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ABSTRACT Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondii. IMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

Microalgae represent a promising source of triglycerides and free fatty acids, synthesized in the form of lipids, for use in renewable fuels and chemicals. One challenge is the ability to efficiently recover the lipids from within the microalgae cell. Although various techniques have been studied individually, a comprehensive study of extraction techniques using consistent experimental and analytical methodology is missing. This study aims to provide this unifying comparison using the common microalgae strain Chlorella vulgaris. The factors that were surveyed and then optimized to achieve maximum extraction efficiency included the solvent type; mechanical pre-treatment using a ball mill at a variety of grinding speeds; microalgae-to-solvent ratio; extraction facilitated by microwave; extraction facilitated by sonication; extraction facilitated using increased temperature; and extraction facilitated by in situ transesterification to convert the lipids into esters prior to extraction. The optimum conditions determined during these studies were utilizing methanol as the solvent, with ball mill pretreatment at a grinding speed of 500 rpm, and a 1:9 microalgae to solvent ratio. When used in combination with microwave-assisted extraction at a temperature of 140 °C, approximately 24 wt% of the initial lipids were recovered. Recoveries of over 70 wt% were obtained without a microwave at extraction temperatures of over 200 °C.