The Role of Metal-Dependent Protein Phosphatase 1H and 1K, in Macrophages

Abstract

Macrophages are key cells of the innate immune system and provide the first line of defense against invading pathogens. Metal-dependent protein phosphatases (PPMs) regulate a myriad of cellular processes, including immune responses. The identification of PPM1A as a regulator of macrophage functions raised the question of whether other PPMs have important roles in macrophages. We identified PPM1H and PPM1K as priority targets given that their expressions were increased upon stimulation. However, these phosphatases have yet to be investigated in the context of infection and immunity. Given that the function of PPM1H and PPM1K are executed via their physiological substrates, this study aimed to identify PPM interactors using a proximity dependent biotinylation screen (i.e. TurboID). Herein, a stable, inducible TurboID screening platform was generated and validated in human THP-1 monocytes/macrophages. Using this system, the PPM1H and PPM1K interactomes were profiled, under resting and infection conditions with the pathogen, Mycobacterium tuberculosis (Mtb). While the screen results demonstrated that TurboID-generated PPM1H and PPM1K interactomes were relatively unchanged by Mtb infection, a repertoire of novel PPM “interactors” were identified. Several hits aligned with our expectations from the literature, with respect to their subcellular localization and functional roles. However, the screen also revealed some previously undescribed processes that these PPMs may impact. Hits of particular interest included MX2 and DLST. MX2 was identified as a PPM1H “interactor” that was unique to the Mtb-infection condition. DLST (2-oxoglutarate dehydrogenase complex E2 subunit) was identified as a PPM1K “interactor”, with a similar estimated sample abundance to PPM1K’s known binding partner, the branched-chain α-keto acid dehydrogenase complex E2 subunit. These hits suggest potentially novel roles for PPM1H and PPM1K, in Mtb infection and TCA cycle regulation, respectively, and should be priority targets for future validation experiments. Further functional assays demonstrated that both PPM1H and PPM1K appear dispensable for the intracellular survival of Mtb as genetic deletion and overexpression did not produce any phenotypes. Nevertheless, this study provides interactome maps to allow for future investigations into novel roles for PPM1H and PPM1K, in macrophage function.