Tau is a microtubule associated protein that is found primarily in neurons, and in pathological conditions such as Alzheimer disease (AD) it accumulates and contributes to the disease process. Since tau plays a fundamental role in the pathogenesis of AD and other tauopathies, and in AD mouse models reducing tau levels improves outcomes, approaches that facilitate tau clearance are being considered as therapeutic strategies. However, fundamental to the development of such interventions is a clearer understanding of the mechanisms that regulate tau clearance. Here we report a novel mechanism of tau degradation mediated by the co-chaperone BAG3. BAG3 has been shown to be an essential component of a complex that targets substrates to the autophagy pathway for degradation. In rat primary neurons, activation of autophagy by inhibition of proteasome activity or treatment with trehalose resulted in significant decreases in tau and phospho-tau levels. These treatments also induced an upregulation of BAG3. Proteasome inhibition activated JNK, which was responsible for the upregulation of BAG3 and increased tau clearance. Inhibiting JNK or knocking down BAG3 blocked the proteasome inhibition-induced decreases in tau. Further, BAG3 overexpression alone resulted in significant decreases in tau and phospho-tau levels in neurons. These results indicate that BAG3 plays a critical role in regulating the levels of tau in neurons, and interventions that increase BAG3 levels could provide a therapeutic approach in the treatment of AD.

No abstract

No abstract

ObjectiveThe goal of this project is to elucidate the mechanism of action of starch dikinases in carbohydrate metabolism using the red alga Cyanidioschyzon merolae (Cm) as a model system.SignificanceHighly regulated reversible starch phosphorylation is key for starch metabolism in photosynthetic organisms such as plants and algae. Defining the enzymes that are involved in reversible starch phosphorylation in C. merolae will allow us to engineer strategies to harness starch production in a more efficient manner for many industrial applications.MethodsBioinformatics to identify conserved functional domains with orthologs from different kingdoms. Radiometric kinase assays to measure specificity and kinetic parameters of starch dikinases. Differential Scanning Fluorimetry (DSF) to characterize glucan binding and protein thermal stability. Cell biology techniques such as gene suppression, synchronized cultures, mRNA extraction, and immunofluorescence. Structural analysis through Small Angle X‐Ray Scattering (SAXS), protein purification, and western analysis.ResultsWe show here that C. merolae GWD shares conserved functional domains with orthologs from many different kingdoms. Validating Cm‐GWD is a functional glucan dikinase, results show robust Cm‐GWD specificity towards phosphorylating the C6 position of glucose residues. Radiometric‐ATP incorporation assays reveals that recombinant Cm‐GWD is as efficient as Arabidopsis thaliana (At)‐GWD at phosphorylating starch. Lastly, we utilize SAXS to structurally characterize each domain of the multi‐domain Cm‐GWD.ConclusionWe have defined C. merolae growth and optimal starch accumulation parameters as well as cell localization of enzymes involved in reversible starch phosphorylation. Furthermore, we have characterized gene expression of Cm‐GWD via mRNA levels during synchronous cultures. Additionally, this study exposes biochemical targets to enhance starch metabolism in planta. The biochemical properties of Cm‐GWD enable increased starch metabolism not only in C. merolae but in exogenous plants.Support or Funding InformationKSEF grants KSEF‐2268RDE‐014 and KSEF‐2971‐RDE‐017; NSF Grants IIA‐1355438 and MCB‐1252345

BackgroundGlucan phosphatases are a unique subset of the Dual Specificity Phosphatase (DSP) family that binds and dephosphorylates carbohydrate substrates. They are found in widely divergent organisms ranging from extremophilic single‐cell red algae to humans and act on different carbohydrate (starch or glycogen) substrates. Red algae, Cyanidioschyzon merolae (Cm) possesses a single glucan phosphatase with three structural domains which are different in family and organization compared to other known glucan phosphatase family members. Further, bioinformatics analysis suggested that it is most similar to a vertebrate glucan phosphatase rather than plant glucan phosphatases. This is intriguing, since its endogenous substrate is starch rather than glycogen.HypothesisCm‐laforin is uniquely stable and active glucan phosphatase and can be an important partner with other catabolic enzymes in the process of starch metabolism.ObjectiveExplore the structural basis for Cm‐laforin's function as a novel glucan phosphatase.MethodsWe expressed and purified the full, tandem, and isolated carbohydrate binding (CBM) and phosphatase (DSP) domains of Cm‐laforin to study their thermal stability, phosphatase function, and domain coupling. We utilized para‐nitrophenylphosphate (p‐NPP) substrate for generic and amylopectin in malachite green assay for specific phosphatase activities determination. Differential scanning fluorimetry (DSF) and starch binding assays were utilized for glucan binding studies. The oligomeric state of laforin was determined by Size Exclusion Chromatography with Multi‐Angle static Light Scattering (SEC‐MALS). X‐ray crystallography was utilized for structural studies, which allowed structure‐guided mutagenesis.ResultsCm‐laforin is found to be a stable and highly active glucan phosphatase. The full three‐domain protein was found to be highly active and have maximum phosphatase activity at 55°C. Further, it possesses 2‐fold higher specific glucan phosphatase activity compared to vertebrate laforin orthologs. Utilizing the tools of structural biology, we explored the importance of different amino acids of the DSP domain required for its stability. We determined the structure of the DSP domain at 1.5Å and found that Cm‐laforin utilizes a significant number of unique electrostatic interactions centered on D89 to maintain its significantly higher stability. We also found that CBM1 and DSP domain can be expressed and function in isolation, but CBM1 and linker region between CBM2 and DSP are required for the proper folding and stability of CBM2. We further demonstrate that Cm‐laforin exists as a monomer, and that the tandem CBM domains are required for high affinity glucan binding and enzymatic coupling during starch degradation.ConclusionCm‐laforin is enzymatically highly active and stable phosphatase with unique structural and functional features that underlie its role as a specific glucan phosphatase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.