AMP-activated protein kinase and creatine transporter as key players in metabolism and modulation of AMPK action by designed ankyrin repeat proteins [thesis]

Nadine Muriel Straumann, Ari Helenius, Theo Wallimann
SUMMARY Mammalian S'-AMP-activated protein kinase (AMPK) belongs to a subfamily of protein serine/threonine-kinases that is highly conserved in animals, plants and yeast. The AMPK signalling cascade is thought to save cellular energy charge, and is therefore activated in an ultrasensitivemanner by cellular Stresses that compromise cellular ATP levels. The activation process involves first allosteric activation of AMPK by AMP, triggered by lowered ATP/AMP ratios, and second activation by
more » ... ylation of AMPK within the activation loop of the a-subunit kinase domain by one or more upstream kinases. AMPK is a heterotrimeric protein complex consisting of three different subunits that occur in different isoforms. Combinationof the different isoforms of the catalytical a-subunit (al, a2) and the two regulatory ßand y-subunits (ßl, ß2, yl, y2 and y3) can result in up to 12 differentAMPK isoenzymes. Severalchemical Compounds that activate or inhibit AMPK have been described. Their applicability is restricted to in vivo experiments, because they do not interact directly with AMPK but need to be metabolized or perform activation of AMPK via unknown signalling events. This also implies a certain degree of unspecificity of these Compounds, and side effects are difficult to rule out. In the thesis presentedhere, we sought for an alternativeto chemical Compounds to modulate AMPK action. In the approach described, a synthetic library construeted by Binz et al. (170) was used, which was based on the ankyrin repeat (AR) scaffold. The library molecules with a theoretical diversity of 7.2 x 107 for each module were named designed ankyrin repeat proteins (DARPins). From the synthetic DARPin library with molecules consisting of two (N2C) or three (N3C) ankyrin-repeats, we selected molecules binding to recombinant AMPKalßlyl (rAMPK) produced in E. coli by ribosomal display. Selected pools of binders were further enriched for specific binding to rAMPK with enzyme-linked immunosorbent Screening techniques. The best binders were expressed in large scale, purified by nickel-ion chromatography and used for further biochemical characterisations. Specificity and affinity of the selected binders for rAMPK were assessed by competition ELISA experiments with free rAMPK and surface plasmon resonance measurements, which revealed binding affinities in the nanomolar ränge. We further investigated the binding capability of selected DARPins to AMPK produced in mammalian Systems. Experiments that followed Standard immunopreeipitationprocedures used immobilized DARPins to enrich AMPK from 2 Summary mammalian cell extracts, thus confirming the ability of these binders to interact with AMPK
doi:10.3929/ethz-a-005068124 fatcat:tutplatsxvektkes3ys44uibge