Protein oxidation increases SAMHD1 binding ssDNA via its regulatory site.

SAMHD1 dNTP hydrolase activity places it at the crossroad of several important biological pathways, such as viral restriction, cell cycle regulation, and innate immunity. Recently, a dNTPase independent function for SAMHD1 in homologous recombination (HR) of DNA double-strand breaks has been identified. SAMHD1 function and activity is regulated by several post-translational modifications, including protein oxidation. Here, we showed that oxidation of SAMHD1 increases ssDNA binding affinity and occurs in a cell cycle-dependent manner during S phase consistent with a role in HR. We determined the structure of oxidized SAMHD1 in complex with ssDNA. The enzyme binds ssDNA at the regulatory sites at the dimer interface. We propose a mechanism that oxidation of SAMHD1 acts as a functional switch to toggle between dNTPase activity and DNA binding.

Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

SAMHD1 Phosphorylation at T592 Regulates Cellular Localization and S-phase Progression.

SAMHD1 activity is regulated by a network of mechanisms including phosphorylation, oxidation, oligomerization, and others. Significant questions remain about the effects of phosphorylation on SAMHD1 function and activity. We investigated the effects of a SAMHD1 T592E phosphorylation mimic on its cellular localization, catalytic activity, and cell cycle progression. We found that the SAMHD1 T592E is a catalytically active enzyme that is inhibited by protein oxidation. SAMHD1 T592E is retained in the nucleus at higher levels than the wild-type protein during growth factor-mediated signaling. This nuclear localization protects SAMHD1 from oxidation by cytoplasmic reactive oxygen species. The SAMHD1 T592E phosphomimetic further inhibits the cell cycle S/G2 transition. This has significant implications for SAMHD1 function in regulating innate immunity, antiviral response and DNA replication.

Novel translocation responses of cytosolic phospholipase A2alpha fluorescent proteins.

Cytosolic phospholipase A2 (cPLA2)alpha responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2alpha translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2alpha or cPLA2alpha's C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA(2)alpha translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+](i) rises and did not translocate the proteins to the Golgi. AA's action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2alpha translocation and that lipid bodies are common targets of cPLA2alpha and contributors to stimulus-induced lipid mediator synthesis.

Blocking LPA-dependent signaling increases ovarian cancer cell death in response to chemotherapy.

The paradoxical role of reactive oxygen species in cell death versus cell survival establishes a delicate balance between chemotherapy efficacy and management of detrimental side effects. Normal proliferative signaling requires that cells remain inside a redox range that allows reversible protein oxidation to occur. Shifting the redox environment toward highly reducing or oxidizing states leads to cellular stress and cell death. Reactive oxygen species produced in response to Taxol and cisplatin treatment are necessary for effective cancer cell killing but the same ROS leads to damaging side effects in normal tissues. Combining antioxidants with chemotherapeutics to alleviate the unwanted side effects produces variable and often undesirable effects on cancer treatment. Here, we describe a more targeted method to improve ovarian cancer cell killing without the need for antioxidants. In ovarian cancer cells, lysophosphatidic acid (LPA) is a prominent growth factor that contributes to tumor survival and proliferation. We find that blocking LPA-dependent signaling with a specific receptor antagonist consistently increases cell death in response to both Taxol and cisplatin. We propose that inhibiting the upregulated growth factor-dependent signaling in cancer cells will target chemo-insensitivity, potentially lowering the necessary dose of the drugs and preventing harmful side effects.

Endogenous, regulatory cysteine sulfenylation of ERK kinases in response to proliferative signals.

ERK-dependent signaling is key to many pathways through which extracellular signals are transduced into cell-fate decisions. One conundrum is the way in which disparate signals induce specific responses through a common, ERK-dependent kinase cascade. While studies have revealed intricate ways of controlling ERK signaling through spatiotemporal localization and phosphorylation dynamics, additional modes of ERK regulation undoubtedly remain to be discovered. We hypothesized that fine-tuning of ERK signaling could occur by cysteine oxidation. We report that ERK is actively and directly oxidized by signal-generated H2O2 during proliferative signaling, and that ERK oxidation occurs downstream of a variety of receptor classes tested in four cell lines. Furthermore, within the tested cell lines and proliferative signals, we observed that both activation loop-phosphorylated and non-phosphorylated ERK undergo sulfenylation in cells and that dynamics of ERK sulfenylation is dependent on the cell growth conditions prior to stimulation. We also tested the effect of endogenous ERK oxidation on kinase activity and report that phosphotransfer reactions are reversibly inhibited by oxidation by as much as 80-90%, underscoring the importance of considering this additional modification when assessing ERK activation in response to extracellular signals.

The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

AIMS: Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. RESULTS: Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.

5-Oxo-ETE analogs and the proliferation of cancer cells.

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

5(S)-Hydroxy-6,8,11,14-E,Z,Z,Z-eicosatetraenoate stimulates PC3 cell signaling and growth by a receptor-dependent mechanism.

5(S)-Hydroxy-6,8,11,14-E,Z,Z,Z-eicosatetraenoate (5-HETE) causes PC3 cells to grow by an unknown mechanism. We find that it also induces the cells to activate extracellular signal-regulated kinases and Akt. Pertussis toxin inhibits both responses. 5-HETE, 5-oxo-6,8,11,14-E,Z,Z,Z-eicosatetraenoate, and 5-oxo-15-hydroxy-eicosatetraenoate are known to stimulate leukocytes by a receptor coupled to pertussis toxin-sensitive G proteins. Their respective relative potencies in leukocytes are 1, 10, and 3. In PC3 cells, however, these values are 10, 1, and 0. PC3 cells, we propose, express a non-leukocyte-type, G protein-coupled, 5-HETE receptor. This novel receptor and the extracellular signal-regulated kinase and Akt pathways it recruits may contribute to the progression of prostate adenocarcinoma.

Endosomal H2O2 production leads to localized cysteine sulfenic acid formation on proteins during lysophosphatidic acid-mediated cell signaling.

Lysophosphatidic acid (LPA) is a growth factor for many cells including prostate and ovarian cancer-derived cell lines. LPA stimulates H2O2 production which is required for growth. However, there are significant gaps in our understanding of the spatial and temporal regulation of H2O2-dependent signaling and the way in which signals are transmitted following receptor activation. Herein, we describe the use of two reagents, DCP-Bio1 and DCP-Rho1, to evaluate the localization of active protein oxidation after LPA stimulation by detection of nascent protein sulfenic acids. We found that LPA stimulation causes internalization of LPA receptors into early endosomes that contain NADPH oxidase components and are sites of H2O2 generation. DCP-Rho1 allowed visualization of sulfenic acid formation, indicative of active protein oxidation, which was stimulated by LPA and decreased by an LPA receptor antagonist. Protein oxidation sites colocalized with LPAR1 and the endosomal marker EEA1. Concurrent with the generation of these redox signaling-active endosomes (redoxosomes) is the H2O2- and NADPH oxidase-dependent oxidation of Akt2 and PTP1B detected using DCP-Bio1. These new approaches therefore enable detection of active, H2O2-dependent protein oxidation linked to cell signaling processes. DCP-Rho1 may be a particularly useful protein oxidation imaging agent enabling spatial resolution due to the transient nature of the sulfenic acid intermediate it detects.

Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells.

Lysophosphatidic acid (LPA) is produced by tumor cells and is present in the ascites fluid of ovarian cancer patients. To determine the role of endogenous LPA in the ovarian cancer cell line SKOV3, we treated cells with the LPA receptor antagonist VPC32183 and found that it inhibited cell growth and induced apoptosis. Exogenous LPA further stimulated ERK and Akt phosphorylation and NF-κB activity. To determine if reactive oxygen species (ROS), which have been implicated as second messengers in cell signaling, were also involved in LPA signaling, we treated cells with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), and antioxidants N-acetyl cysteine, EUK-134 and curcumin, and showed that all blocked LPA-dependent NF-κB activity and cell proliferation. DPI and EUK-134 also inhibited Akt and ERK phosphorylation. LPA was shown to stimulate dichlorofluorescein fluorescence, though not in the presence of DPI, apocynin (an inhibitor of NADPH oxidase), VPC32183, or PEG-catalase. Akt phosphorylation was also inhibited by PEG-catalase and apocynin. These data indicate that NADPH oxidase is a major source of ROS and H(2)O(2) is critical for LPA-mediated signaling. Thus, LPA acts as a growth factor and prevents apoptosis in SKOV3 cells by signaling through redox-dependent activation of ERK, Akt, and NF-κB-dependent signaling pathways.

Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.

Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.