David Emergy Skarda and Dr. Roger L. Kaspar, Chemistry & Biochemistry
The polypeptide Interleukin- 1 (IL-1) is one of the key mediators of the body’s response to microbial invasion, inflammation, immunological reactions, and tissue injury.1 IL- 1 gene expression regulation occurs at many steps. One of the steps currently being researched is translation. Recent research indicates that the translational regulation mechanisms of IL- 1 include an AU-rich element in the 3′ untranslated region (Fig. 1).2 In this mechanism, an induced protein binds to the AU-rich element (ARE) causing a decrease in translation efficiency. Isolation and subsequent identification of the ARE binding protein may lead to effective new drugs that induce the ARE binding protein to bind to the mRNA, turning off translation and inhibiting IL- 1 gene expression. These drugs could be used to treat pathologies such as diabetes, rheumatoid arthritis, and autoimmune disorders.
Previous UV cross-linking experiments demonstrated that a 55 kD protein binds to the ARE (Fig. 2). UV cross-linking experiments are performed by incubating MM6 cell extracts with 32P-labeled RNA containing the ARE. During incubation the radio-labeled RNA binds proteins in the cell extract for which affinity exists. The extracts are then subjected to UV light. The UV light causes covalent bonds to form between the RNA and bound protein(s) in much the same way it causes thyminethymine dimers in DNA. The cell extracts are then treated with RNase which degrades the unbound RNA leaving only the covalently bound RNA segments. This effectively radio-labels the proteins that specifically bind the RNA. The proteins are visualized by running them on an SDS-PAGE, which separates proteins by size, and autoradiography.
I used affinity chromatography to isolate the 55 kD protein. I created an affinity column using RNA. The RNA that I used was forty bases long and contained both the ARE and an amino group on the 3′ end. This amino group allowed me to create a covalent bond between RNA and Sepharose, a common column packing material. I then ran MM6 cell extracts over the column at low salt concentrations (20 mM KCI). At low salt concentrations the protein with an affinity to the ARE bound the Sepharose-RNA complex. This caused the ARE-binding protein to “stick” to the column while I washed away all other proteins by running more low salt concentration buffer over the column. Higher salt concentration disrupts the affinity between the ARE-binding protein and the RNA-Sepharose complex. I step-wise increased the salt concentration of the running buffers and collected each fraction. This allowed me to collect a fraction that contained only the ARE-binding protein.
I concentrated the fractions, ran them on an SDS-PAGE, and visualized the proteins by coomassie stain. Figure 3 shows the gel with an arrow pointing to the single protein band isolated in the 0.6 M KCI fraction. By comparison to protein standards also run on the gel, the isolated protein has a mass of 55 kD. 55 kD is the same mass as the protein shown to bind the ARE in the UV crosslinking experiments.
Future research includes using the amino acid sequence of the isolated protein to create degenerate DNA probes which will allow me to isolate, clone, and sequence the ARE-binding protein gene. 210 Continued research of the ARE-binding protein may lead to effective new drugs that induce the AREbinding protein to bind to the MRNA, turning off translation and inhibiting IL-1 gene expression. These drugs could be used to treat pathologies such as diabetes, rheumatoid arthritis, and autoimmune disorders.
References
- Dinarello, C. A., Biology of Interleukin 1, (1989) FASEB J 2, 108-115.
- Kruys, V. et al., Translational Blockade Imposed by Cytokine-Derived UA-Rich Sequences. (1989) Science. 245, 852-855.
- The aid of Dr. Masauki Nashimoto is gratefully acknowledged.