DnaK promotes autophosphorylation of DYRK1A and its family kinases in Escherichia coli-based cell-free protein expression.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.

Expression and purification of DYRK1A kinase domain in complex with its folding intermediate-selective inhibitor FINDY.

The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.

Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.

DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.

Regulation of primary metabolic pathways in oyster mushroom mycelia induced by blue light stimulation: accumulation of shikimic acid.

Shikimic acid is a key intermediate in the aromatic amino acid pathway as well as an important starting material for the synthesis of Tamiflu, a potent and selective inhibitor of the neuraminidase enzyme of influenza viruses A and B. Here we report that in oyster mushroom (Pleurotus ostreatus) mycelia cultivated in the dark, stimulation with blue light-emitting diodes induces the accumulation of shikimic acid. An integrated analysis of primary metabolites, gene expression and protein expression suggests that the accumulation of shikimic acid caused by blue light stimulation is due to an increase in 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, EC2.5.1.54), the rate-determining enzyme in the shikimic acid pathway, as well as phosphofructokinase (PFK, EC2.7.1.11) and glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), the rate-determining enzymes in the glycolysis and pentose phosphate pathways, respectively. This stimulation results in increased levels of phosphoenolpyruvic acid (PEP) and erythrose-4-phosphate (E4P), the starting materials of shikimic acid biosynthesis.