Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers.

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-ß-phosphate of inositol pyrophosphates and only minor activity against the ß-phosphates of 4-InsP7 and 6-InsP7. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-ß-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

Dual Bioorthogonal Labeling of the Amyloid-ß Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products.

The amyloid-ß protein precursor (AßPP) is critical in the pathophysiology of Alzheimer's disease (AD), since two-step proteolytic processing of AßPP generates the neurotoxic amyloid-ß peptide (Aß). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AßPP. The C-terminal tail of AßPP was fluorescently labeled using a SNAP-tag, while the Aß region of AßPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AßPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AßPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AßPP variants was validated with ELISA and immunoblotting, and seven AßPP mutants that showed similar cleavage pattern as wild-type AßPP were identified. The AßPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AßPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aß and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aß with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AßPP.