Reactive oxygen species (ROS) are a family of molecules that are continuously generated, transformed and consumed in all living organisms as a consequence of aerobic life. The traditional view of these reactive oxygen metabolites is one of oxidative stress and damage that leads to decline of tissue and organ systems in aging and disease. However, emerging data show that ROS produced in certain situations can also contribute to physiology and increased fitness. This Perspective provides a focused discussion on what factors lead ROS molecules to become signal and/or stress agents, highlighting how increasing knowledge of the underlying chemistry of ROS can lead to advances in understanding their disparate contributions to biology. An important facet of this emerging area at the chemistry-biology interface is the development of new tools to study these small molecules and their reactivity in complex biological systems.
Hydrogen peroxide (H 2 O 2 ) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H 2 O 2 to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H 2 O 2 into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H 2 O 2 , directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H 2 O 2 specifically through membranes in mammalian cells. Moreover, we show that intracellular H 2 O 2 accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Noxderived H 2 O 2 signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H 2 O 2 can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions.growth factor signaling | redox biology | reactive oxygen species | fluorescent sensor | membrane regulation H ydrogen peroxide (H 2 O 2 ) is garnering increased attention as a molecule involved not only in immune response and oxidative stress, but also as a physiological effector in essential cellular processes (1-5). Seminal contributions have elucidated ligand stimulants (6-10) and enzymatic sources (11-13) for cellular H 2 O 2 production as well as putative downstream targets (14-24), but principles of how this reactive oxygen species (ROS) is spatially and temporally regulated to promote redox signaling over oxidative stress pathways remain insufficiently understood. Because many of the signaling functions of H 2 O 2 rely on its generation by nonphagocytic forms of NADPH (Nox) proteins on the extracellular side of cell membranes, understanding how cells funnel H 2 O 2 toward beneficial pathways in these locales is of significant interest. Despite its reactive nature, H 2 O 2 has been long thought to be freely diffusible across biological membranes in a manner akin to the related canonical small-molecule signal nitric oxide (NO) (25). More recent studies implicate the role of AQP water channels, a class of membrane-spanning proteins that facilitate the diffusion of water and other substrates with varying specificity (26-28), in mediating H 2 O 2 passage across the plasma membrane of reconstituted yeast (29) and plant (30) cells. However, given that the experiments described in these reports utilized nonspecific chemical reagents for determination of the redox signal that was passed into the cell, direct evidence that aquaporins influence the cellular uptake of H 2 O 2 in a na...
The pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.
We present the design, synthesis, and biological applications of Mitochondria Peroxy Yellow 1 (MitoPY1), a new type of bifunctional fluorescent probe for imaging hydrogen peroxide levels within the mitochondria of living cells. MitoPY1 combines a chemoselective boronate-based switch and a mitochondrial-targeting phosphonium moiety for detection of hydrogen peroxide localized to cellular mitochondria. Confocal microscopy and flow cytometry experiments in a variety of mammalian cell types show that MitoPY1 can visualize localized changes in mitochondrial hydrogen peroxide concentrations generated by situations of oxidative stress.Hydrogen peroxide (H 2 O 2 ) is an increasingly recognized small-molecule mediator of physiology, aging, and disease in living organisms. [1][2][3][4][5][6] In this regard, aberrant production or accumulation of H 2 O 2 within cellular mitochondria over time due to environmental stress(es) and/or genetic mutation(s) is connected to serious diseases where age is a risk factor, including cancer 7 and neurodegenerative Alzheimer's, Parkinson's, and Huntington's diseases.8 , 9 Indeed, overexpression and mitochondrial targeting of catalase, a peroxide-detoxifying enzyme, can increase life span in mouse models.10 On the other hand, newer data suggest that controlled bursts of mitochondrial H 2 O 2 can also serve beneficial roles for cell survival, growth, differentiation, and maintenance. [3][4][5][6] New imaging methods that allow visualization of localized production and accumulation of mitochondrial H 2 O 2 in living samples are potentially useful for disentangling the complex contributions of this reactive oxygen species (ROS) to both healthy and diseased states. Synthetic fluorescent H 2 O 2 indicators that can be targeted to precise subcellular locations offer one approach to this goal and do not require transfection like their protein counterparts,11 -12 but traditional ROS indicators such as dihydrorhodamine (DHR) are uncharged and hence not preferentially localized in cells before oxidation. 13 Our overall strategy for fluorescence imaging of mitochondrial H 2 O 2 in living systems is to create bifunctional dyes that contain both a peroxide-responsive element and a mitochondrialtargeting moiety. For the latter purpose, we were inspired by the use of phosphonium head groups by Murphy and others to deliver antioxidants, electrophiles, and EPR and optical probes to mitochondria, as these and related lipophilic cations selectively accumulate in this organelle due to proton gradient considerations. [14][15][16] In addition, we sought a modular synthetic route that would allow facile introduction of a phosphonium or any other desired targeting group after installation of the boronate switch, which circumvents potential complications arising from sensitive functionalities that are incompatible with palladium-catalyzed Miyaura-Suzuki reactions typically used to introduce the H 2 O 2 -cleavable boronate cage. Both of these design criteria can be met by the approach outlined in Scheme 1...
We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H 2 O 2 ) generated at physiological cell signaling levels. This structurally homologous series of fluorescein-and rhodol-based reporters relies on a chemospecific boronate-tophenol switch to respond to H 2 O 2 over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H 2 O 2 signals, as shown by studies in RAW 264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H 2 O 2 -specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H 2 O 2 and hypochlorous acid (HOCl) levels in live RAW 264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H 2 O 2 , and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.
Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. In this context, NAPDH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke, and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins exhibit cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H2O2), we show that adult hippocampal stem/progenitor cells (AHPs) generate H2O2 through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations.
Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies often suffer from a lack of signal-to-noise, requirements for extensive optimization for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting “activity-responsive RNAP” (AR) system to create light and small molecule activated biosensors, demonstrating the “plug-and-play” nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications.
Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.
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