteins and ligands. Each of these method bears its’ potential advantages and disadvantages. Although X-ray crystallography has numerous approaches that are used to obtain such as antibody fragments, lipids, carrier proteins, etc. can be used to enhance chances of obtaining a protein crystal the success unpredictability of one single method still hinders protein crystallization attempts. In our study we selected X-ray crystallography as a starting point to solve the 3D structure of Nogo-B receptor ((NgBR) protein. Our study successfully expressed and purified NgBR (79-293). This construct was then used to perform circular dichroism. We further confirmed our previous SAX analysis results that showed NgBR to be well folded in solution and resembles UPPS secondary structure content. These findings are contradictory to the currently acceptable topology of NgBR which classifies it as type I transmembrane protein. To enhance the solubility and expression of NgBR (55-293) we designed and performed small scale expression test on fusion construct MBP-NgBR (55-293). These newly expressed constructs open novel opportunities to obtain crystalize and solve the macromolecular structure of NgBR.

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