Plant dehydroascorbate reductase moonlights as membrane integrated ion channel.

Plant dehydroascorbate reductases (DHARs) are only known as soluble antioxidant enzymes of the ascorbate-glutathione pathway. They recycle ascorbate from dehydroascorbate, thereby protecting plants from oxidative stress and the resulting cellular damage. DHARs share structural GST fold with human chloride intracellular channels (HsCLICs) which are dimorphic proteins that exists in soluble enzymatic and membrane integrated ion channel forms. While the soluble form of DHAR has been extensively studied, the existence of a membrane integrated form remains unknown. We demonstrate for the first time using biochemistry, immunofluorescence confocal microscopy, and bilayer electrophysiology that Pennisetum glaucum DHAR (PgDHAR) is dimorphic and is localized to the plant plasma membrane. In addition, membrane translocation increases under induced oxidative stress. Similarly, HsCLIC1 translocates more into peripheral blood mononuclear cells (PBMCs) plasma membrane under induced oxidative stress conditions. Moreover, purified soluble PgDHAR spontaneously inserts and conducts ions in reconstituted lipid bilayers, and the addition of detergent facilitates insertion. In addition to the well-known soluble enzymatic form, our data provides conclusive evidence that plant DHAR also exists in a novel membrane-integrated form. Thus, the structure of DHAR ion channel form will help gain deeper insights into its function across various life forms.

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plants and humans.

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.