Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein–protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.

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BackgroundSaccharomyces cerevisiae ScGdt1 and mammalian TMEM165 are two members of the UPF0016 membrane protein family that is likely to form a new group of Ca2+/H+ antiporter and/or a Mn2+ transporter in the Golgi apparatus. We have previously shown that Candida albicans CaGDT1 is a functional ortholog of ScGDT1 in the response of S. cerevisiae to calcium stress. However, how CaGdt1 together with the Golgi calcium pump CaPmr1 regulate calcium homeostasis and cell wall integrity in this fungal pathogen remains unknown.MethodsChemical sensitivity was tested by dilution assay. Cell survival was examined by measuring colony-forming units and staining with Annexin V-FITC and propidium iodide. Calcium signaling was examined by expression of downstream target gene CaUTR2, while cell wall integrity signaling was revealed by detection of phosphorylated Mkc1 and Cek1. Subcellular localization of CaGdt1 was examined through direct and indirect immunofluorescent approaches. Transcriptomic analysis was carried out with RNA sequencing.ResultsThis study shows that Candida albicans CaGDT1 is also a functional ortholog of ScGDT1 in the response of S. cerevisiae to cell wall stress. CaGdt1 is localized in the Golgi apparatus but at distinct sites from CaPmr1 in C. albicans. Loss of CaGDT1 increases the sensitivity of cell lacking CaPMR1 to cell wall and ER stresses. Deletion of CaGDT1 and/or CaPMR1 increases calcium uptake and activates the calcium/calcineurin signaling. Transcriptomic profiling reveals that core functions shared by CaGdt1 and CaPmr1 are involved in the regulation of cellular transport of metal ions and amino acids. However, CaGdt1 has distinct functions from CaPmr1. Chitin synthase gene CHS2 is up regulated in all three mutants, while CHS3 is only up regulated in the pmr1/pmr1 and the gdt1/gdt1 pmr1/pmr1 mutants. Five genes (DIE2, STT3, OST3, PMT1 and PMT4) of glycosylation pathway and one gene (SWI4) of the cell wall integrity (CWI) pathway are upregulated due to deletion of CaGDT1 and/or CaPMR1. Consistently, deletion of either CaPMR1 or CaGDT1 activates the CaCek1-mediated CWI signaling in a cell wall stress-independent fashion. Calcineurin function is required for the integrity of the cell wall and vacuolar compartments of cells lacking both GDT1 and CaPMR1.ConclusionsCaPmr1 is the major player in the regulation of calcium homeostasis and cell wall stress, while CaGdt1 plays a compensatory role for CaPmr1 in the Golgi compartment in C. albicans.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0246-x) contains supplementary material, which is available to authorized users.

Gene knockout and knockdown strategies have been immensely successful probes of gene function, but small molecule inhibitors (SMIs) of gene products allow much greater time resolution and are particularly useful when the targets are essential for cell replication or survival. SMIs also serve as lead compounds for drug discovery. However, discovery of selective SMIs is costly and inefficient. The action of SMIs can be modeled simply by tagging gene products with an auxin-inducible degron (AID) that triggers rapid ubiquitylation and proteasomal degradation of the tagged protein upon exposure of live cells to auxin. To determine if this approach is broadly effective, we AID-tagged over 750 essential proteins in Saccharomyces cerevisiae and observed growth inhibition by low concentrations of auxin in over 66% of cases. Polytopic transmembrane proteins in the plasma membrane, Golgi complex, and endoplasmic reticulum were efficiently depleted if the AID-tag was exposed to cytoplasmic OsTIR1 ubiquitin ligase. The auxin analog 1-napthylacetic acid (NAA) was as potent as auxin on AID-tags, but surprisingly NAA was more potent than auxin at inhibiting target of rapamycin complex 1 (TORC1) function. Auxin also synergized with known SMIs when acting on the same essential protein, indicating that AID-tagged strains can be useful for SMI screening. Auxin synergy, resistance mutations, and cellular assays together suggest the essential GMP/GDP-mannose exchanger in the Golgi complex ( Vrg4 ) as the target of a natural cyclic peptide of unknown function (SDZ 90-215). These findings indicate that AID-tagging can efficiently model the action of SMIs before they are discovered and can facilitate SMI discovery.

Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H+) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H+ for cytoplasmic Ca2+, respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H+/Ca2+ exchangers, Gdt1 also promoted growth in high Ca2+ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together with Gdt1.