What is the function of polyQ stretches in the C-terminal tails of yeast 14-3-3 isoforms?

 

D. Urychova1,2, P. Herman3, J. Vecer3, T. Obsil1,2, V. Obsilova1

 

1Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague 4, Czech Republic

2Faculty of Science, Charles University, 12843 Prague, Czech Republic

3Faculty of Mathematics and Physics, Institute of Physics, Charles University; 12116 Prague, Czech Republic

dana_urychova@post.cz

 

The polyglutamine (polyQ) sequences of certain proteins are known to induce protein aggregation that underlies the cytotoxicity of these molecules. It has been shown that peptides containing polyQ stretches adopt β-structure and can form β-sheets (both antiparallel and parallel), β-hairpins, and also highly compact random coil [1,2].

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. While lower eukaryotes, e.g. yeast, contain only two 14-3-3 genes, higher eukaryotes possess up to 15 14-3-3 genes. For example, in mammals seven isoforms have been identified to date. The presence of multiple 14-3-3 isoforms over the wide range of species suggests that individual isoforms can interact with different targets. Maximal isoform sequence variability occurs within the C-terminal stretch, a region that is believed to be flexible. The structure of this part of 14-3-3 molecule is unknown because it cannot be seen in any of the available 14-3-3 x-ray structures presumably due to disorder. It has been shown that 14-3-3 C-terminal stretch is involved in the regulation of ligand binding and in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 molecule [3].

Yeast 14-3-3 isoforms (BMH1 and BMH2) compared to mammalian and plant isoforms, posses significantly longer C-terminal tails that contain polyQ sequences with unknown function. In this work, we used methods of fluorescence spectroscopy and studied conformational properties of these segments. Site-directed mutagenesis was used to generate single-tryptophan mutants of BMH proteins with the Trp residue located at both sides of polyQ stretches. Time-resolved fluorescence measurements revealed that ligand (phosphopeptide) binding does not affect the conformation of C-terminal segments of BMH proteins. It seems, therefore, that the C-terminal tails of BMH proteins do not function as autoinhibitors which are ejected from the ligand binding groove during the ligand binding (as has been observed for mammalian isoforms). In addition, fluorescence anisotropy measurements revealed that BMH proteins form oligomers bigger than expected dimmers. The presence of these oligomers was also confirmed using DLS. Therefore we speculate that polyQ stretches located within the C-terminal tails of BMH proteins induce their oligomerization.         

 

References

1.     E. Scherzinger, R. Lurz, M. Turmaine, L. Mangiarini, B. Hollenbach, R. Hasenbank, G.P. Bates, S.W. Davies, H. Lerach, E.E. Wanker, Cell., 90, (1997), 549.

2.     C.A. Ross, M.A. Poirier, E.E. Wanker, M. Amzel P.N.A.S. USA., 100, (2003), 1.

4.     V. Obsilova, P. Herman, J. Vecer, M. Sulc, J. Teisinger, T. Obsil, J. Biol. Chem., 279, (2004), 4531.

 

Acknowledgements.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.