Mechanistic insights into RNase L through use of an MDMX-derived multi-functional protein domain

作者：Garvie Colin W^*; Vasanthavada Keshav; Xiang Qing

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2013, 1834(8): 1562-1571.

DOI：10.1016/j.bbapap.2013.04.010

• RNase L
• Dimerization
• MDMX
• Affinity purification
• Ribonuclease
• FRET assay

RNase L is part of the innate immune response to viral infection. It is activated by a small oligonucleotide (2-5A) whose synthesis is initiated as part of the interferon response. Binding of 2-5A to the N-terminal regulatory region, the ANK domain, of RNase L activates its ribonuclease activity and results in cleavage of RNA in the cell, which ultimately leads to apoptosis of the infected cell. The mechanism by which 2-5A activates the ribonuclease activity of RNase L is currently unclear but 2-5A has been shown to induce dimerization of RNase L To investigate the importance of dimerization of RNase L, we developed a 15 kDa dimerization-inducing protein domain that was fused to the N-terminus of RNase L From these studies we provide direct evidence that dimerization of RNase L occurs at physiologically relevant protein concentrations and correlates with activation of ribonuclease activity. We also show that the binding of 2-5A to RNase L promotes dimerization of the ANK domain and suggest how this could transmit a signal to the rest of the protein to activate ribonuclease activity. Finally, we show that the dimerization-inducing domain can be used as a general fusion partner to aid in protein expression and purification.

• 出版日期2013-8