Perego, C. | Search bioRxiv

bioRxiv2021

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80 % of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinsons disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knockin mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular relocalization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

neuroscience↗

bioRxiv2018

Searching the optimal folding routes of a Complex Lasso protein

Understanding how polypeptides can efficiently and reproducibly attain a self-entangled conformation is a compelling biophysical challenge, which might shed new light on our general knowledge of protein folding. Complex Lassos, namely self-entangled protein structures characterized by a covalent loop sealed by a cysteine bridge, represent an ideal test system in the framework of entangled folding. Indeed, as cysteine bridges form in oxidizing conditions, they can be used as on/off switches of the structure topology, to investigate the role played by the backbone entanglement in the process. In the present work we have used molecular dynamics to simulate the folding of a complex lasso glycoprotein, Granulocyte-macrophage colony-stimulating factor, modeling both reducing and oxidizing conditions. Together with a well-established Go-like description, we have employed the elastic folder model, a Coarse-Grained, minimalistic representation of the polypeptide chain, driven by a structure-based angular potential. The purpose of this study is to assess the kinetically optimal pathways, in relation to the formation of the native topology. To this end we have implemented an evolutionary strategy that tunes the elastic folder model potentials to maximize the folding probability within the early stages of the dynamics. The resulting protein model is capable of folding with high success rate, avoiding the kinetic traps that hamper the efficient folding in the other tested models. Employing specifically designed topological descriptors, we could observe that the selected folding routes avoid the topological bottleneck by locking the cysteine bridge after the topology is formed. These results provide valuable insights on the selection of mechanisms in self-entangled protein folding while, at the same time, the proposed methodology can complement the usage of established minimalistic models, and draw useful guidelines for more detailed simulations.

biophysics↗