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Significance Cu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme, and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress; metabolic defects; and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2•−) in a manner that produces an oxidant as byproduct, hydrogen peroxide (H2O2). While much is known about the necessity of scavenging O2•−, it is less clear what the physiological roles of Sod1-derived H2O2 are. We discovered that Sod1-derived H2O2 plays an important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for reactive oxygen species scavenging enzymes, as well as redox regulating a large network of enzymes., Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2•−) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2•− to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2•− toxicity, which only requires

The inherited form of open angle glaucoma arises due to a toxic gain-of-function intracellular misfolding event involving a mutated myocilin olfactomedin domain (OLF). Mutant myocilin is recognized by the endoplasmic reticulum (ER)-resident heat shock protein 90 paralog, glucose regulated protein 94 (Grp94), but their co-aggregation precludes mutant myocilin clearance by ER-associated degradation. When the Grp94-mutant myocilin interaction is abrogated by inhibitors or siRNA, mutant myocilin is efficiently degraded. Here we dissected Grp94 into component domains (N, NM, MC) to better understand the molecular factors governing its interaction with OLF. We show that the Grp94 N-terminal nucleotide-binding N domain is responsible for accelerating OLF aggregation in vitro. Upon inhibiting the isolated N domain pharmacologically or removing the Pre-N terminal 57 residues from full-length Grp94, OLF aggregation rates revert to those seen for OLF alone, but only pharmacological inhibition rescues co-aggregation. The Grp94-OLF interaction is below the detection limit of fluorescence polarization measurements, but chemical crosslinking paired with mass spectrometry analyses traps a reproducible interaction between OLF and the Grp94 N domain, as well as between OLF and the Grp94 M domain. The emerging molecular-level picture of quinary interactions between Grp94 and myocilin points to a role for the far N-terminal sequence of the Grp94 N domain and a cleft in the M domain. Our work further supports drug discovery efforts to inhibit these interactions as a strategy to treat myocilin-associated glaucoma.

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2⸱-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major new aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is exclusively dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2⸱- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this new mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2⸱- toxicity, which only requires < 1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.Significance StatementCu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress, metabolic defects, and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2⸱-) in a manner that produces an oxidant as a byproduct, hydrogen peroxide (H2O2). While much is known about the necessity of scavenging O2⸱-, it is less clear what the physiological roles of Sod1-derived H2O2 are. Herein, we discovered that Sod1-derived H2O2 plays a very important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for ROS scavenging enzymes, as well as redox regulating a large network of enzymes.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His–Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr–Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aβ42 peptide (Ala–Thr) and the other to the non-pathogenic Aβ48 (Thr–Leu). For the former site, we observed more favorable kinetics in lipid bilayer–mimicking bicelles than in detergent solution, indicating that substrate–lipid and substrate–enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

We describe the DRILL (dry ion localization and locomotion) device, which is an interface for electrospray ionization (ESI)-mass spectrometry (MS) that exploits a swirling flow to enable the use of inertial separation to prescribe different fates for electrosprayed droplets based on their size. This source adds a new approach to charged droplet trajectory manipulation which, when combined with hydrodynamic drag forces and electric field forces, provides a rich range of possible DRILL operational modes. Here, we experimentally demonstrate sensitivity improvement obtained via vortex-induced inertial sorting of electrosprayed droplets/ions: one possible mode of DRILL operation. In this mode, DRILL removes larger droplets while accelerating the remainder of the ESI plume, producing a high velocity stream of gas-enriched spray with small, highly charged droplets and ions and directing it toward the MS inlet. The improved signal-to-noise ratio (10-fold enhancement) in the detection of angiotensin I is demonstrated using the DRILL interface coupled to ESI-MS along with an improved limit of detection (10-fold enhancement, 100 picomole) in the detection of angiotensin II. The utility of DRILL has also been demonstrated by liquid chromatography (LC)-MS: a stable isotope labeled peptide cocktail was spiked into a complex native tissue extract and quantified by unscheduled multiple reaction monitoring on a TSQ Vantage. DRILL demonstrated improved signal strength (up to a 700-fold) for 8 out of 9 peptides and had no effects on the peak shape of the transitions.

Double strand-breaks (DSBs) of genomic DNA caused by ionizing radiation or mutagenic chemicals are a common source of mutation, recombination, chromosomal aberration, and cell death. Linker histones are DNA packaging proteins with established roles in chromatin compaction, gene transcription, and in homologous recombination (HR)-mediated DNA repair. Using a machine-learning model for functional prioritization of eukaryotic post-translational modifications (PTMs) in combination with genetic and biochemical experiments with the yeast linker histone, Hho1, we discovered that site-specific phosphorylation sites regulate HR and HR-mediated DSB repair. Five total sites were investigated (T10, S65, S141, S173, and S174), ranging from high to low function potential as determined by the model. Of these, we confirmed S173/174 are phosphorylated in yeast by mass spectrometry and found no evidence of phosphorylation at the other sites. Phospho-nullifying mutations at these two sites results in a significant decrease in HR-mediated DSB repair templated either with oligonucleotides or a homologous chromosome, while phospho-mimicing mutations have no effect. S65, corresponding to a mammalian phosphosite that is conserved in yeast, exhibited similar effects. None of the mutations affected base- or nucleotide-excision repair, nor did they disrupt non-homologous end joining or RNA-mediated repair of DSBs when sequence heterology between the break and repair template strands was low. More extensive analysis of the S174 phospho-null mutant revealed that its repression of HR and DSB repair is proportional to the degree of sequence heterology between DSB ends and the HR repair template. Taken together, these data demonstrate the utility of machine learning for the discovery of functional PTM hotspots, reveal linker histone phosphorylation sites necessary for HR and HR-mediated DSB repair, and provide insight into the context-dependent control of DNA integrity by the yeast linker histone Hho1.

Polyaniline (PANI) is a conducting polymer that has been extensively studied and characterized and has been used in sensors, catalysis and other electrochemical applications1. Incorporating metal clusters into the polymer matrix creates a composite material that has improved activity over the polymer itself. The properties of the metal clusters are size-dependent, where inert metals can even become active when they are scaled down from bulk to nanometer or sub-nanometer sizes2. An important factor with nanocluster creation is controlling the size of the final metal cluster, which is typically done synthetically with capping agents. However, taking advantage of the properties and structure of PANI can allow for electrochemically controlled growth of metal clusters at the imine sites while PANI is acting as an aggregation barrier, thus eliminating the need for capping agents3. Cluster formation begins with metal anions, such as AuX4 - and PdX4 2- (X = halide), that have an affinity for protonated imine sites of the PANI; these anions can be spontaneously reduced to neutral metal atoms by the emeraldine salt form of PANI. Previous work used this process with an attempt to control cluster sizes in a “top-down” manner by controlling time and concentration variables4. In this work, a controlled deposition and “bottom-up” approach of one metal atom per imine site is explored. The controlled process is completed by holding the PANI at a potential where it is in the oxidized state until excess metal anions are rinsed away and the potential is dropped and the metal is reduced3. A flow-cell connected to a flow-injection system allows for uninterrupted solution and potential changes. Our recent interest is on understanding the PANI-metal interaction and how to tune the interaction5 to build atomic-sized clusters. The most recent work involves the study of the intercalation of Pd in PANI. PdN (N=0-7) clusters are inserted into a polyaniline matrix through this controlled process using the PdCl4 2- anion. Our recent findings show that the speciation of the metal anion is of critical importance for a specific PANI-metal anion interaction. The mechanism is believed to be dependent on the Bronsted-like interactions and/or charge transfer Results have been confirmed using Raman spectroscopy, XPS, and electrochemical methods. In this presentation we will explain the fundamental steps of the formation of the atomic Pd clusters in PANI and how that Pd-PANI interaction is affected and/or tuned by controlling various conditions such as concentration, potential, and degree of PANI protonation. (1) Huang, W.-S.; Humphrey, B. D.; MacDiarmid, A. G. J. Chem. Soc. Faraday Trans. 1 1986, 82 (8), 2385. (2) Schwartz, I.; Jonke, A. P.; Josowicz, M.; Janata, J. Catal. Letters 2012, 142 (11), 1344. (3) Jonke, A. P.; Josowicz, M.; Janata, J.; Engelhard, M. H. J. Electrochem. Soc. 2010, 157 (10), P83. (4) Saheb, A.; Smith, J. A.; Josowicz, M.; Janata, J.; Baer, D. R.; Engelhard, M. H. J. Electroanal. Chem. 2008, 621 (2), 238. (5) Harbottle, A. M.; Hira, S. M.; Josowicz, M.; Janata, J. Langmuir 2016, 32 (33), 8315.

Under precisely controlled conditions, atomic gold electrodes with even or odd number of Au atoms per polyaniline repeat unit (Pt/PANI/Au N for 0

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