Protein Interaction Module–assisted Function X (PIMAX) Approach to Producing Challenging Proteins Including Hyperphosphorylated Tau and Active CDK5/p25 Kinase Complex

Abstract

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction moduleassisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3␤ and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research. Molecular & Cellular Proteomics

Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death

“…The plasmids pMK1013-GSK-tau(1N4R) and pMK1013-tau(1N4R) for the expression of the 1N4R tau and p-tau were previously described (43). This p-tau was used throughout the main portion of this study.…”

“…We wished to use the recombinant DNA technology for the synthesis of hyperphosphorylated tau to support both mechanistic and drug development research. To this end, we used the PIMAX method (43), in which the Fos and Jun leucine zipper domains were respectively fused to the GSK-3β kinase and the human 1N4R isoform of tau. Upon bacterial coexpression, Fos-Jun heterodimerization brought GSK-3β and tau to close proximity, resulting in e cient phosphorylation of the latter (43).…”

“…However, most of these works shared one limitation, that is, the nearly exclusive use of unmodi ed tau lacking altogether the pathological feature of phosphorylation. Unlike hyperphosphorylated tau that aggregates spontaneously (43), unmodi ed tau relies on an arti cial inducer such as heparin or micelles of fatty acids to accelerate brillization (44,45). The pathophysiological signi cance of these inducers has not been fully explored.…”

“…To facilitate mechanistic studies of the molecular mechanisms underlying neurodegeneration of tauopathies, and to enable neuro brillary tangle-based drug development, we report here the procurement of hyperphosphorylated tau (p-tau) via the use of the PIMAX system (Protein Interaction Modules-Assisted function X) (43). This p-tau exhibited multiple disease-relevant molecular characters.…”

Traditional Chinese Medicine Huannao Yicong Decoction Extract Decreases Tau Hyperphosphorylation in the Brain of Alzheimer’s Disease Model Rats Induced by Aβ1–42

“…Increased A β could induce tau protein phosphorylation by disrupting the balance of kinases and phosphatases. The kinases involved in the phosphorylation of tau protein include GSK-3 and CDK5 [12, 13]. Interestingly, in a Drosophila model of AD, aberrant expression of GSK-3 and inhibition of GSK-3 both could significantly improve A β -induced neurotoxicity [14].…”