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Access Type
WSU Access
Date of Award
January 2022
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Young-Hoon Ahn
Abstract
SMYD2, a protein lysine methyltransferase, is one of the proteins reported to be involved in the development and organization of striated muscle. We recently reported the loss of sarcomere alignment in rat cardiomyocytes during oxidative stress. This disorganization was associated with the loss of SMYD2’s interaction resulting from its glutathionylation. Further in vitro analysis indicated that titin was digested in myofibrils treated by the protease MMP2, and the digestion was reduced in the presence of SMYD2. Also, a unique domain of titin known as the N2A region was protected from MMP2 and calpain-1 in the presence of SMYD2. MMP2 and calpain-1 are proteases upregulated during stress in muscle cells.The importance of the N2A titin domain lies in its propensity for protein-protein interactions that may modulate signaling cascades. N2A consists of four immunoglobulin (Ig) sub-domains: Ig80, Ig81, Ig82, Ig83, and a domain called the unique insertion (uN2A), sandwiched between Ig80 and Ig81. Although SMYD2’s binding to N2A and its protection has been shown, identifying the sub-domains of N2A mediating its interaction with SMYD2 will help understand how SMYD2 protects it from proteases. Using molecular cloning techniques, we made various sub-domain constructs of the N2A domain and quantitatively studied their binding with SMYD2 by bio-layer interferometry (BLI). We have identified Ig80 as the N2A domain facilitating SMYD2’s binding to N2A. We also mapped the amino acid sequences in Ig80 that enable binding to SMYD2, which was found as a PAVAP motif in the proximity of its N-terminus. Furthermore, we identified an additional motif within Ig80 that interacts with SMYD2. Our subsequent analysis revealed a 13-sequence residue within Ig80 that may be exposed to bind to SMYD2 during the unfolding of Ig80. As Ig80 unfolding may occur during the muscle contraction and relaxation or pathologic conditions, our data may find a novel functional role of SMYD2 associated with an unfolded state of Ig80. Finally, we found that SMYD2 was able to bind and protect Ig80 that is susceptible to digestion by calpain-1. This indicates that Ig80 may be the segment of N2A predisposing its vulnerability to proteases. Overall, our findings present a new understanding of SMYD2’s interaction in titin. SMYD2, a protein lysine methyltransferase, is one of the proteins reported to be involved in the development and organization of striated muscle. We recently reported the loss of sarcomere alignment in rat cardiomyocytes during oxidative stress. This disorganization was associated with the loss of SMYD2’s interaction resulting from its glutathionylation. Further in vitro analysis indicated that titin was digested in myofibrils treated by the protease MMP2, and the digestion was reduced in the presence of SMYD2. Also, a unique domain of titin known as the N2A region was protected from MMP2 and calpain-1 in the presence of SMYD2. MMP2 and calpain-1 are proteases upregulated during stress in muscle cells. The importance of the N2A titin domain lies in its propensity for protein-protein interactions that may modulate signaling cascades. N2A consists of four immunoglobulin (Ig) sub-domains: Ig80, Ig81, Ig82, Ig83, and a domain called the unique insertion (uN2A), sandwiched between Ig80 and Ig81. Although SMYD2’s binding to N2A and its protection has been shown, identifying the sub-domains of N2A mediating its interaction with SMYD2 will help understand how SMYD2 protects it from proteases. Using molecular cloning techniques, we made various sub-domain constructs of the N2A domain and quantitatively studied their binding with SMYD2 by bio-layer interferometry (BLI). We have identified Ig80 as the N2A domain facilitating SMYD2’s binding to N2A. We also mapped the amino acid sequences in Ig80 that enable binding to SMYD2, which was found as a PAVAP motif in the proximity of its N-terminus. Furthermore, we identified an additional motif within Ig80 that interacts with SMYD2. Our subsequent analysis revealed a 13-sequence residue within Ig80 that may be exposed to bind to SMYD2 during the unfolding of Ig80. As Ig80 unfolding may occur during the muscle contraction and relaxation or pathologic conditions, our data may find a novel functional role of SMYD2 associated with an unfolded state of Ig80. Finally, we found that SMYD2 was able to bind and protect Ig80 that is susceptible to digestion by calpain-1. This indicates that Ig80 may be the segment of N2A predisposing its vulnerability to proteases. Overall, our findings present a new understanding of SMYD2’s interaction in titin.
Recommended Citation
Adewale, Adeleye Olushola, "Biophysical Characterization Of Smyd2’s Binding With N2a Titin" (2022). Wayne State University Dissertations. 3586.
https://digitalcommons.wayne.edu/oa_dissertations/3586