Structural Insights Into Histone Modifying Enzymes

Author

Shruti Amle, Wayne State UniversityFollow

Access Type

WSU Access

Date of Award

January 2019

Degree Type

Thesis

Degree Name

M.S.

Department

Biochemistry and Molecular Biology

First Advisor

Zhe Yang

Abstract

X-ray crystallography is a powerful tool in the elucidation of the three-dimensional structure of bio molecules such as proteins and nucleic acids. Obtaining their structures will allow deep understanding of their biochemical mechanisms and how the alterations in the structures influence human health. X-crystallography has been used as an important approach to understand the structural and biochemical properties of histone modifying enzymes and shed light on poorly understood mechanisms.

SMYD5, one of the members of SMYD protein family, is identified as a histone lysine methyltransferase. Lysine methylation is known to modulate various biological processes including DNA damage response and gene regulation. SMYD5 is involved in hematopoiesis regulation, immune response and tumorigenesis. So far, the biochemical and structural features required to understand its methyl transferase function remains elusive. Solving the crystal structure and identifying the new substrates of SMYD5 will provide a molecular window to visualize how these biological molecules influence human health. In this study, we conducted the crystallization attempts and various biochemical assays to understand the function of SMYD5. To identify if SMYD5 can methylate non histone substrates, an artificial peptide, assumed to be the best peptide substrate for SMYD5 was designed based on the substrate selectivity profile of SMYD5. We found that SMYD5 is much more active on this peptide than its known histone substrate H4K20 in both enzyme kinetics assay and dot blot assay.

Methylation controls chromatin regulation which is catalyzed by SET domain-containing methyltransferases. SET5 was identified as the first histone methyltransferase enzyme in budding yeast. It is known to monomethylate the important H4 lysine residues, H4K5, H4K8 and H4K12. Studies have been demonstrated a functional relationship between histone H3 and histone H4 methyltransferases, whose combined activities play roles in preserving genomic integrity. Also, SET5 is recognized as human SMYD3 ortholog, which also catalyzes the methylation at H4K5. Therefore, SET5 functional studies might reveal previously unknown conserved mechanisms that may contribute to SMYD3 dependent oncogenesis in human cells. In this study, X-ray crystallographic studies were utilized to solve the crystal structure of SET5. Our initial crystallization screening has successfully resulted in Set5 crystallization. Solving its crystal structure will shed light on poorly understood mechanisms of SET5-mediated histone H4 methylation.

Rad6B plays a key role in breast cancer cell growth. It is a ubiquitin conjugating enzyme (E2) which mediates ubiquitination. It is essential for the post-replication, DNA repair and genomic integrity maintenance via its ubiquitin-conjugating activity. Alterations in ubiquitination occur frequently in cancer. Because Rad6B is overexpressed in breast cancer cells, targeting Rad6B would be a viable approach for breast cancer treatment. SMI9 is a specific small molecule inhibitor of Rad6B. It is known to inhibit Rad6B-induced histone H2A ubiquitination, induce G2–M arrest and apoptosis, downregulate intracellular β-catenin and inhibit proliferation and migration of metastatic human breast cancer cells. In our study, the crystals were obtained for Rad6B which will serve as a good starting point to and solve macromolecular structure of Rad6B in complex with SMI9. Solving the crystal structure will facilitate the development of new Rad6B inhibitors which can be used to inhibit the overexpression of Rad6B in breast cancer.

Recommended Citation

Amle, Shruti, "Structural Insights Into Histone Modifying Enzymes" (2019). Wayne State University Theses. 693.
https://digitalcommons.wayne.edu/oa_theses/693