WIPI3 and WIPI4 ß-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy.

Autophagy is controlled by AMPK and mTOR, both of which associate with ULK1 and control the production of phosphatidylinositol 3-phosphate (PtdIns3P), a prerequisite for autophagosome formation. Here we report that WIPI3 and WIPI4 scaffold the signal control of autophagy upstream of PtdIns3P production and have a role in the PtdIns3P effector function of WIPI1-WIPI2 at nascent autophagosomes. In response to LKB1-mediated AMPK stimulation, WIPI4-ATG2 is released from a WIPI4-ATG2/AMPK-ULK1 complex and translocates to nascent autophagosomes, controlling their size, to which WIPI3, in complex with FIP200, also contributes. Upstream, WIPI3 associates with AMPK-activated TSC complex at lysosomes, regulating mTOR. Our WIPI interactome analysis reveals the scaffold functions of WIPI proteins interconnecting autophagy signal control and autophagosome formation. Our functional kinase screen uncovers a novel regulatory link between LKB1-mediated AMPK stimulation that produces a direct signal via WIPI4, and we show that the AMPK-related kinases NUAK2 and BRSK2 regulate autophagy through WIPI4.

Fluorescence-based imaging of autophagy progression by human WIPI protein detection.

Central to the process of macroautophagy (hereafter autophagy) is the formation of autophagosomes, double-membrane vesicles that sequester cytoplasmic cargo, including proteins, lipids and organelles, for lysosomal degradation and macromolecule recycling. Tight regulation of both autophagic activity and capacity is crucial to secure cellular homeostasis and aberrant autophagy is tightly linked to the development of many human diseases. Hence it is of great importance to accurately measure autophagy progression in health and disease. Members of the human WIPI ß-propeller proteins associate with autophagosomal membranes due to specific phosphatidylinositol 3-phosphate (PtdIns3P) binding at the onset of autophagy. The specific autophagosomal localization of both WIPI1 and WIPI2 (refered to as WIPI puncta) has been employed to assess autophagy using fluorescence microscopy methods, such as confocal and live-cell video microscopy and was extended for automated high-throughput image acquisition and analyses procedures. We here provide an overview on the employment of human WIPI members for the assessment of autophagy in higher eukaryotic cells, suitable for systems biology approaches such as mathematical modelling.

Structural characterization of the PPIase domain of FKBP51, a cochaperone of human Hsp90.

Steroid hormone receptors are key components of mammalian stress and sex hormone systems. Many of them rely on the Hsp90 chaperone system for full function and are further fine-tuned by Hsp90-associated peptidyl-prolyl isomerases such as FK506-binding proteins 51 and 52. FK506-binding protein 51 (FKBP51) has been shown to reduce glucocorticoid receptor signalling and has been genetically associated with human stress resilience and with numerous psychiatric disorders. The peptidyl-prolyl isomerase domain of FKBP51 contains a high-affinity binding site for the natural products FK506 and rapamycin and has further been shown to convey most of the inhibitory activity on the glucocorticoid receptor. FKBP51 has therefore become a prime new target for the treatment of stress-related affective disorders that could be amenable to structure-based drug design. Here, a series of high-resolution structures of the peptidyl-prolyl isomerase domain of FKBP51 as well as a cocrystal structure with the prototypic ligand FK506 are described. These structures provide a detailed picture of the drug-binding domain of FKBP51 and the molecular binding mode of its ligand as a starting point for the rational design of improved inhibitors.