RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways

The conserved lid tryptophan, W211, potentiates thermostability and thermoactivity in bacterial thermoalkalophilic lipases

We hypothesize that aggregation of thermoalkalophilic lipases could be a thermostability mechanism. The conserved tryptophans (W211, W234) in the lid are of particular interest owing to their previous involvements in aggregation and thermostability mechanisms in many other proteins. The thermoalkalophilic lipase from Bacillus thermocatenulatus (BTL2) and its mutants (W211A, W234A) were expressed and purified to homogeneity. We found that, when aggregated, BTL2 is more thermostable than its non-aggregating form, showing that aggregation potentiates thermostability in the thermoalkalophilic lipase. Among the two lid mutants, the W211A lowered aggregation tendency drastically and resulted in a much less thermostable variant of BTL2, which indicated that W211 stabilizes the intermolecular interactions in BTL2 aggregates. Further thermoactivity and CD spectroscopy analyses showed that W211A also led to a strong decrease in the optimal and the melting temperature of BTL2, implying stabilization by W211 also to the intramolecular interactions. The other lid mutant W234A had no effects on these properties. Finally, we analyzed the molecular basis of these experimental findings in-silico using the dimer (PDB ID: 1KU0) and the monomer (PDB ID: 2W22) lipase structures. The computational analyses confirmed that W211 stabilized the intermolecular interactions in the dimer lipase and it is critical to the stability of the monomer lipase. Explicitly W211 confers stability to the dimer and the monomer lipase through distinct aromatic interactions with Y273-Y282 and H87-P232 respectively. The insights revealed by this work shed light not only on the mechanism of thermostability and its relation to aggregation but also on the particular role of the conserved lid tryptophan in the thermoalkalophilic lipases

Direct in vitro selection of titanium-binding epidermal growth factor

Epidermal growth factor (EGF) with affinity to TiO2 surfaces was obtained by direct in vitro selection. A random peptide library was generated for fusion to the N-terminal of EGF, and polypeptides exhibiting affinity were selected in vitro by ribosome display. The best-performing polypeptide sequence was selected for synthesis using a solid-phase method and showed high affinity to TiO2 after refolding. Molecular dynamic simulations indicated that the interaction of the selected peptide segment with the TiO2 surface was comparable to that of a previously reported titanium-binding peptide, TBP-1. The hydroxyl groups in the selected peptide segment were found to be critical for the binding interaction. NIH3T3 cell culture for two days in the presence of the TiO2-binding EGF showed that it was able to enhance cell proliferation as much as unmodified EGF in solution. As a result, the selected EGF construct was able to induce cell proliferation on titanium surfaces. This direct in vitro selection technique should extend the possibilities for the design of other surface-binding growth factors