Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients.

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90ß, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1-/- cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Discerning Dynamic Signatures of Membrane-Bound α-Synuclein Using Site-Specific Fluorescence Depolarization Kinetics.

α-Synuclein is an intrinsically disordered protein that adopts an α-helical structure upon binding to the negatively charged lipid membrane. Binding-induced conformational change of α-synuclein plays a crucial role in the regulation of synaptic plasticity. In this work, we utilized the fluorescence depolarization kinetics methodology to gain the site-specific dynamical insights into the membrane-bound α-synuclein. We took advantage of the nonoccurrence of Cys in α-synuclein and created single-Cys variants at different sites for us to be able to label it with a thiol-active fluorophore. Our fluorescence depolarization results reveal the presence of three dynamically distinct types of motions of α-synuclein on POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) small unilamellar vesicles (SUVs): (i) the (local) wobbling-in-cone motion of the fluorophore on the subnanosecond timescale, (ii) the backbone segmental mobility on the nanosecond timescale, and (iii) a slow depolarization component with a characteristic long rotational correlation time (∼60 ns) that is independent of the residue position. This characteristic timescale could potentially arise due to global tumbling of the protein-membrane complex, the global reorientation of only the protein within the membrane, and/or the translation diffusion of the protein on the curved membrane surface that could result in fluorescence depolarization due to the angular displacement of the transition dipole. In order to discern the molecular origin of the characteristic long rotational correlation time, we then carried our depolarization experiments varying the curvature of the membrane and varying the binding affinity by changing the lipid headgroup. These experiments revealed that the long rotational correlation time primarily arises due to the translational diffusion of α-synuclein on the curved membrane surface with a diffusion coefficient of ∼8.7 × 10-10 m2/s. The site-specific fluorescence depolarization methodology will find broad application in quantifying diffusion of a wide range of membrane-associated proteins involved in functions and diseases.

Cancerous perturbations within the ERK, PI3K/Akt, and Wnt/ß-catenin signaling network constitutively activate inter-pathway positive feedback loops.

Perturbations in molecular signaling pathways are a result of genetic or epigenetic alterations, which may lead to malignant transformation of cells. Despite cellular robustness, specific genetic or epigenetic changes of any gene can trigger a cascade of failures, which result in the malfunctioning of cell signaling pathways and lead to cancer phenotypes. The extent of cellular robustness has a link with the architecture of the network such as feedback and feedforward loops. Perturbation in components within feedback loops causes a transition from a regulated to a persistently activated state and results in uncontrolled cell growth. This work represents the mathematical and quantitative modeling of ERK, PI3K/Akt, and Wnt/ß-catenin signaling crosstalk to show the dynamics of signaling responses during genetic and epigenetic changes in cancer. ERK, PI3K/Akt, and Wnt/ß-catenin signaling crosstalk networks include both intra and inter-pathway feedback loops which function in a controlled fashion in a healthy cell. Our results show that cancerous perturbations of components such as EGFR, Ras, B-Raf, PTEN, and components of the destruction complex cause extreme fragility in the network and constitutively activate inter-pathway positive feedback loops. We observed that the aberrant signaling response due to the failure of specific network components is transmitted throughout the network via crosstalk, generating an additive effect on cancer growth and proliferation.