Stepwise oxidations play key roles in the structural and functional regulations of DJ-1.

DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. Human DJ-1 has three cysteine residues (Cys46, Cys53 and Cys106). We found that, in addition to Cys106, Cys46 is the most reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe (Ctag) that selectively reacts with redox-sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with adjacent resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. Mutants (C46A and C53A), not forming Cys46-Cys53 disulfide cross-linking, increased oxidation of Cys106 to sulfinic and sulfonic acids. Furthermore, we found that DJ-1 C46A mutant has distorted unstable structure identified by biochemical assay and employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike WT DJ-1. These findings suggest that all three Cys residues including Cys46-Cys53 disulfide cross-linking are required for maintaining the structural integrity, the regulation process and cellular function as an antioxidant protein. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

Proteomic Analysis of Hippocampus in a Mouse Model of Depression Reveals Neuroprotective Function of Ubiquitin C-terminal Hydrolase L1 (UCH-L1) via Stress-induced Cysteine Oxidative Modifications.

Chronic physical restraint stress increases oxidative stress in the brain, and dysregulation of oxidative stress can be one of the causes of major depressive disorder. To understand the underlying mechanisms, we undertook a systematic proteomic analysis of hippocampus in a chronic restraint stress mouse model of depression. Combining two-dimensional gel electrophoresis (2D-PAGE) for protein separation with nanoUPLC-ESI-q-TOF tandem mass spectrometry, we identified sixty-three protein spots that changed in the hippocampus of mice subjected to chronic restraint stress. We identified and classified the proteins that changed after chronic stress, into three groups respectively functioning in neural plasticity, metabolic processes and protein aggregation. Of these, 5 proteins including ubiquitin C-terminal hydrolase L1 (UCH-L1), dihydropyrimidinase-related protein 2 (DPYL2), haloacid dehalogenase-like hydrolase domain-containing protein 2 (HDHD2), actin-related protein 2/3 complex subunit 5 (ARPC5) and peroxiredoxin-2 (PRDX2), showed pI shifts attributable to post-translational modifications. Further analysis indicated that UCH-L1 underwent differential oxidations of 2 cysteine residues following chronic stress. We investigated whether the oxidized form of UCH-L1 plays a role in stressed hippocampus, by comparing the effects of UCH-L1 and its Cys mutants on hippocampal cell line HT-22 in response to oxidative stress. This study demonstrated that UCH-L1 wild-type and cysteine to aspartic acid mutants, but not its cysteine to serine mutants, afforded neuroprotective effects against oxidative stress; there were no discernible differences between wild-type UCH-L1 and its mutants in the absence of oxidative stress. These findings suggest that cysteine oxidative modifications of UCH-L1 in the hippocampus play key roles in neuroprotection against oxidative stress caused in major depressive disorder.

Cataract-Associated New Mutants S175G/H181Q of ßΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes.

ß/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of ß/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of ßΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant ß-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of ß/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

Heterogeneous nuclear ribonucleoprotein K inhibits heat shock-induced transcriptional activity of heat shock factor 1.

When cells are exposed to heat shock and various other stresses, heat shock factor 1 (HSF1) is activated, and the heat shock response (HSR) is elicited. To better understand the molecular regulation of the HSR, we used 2D-PAGE-based proteome analysis to screen for heat shock-induced post-translationally modified cellular proteins. Our analysis revealed that two protein spots typically present on 2D-PAGE gels and containing heterogeneous nuclear ribonucleoprotein K (hnRNP K) with trioxidized Cys132 disappeared after the heat shock treatment and reappeared during recovery, but the total amount of hnRNP K protein remained unchanged. We next tested whether hnRNP K plays a role in HSR by regulating HSF1 and found that hnRNP K inhibits HSF1 activity, resulting in reduced expression of hsp70 and hsp27 mRNAs. hnRNP K also reduced binding affinity of HSF1 to the heat shock element by directly interacting with HSF1 but did not affect HSF1 phosphorylation-dependent activation or nuclear localization. hnRNP K lost its ability to induce these effects when its Cys132 was substituted with Ser, Asp, or Glu. These findings suggest that hnRNP K inhibits transcriptional activity of HSF1 by inhibiting its binding to heat shock element and that the oxidation status of Cys132 in hnRNP K is critical for this inhibition.

Ubiquitin C-terminal hydrolase-L1 plays a key role in angiogenesis by regulating hydrogen peroxide generated by NADPH oxidase 4.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), which catalyzes the hydrolysis of ubiquitin esters and amides, is highly expressed in brain. Recently, UCH-L1 has been found to increase cancer cell migration and invasion by modulating hydrogen peroxide generated by NADPH oxidase 4 (NOX4). Because angiogenesis is also mediated by hydrogen peroxide, we explored the role of UCH-L1 in angiogenesis in human umbilical vein endothelial cells (HUVECs). Silencing UCH-L1 suppressed tubule formation in HUVECs, indicating that UCH-L1 promotes angiogenesis in vitro. This was confirmed using in vivo Matrigel plug studies of HUVECs, after overexpressing or silencing UCH-L1. Silencing UCH-L1 significantly suppressed VEGF-induced ROS levels as well as activation of VEGFR, both of which are required for angiogenesis. This study also showed that UCH-L1 promotes angiogenesis of HUVECs, as well as invasion in cancer cells, by up-regulating ROS by deubiquitination of NOX4, suggesting that UCH-L1 plays a key role in angiogenesis of HUVECS by regulating ROS levels by deubiquitination of NOX4.

Secretagogin affects insulin secretion in pancreatic ß-cells by regulating actin dynamics and focal adhesion.

Secretagogin (SCGN), a Ca(2+)-binding protein having six EF-hands, is selectively expressed in pancreatic ß-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca(2+)-sensor protein, but the underlying mechanism has not been elucidated. The present study explored the mechanism by which SCGN enhances glucose-induced insulin secretion in NIT-1 insulinoma cells. To determine whether SCGN influences the first or second phase of insulin secretion, we examined how SCGN affects the kinetics of insulin secretion in NIT-1 cells. We found that silencing SCGN suppressed the second phase of insulin secretion induced by glucose and H2O2, but not the first phase induced by KCl stimulation. Recruitment of insulin granules in the second phase of insulin secretion was significantly impaired by knocking down SCGN in NIT-1 cells. In addition, we found that SCGN interacts with the actin cytoskeleton in the plasma membrane and regulates actin remodelling in a glucose-dependent manner. Since actin dynamics are known to regulate focal adhesion, a critical step in the second phase of insulin secretion, we examined the effect of silencing SCGN on focal adhesion molecules, including FAK (focal adhesion kinase) and paxillin, and the cell survival molecules ERK1/2 (extracellular-signal-regulated kinase 1/2) and Akt. We found that glucose- and H2O2-induced activation of FAK, paxillin, ERK1/2 and Akt was significantly blocked by silencing SCGN. We conclude that SCGN controls glucose-stimulated insulin secretion and thus may be useful in the therapy of Type 2 diabetes.

ROSics: chemistry and proteomics of cysteine modifications in redox biology.

Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels.

Mechanism of 1-Cys type methionine sulfoxide reductase A regeneration by glutaredoxin.

Glutaredoxin (Grx), a major redox regulator, can act as a reductant of methionine sulfoxide reductase A (MsrA). However, the biochemical mechanisms involved in MsrA activity regeneration by Grx remain largely unknown. In this study, we investigated the regeneration mechanism of 1-Cys type Clostridium oremlandii MsrA (cMsrA) lacking a resolving Cys residue in a Grx-dependent assay. Kinetic analysis showed that cMsrA could be reduced by both monothiol and dithiol Grxs as efficiently as by in vitro reductant dithiothreitol. Our data revealed that the catalytic Cys sulfenic acid intermediate is not glutathionylated in the presence of the substrate, and that Grx instead directly formed a complex with cMsrA. Mass spectrometry analysis identified a disulfide bond between the N-terminal catalytic Cys of the active site of Grx and the catalytic Cys of cMsrA. This mixed disulfide bond could be resolved by glutathione. Based on these findings, we propose a model for regeneration of 1-Cys type cMsrA by Grx that involves no glutathionylation on the catalytic Cys of cMsrA. This mechanism contrasts with that of the previously known 1-Cys type MsrB.

Complex of Fas-associated factor 1 (FAF1) with valosin-containing protein (VCP)-Npl4-Ufd1 and polyubiquitinated proteins promotes endoplasmic reticulum-associated degradation (ERAD).

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys(48)-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.

Structure of Nm23-H1 under oxidative conditions.

Nm23-H1/NDPK-A, a tumour metastasis suppressor, is a multifunctional housekeeping enzyme with nucleoside diphosphate kinase activity. Hexameric Nm23-H1 is required for suppression of tumour metastasis and it is dissociated into dimers under oxidative conditions. Here, the crystal structure of oxidized Nm23-H1 is presented. It reveals the formation of an intramolecular disulfide bond between Cys4 and Cys145 that triggers a large conformational change that destabilizes the hexameric state. The dependence of the dissociation dynamics on the H2O2 concentration was determined using hydrogen/deuterium-exchange experiments. The quaternary conformational change provides a suitable environment for the oxidation of Cys109 to sulfonic acid, as demonstrated by peptide sequencing using nanoUPLC-ESI-q-TOF tandem MS. From these and other data, it is proposed that the molecular and cellular functions of Nm23-H1 are regulated by a series of oxidative modifications coupled to its oligomeric states and that the modified cysteines are resolvable by NADPH-dependent reduction systems. These findings broaden the understanding of the complicated enzyme-regulatory mechanisms that operate under oxidative conditions.

Novel oxidative modifications in redox-active cysteine residues.

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODi and MODmap algorithm. We observed unexpected mass shifts (Δm=-16, -34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of -16, -34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO2-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO2-Cys) either to DHA (Δm=-34 Da) or serine along with Cys-SO2-SH (Δm=+64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO2-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Long lifespan in worms with long telomeric DNA.

Telomere length is a crucial factor in senescence, but it has not been determined whether animals with long telomeres live longer than those with normal-length telomeres in the isogenic background of a given species. Here we show the effect of long telomeres on lifespan in the nematode Caenorhabditis elegans. We examined the effect of telomere length on lifespan by overexpressing HRP-1, a telomere-binding protein, which gradually increased telomere length in worms. Worms with longer telomeres lived longer. We confirmed that the extension of lifespan was due to the increased telomere length, and not to the overexpression of HRP-1 per se, by examining the lifespans of nontransgenic progeny of the transgenic worms, who retained the longer telomeres. The lifespan-extending effect of long telomeres was dependent on daf-16. The number of germ stem cells was not affected in worms with long telomeres, indicating that the telomere effect on lifespan is independent of germ stem cell cycling. Worms with long telomeres were more resistant to heat stress. Taken together, our results suggest that signaling may be initiated in postmitotic somatic cells by telomere length to regulate organismal lifespan.

Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6.

Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MOD(i) and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO(2) -SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses.

Monoisotopic mass determination algorithm for selenocysteine-containing polypeptides from mass spectrometric data based on theoretical modeling of isotopic peak intensity ratios.

Selenoproteins, containing selenocysteine (Sec, U) as the 21st amino acid in the genetic code, are well conserved from bacteria to human, except yeast and higher plants that miss the Sec insertion machinery. Determination of Sec association is important to find substrates and to understand redox action of selenoproteins. While mass spectrometry (MS) has become a common and powerful tool to determine an amino acid sequence of a protein, identification of a protein sequence containing Sec was not easy using MS because of the limited stability of Sec in selenoproteins. Se has six naturally occurring isotopes, 74Se, 76Se, 77Se, 78Se, 8°Se, and 8²Se, and 8°Se is the most abundant isotope. These characteristics provide a good indicator for selenopeptides but make it difficult to detect selenopeptides using software analysis tools developed for common peptides. Thus, previous reports verified MS scans of selenopeptides by manual inspection. None of the fully automated algorithms have taken into account the isotopes of Se, leading to the wrong interpretation for selenopeptides. In this paper, we present an algorithm to determine monoisotopic masses of selenocysteine-containing polypeptides. Our algorithm is based on a theoretical model for an isotopic distribution of a selenopeptide, which regards peak intensities in an isotopic distribution as the natural abundances of C, H, N, O, S, and Se. Our algorithm uses two kinds of isotopic peak intensity ratios: one for two adjacent peaks and another for two distant peaks. It is shown that our algorithm for selenopeptides performs accurately, which was demonstrated with two LC-MS/MS data sets. Using this algorithm, we have successfully identified the Sec-Cys and Sec-Sec cross-linking of glutaredoxin 1 (GRX1) from mass spectra obtained by UPLC-ESI-q-TOF instrument.

Tandem use of selenocysteine: adaptation of a selenoprotein glutaredoxin for reduction of selenoprotein methionine sulfoxide reductase.

Several engineered selenocysteine (Sec)-containing glutaredoxins (Grxs) and their enzymatic properties have been reported, but natural selenoprotein Grxs have not been previously characterized. We expressed a bacterial selenoprotein Grx from Clostridium sp. (also known as Alkaliphilus oremlandii) OhILAs in Escherichia coli and characterized this selenoenzyme and its natural Cys homologues in Clostridium and E. coli. The selenoprotein Grx had a 200-fold higher activity than its Sec-to-Cys mutant form, suggesting that Sec is essential for catalysis by this thiol-disulfide oxidoreductase. Kinetic analysis also showed that the selenoprotein Grx had a 10-fold lower K(m) than Cys homologues. Interestingly, this selenoenzyme efficiently reduced a Clostridium selenoprotein methionine sulfoxide reductase A (MsrA), suggesting that it is the natural reductant for the protein that is not reducible by thioredoxin, a common reductant for Cys-containing MsrAs. We also found that the selenoprotein Grx could not efficiently reduce a Cys version of Clostridium MsrA, whereas natural Clostridium and E. coli Cys-containing Grxs, which efficiently reduce Cys-containing MsrAs, poorly acted on the selenoprotein MsrA. This specificity for MsrA reduction could explain why Sec is utilized in Clostridium Grx and more generally provides a novel example of the use of Sec in biological systems.

Building a Korean morphological analyzer using two Korean BERT models.

A morphological analyzer plays an essential role in identifying functional suffixes of Korean words. The analyzer input and output differ from each other in their length and strings, which can be dealt with by an encoder-decoder architecture. We adopt a Transformer architecture, which is an encoder-decoder architecture with self-attention rather than a recurrent connection, to implement a Korean morphological analyzer. Bidirectional Encoder Representations from Transformers (BERT) is one of the most popular pretrained representation models; it can present an encoded sequence of input words, considering contextual information. We initialize both the Transformer encoder and decoder with two types of Korean BERT, one of which is pretrained with a raw corpus, and the other is pretrained with a morphologically analyzed dataset. Therefore, implementing a Korean morphological analyzer based on Transformer is a fine-tuning process with a relatively small corpus. A series of experiments proved that parameter initialization using pretrained models can alleviate the chronic problem of a lack of training data and reduce the time required for training. In addition, we can determine the number of layers required for the encoder and decoder to optimize the performance of a Korean morphological analyzer.

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

SUMO1 attenuates stress-induced ROS generation by inhibiting NADPH oxidase 2.

Small ubiquitin-like modifier 1 (SUMO1) is a member of the superfamily of ubiquitin-like proteins. Despite its structural similarity with ubiquitin, SUMO1 does not seem to play any role in protein degradation and its precise biological function is poorly understood. During our studies on heat-shock responses, we found that heat-shock stress increased SUMO1 conjugation in a dose-dependent manner. Intriguingly, SUMO1 conjugation resulted in decrease of intracellular ROS generation and protection cells from death under heat-shock stress. We showed that NADPH oxidase 2 (NOX2) is a target protein of sumoylation by SUMO1 using immunoprecipitation and is colocalized with SUMO1 at plasma membrane. Additionally, we demonstrated that the attenuation in intracellular ROS generation resulted from inhibition of NADPH oxidase complex (NOX) activity. These results suggested that SUMO1 plays an important role in modulation of NOX activity required for ROS generation.

Activation of Nm23-H1 to suppress breast cancer metastasis via redox regulation.

Non-metastatic protein 23 H1 (Nm23-H1), a housekeeping enzyme, is a nucleoside diphosphate kinase-A (NDPK-A). It was the first identified metastasis suppressor protein. Nm23-H1 prolongs disease-free survival and is associated with a good prognosis in breast cancer patients. However, the molecular mechanisms underlying the role of Nm23-H1 in biological processes are still not well understood. This is a review of recent studies focusing on controlling NDPK activity based on the redox regulation of Nm23-H1, structural, and functional changes associated with the oxidation of cysteine residues, and the relationship between NDPK activity and cancer metastasis. Further understanding of the redox regulation of the NDPK function will likely provide a new perspective for developing new strategies for the activation of NDPK-A in suppressing cancer metastasis.

Nm23-H1 activator phenylbutenoid dimer exerts cytotoxic effects on metastatic breast cancer cells by inducing mitochondrial dysfunction only under glucose starvation.

Mitochondrial oxidative phosphorylation (OXPHOS) has become an attractive target in anti-cancer studies in recent years. In this study, we found that a small molecule phenylbutenoid dimer NMac1 (Nm23-H1 activator 1), (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, a previously identified anti-metastatic agent, has novel anti-proliferative effect only under glucose starvation in metastatic breast cancer cells. NMac1 causes significant activation of AMPK by decreasing ATP synthesis, lowers mitochondrial membrane potential (MMP, ΔΨm), and inhibits oxygen consumption rate (OCR) under glucose starvation. These effects of NMac1 are provoked by a consequence of OXPHOS complex I inhibition. Through the structure-activity relationship (SAR) study of NMac1 derivatives, NMac24 was identified as the most effective compound in anti-proliferation. NMac1 and NMac24 effectively suppress cancer cell proliferation in 3D-spheroid in vivo-like models only under glucose starvation. These results suggest that NMac1 and NMac24 have the potential as anti-cancer agents having cytotoxic effects selectively in glucose restricted cells.