Protein interaction studies on the titin A150 domain using proximity-dependent biotinylation

Titin is an enormous protein that spans over 1 µm in the sarcomere, making it the largest protein in the human body. A single missense variant within the titin A150 domain, also called FN3 119, is sufficient to cause a dominant myopathy known as Hereditary Myopathy with Early Respiratory Failure (HMERF), with the most prevalent disease-causing variant being the c.95134T>C (p.C31712R) variant. HMERF is characterized by weakness in proximal and distal skeletal muscles and severe, life-threatening, respiratory insufficiency. Despite its clinical significance, the pathomechanism behind the disease remains unknown. The aim of this thesis was to screen for protein-protein interactions of both the wild-type titin A150 domain and the A150 domain containing the disease-causing variant p.C31712R, with the goal of gaining deeper insight into the protein interactions of titin and the molecular mechanism behind HMERF. In this study, proximity dependent biotinylation (PDB) was used to screen for protein interactions. PDB was conducted using the biotin-ligase UltraID fused to a titin fragment. This study encompasses the creation of the titin-UltraID fusion genes, the functional validation of the titin UltraID proteins, the development of a PDB workflow and the analysis of identified proteins. The study was conducted in stable, differentiated and electrostimulated C2C12 myotubes grown on a gelatin hydrogel. Although the proteins identified were not statistically significant, this thesis provides insight into potential interactors of the titin A150 domain and the A150 domain with the pathogenic p.C31712R variant. This thesis also offers insight into the use of PDB for studying titinopathies in a C2C12 cell model. However, further validation is needed to fully substantiate the findings in this thesis.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2025

Author dc:creator

• Vainio, Anna Linnéa

• titin
• titinopathy
• Hereditary myopathy with early respiratory failure
• HMERF
• proximity dependent biotinylation
• protein interactomics
• UltraID
• BioID
• biotin labelling
• proximity labelling
• myopathy

Statement dc:rights

• CC BY-NC-ND 4.0

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/591384

OAI identifier oai:identifier

oai:helda.helsinki.fi:10138/591384