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Supplementary Figure 2 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Materials and Methods, Figure Legends 1-7, Table 1 Legend from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 5 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 3 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Table 1 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 1 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 4 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 6 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

Supplementary Figure 7 from Arrest Defective 1 Autoacetylation Is a Critical Step in Its Ability to Stimulate Cancer Cell Proliferation

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors β-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis. Cancer Res; 70(11); 4422–32. ©2010 AACR.

Supplementary Figures 1-2 from Hydrogen Peroxide Produced by Angiopoietin-1 Mediates Angiogenesis

Angiopoietin-1 (Ang1) mediates angiogenesis by enhancing endothelial cell survival and migration. It is also known that Ang1 activates Tie2, an endothelial-specific tyrosine kinase receptor, but the molecular mechanism of this process is not clear. In this study, we investigated whether reactive oxygen species (ROS) production plays a role in Ang1-mediated angiogenesis. We found that human umbilical vein endothelial cells treated with Ang1 produce ROS transiently, which was suppressed by NADPH oxidase inhibitor, diphenylene-iodonium chloride, and rotenone. The Ang1-induced ROS was identified as hydrogen peroxide (H2O2) using adenovirus-catalase infection. Removal of H2O2 by adenovirus-catalase significantly suppressed Ang1-induced in vitro endothelial cell migration, in vivo tubule formation and angiogenesis, and activation of p44/42 mitogen-activated protein kinase (MAPK), involved in cell migration, and delayed the deactivation of Akt phosphorylation involved in cell survival. Supporting to in vitro data, Ang1-induced vascular remodeling in catalase (−/−) mice was more prominent than in catalase (+/+) mice: Ang1-induced increases of the diameter of terminal arterioles and the postcapillary venules in catalase (−/−) mice were significant compared with catalase (+/+) mice. These results show that Ang1-induced H2O2 plays an important role in Ang1-mediated angiogenesis by modulating p44/42 MAPK activity. (Cancer Res 2006; 66(12): 6167-74)

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.

Sentence compression is a natural language-processing task that produces a short paraphrase of an input sentence by deleting words from the input sentence while ensuring grammatical correctness and preserving meaningful core information. This study introduces a graph convolutional network (GCN) into a sentence compression task to encode syntactic information, such as dependency trees. As we upgrade the GCN to activate a directed edge, the compression model with the GCN layers can distinguish between parent and child nodes in a dependency tree when aggregating adjacent nodes. Furthermore, by increasing the number of GCN layers, the model can gradually collect high-order information of a dependency tree when propagating node information through the layers. We implement a sentence compression model for Korean and English, respectively. This model consists of three components: pre-trained BERT model, GCN layers, and a scoring layer. The scoring layer can determine whether a word should remain in a compressed sentence by relying on the word vector containing contextual and syntactic information encoded by BERT and GCN layers. To train and evaluate the proposed model, we used the Google sentence compression dataset for English and a Korean sentence compression corpus containing about 140,000 sentence pairs for Korean. The experimental results demonstrate that the proposed model achieves state-of-the-art performance for English. To the best of our knowledge, this sentence compression model based on the deep learning model trained with a large-scale corpus is the first attempt for Korean.

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.

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., Breast cancer: stopping the spread A small molecule called NMac1 from a plant related to ginger boosts the activity of Nm23-H1, a protein-suppressing metastasis, the spread of cancer cells to other tissues. Metastasis is a primary cause of death in cancer patients. Efforts to develop remedies based on Nm23-H1 treatments have had limited success. Kong-Joo Lee and Bokyung Kim at Ewha Womans University in Seoul, South Korea, reviewed discoveries about the molecular basis of Nm23-H1 action. They report that Nm23-H1 is easily oxidized in cells, preventing it from performing its anti-metastasis function. However, the small molecule NMac1 can stabilize Nm23-H1 by preventing oxidation, and action of NMac1 on Nm23-H1 was shown to prevent breast cancer metastasis in mice. This improved understanding of the molecular mechanism is the first step to potential new anti-metastasis treatments.

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.

Long-term peritoneal dialysis is associated with progressive fibrosis of the peritoneum. Epithelial-mesenchymal transition (EMT) of mesothelial cells is an important mechanism involved in peritoneal fibrosis, and TGF-β1 is considered central in this process. However, targeting currently known TGF-β1-associated pathways has not proven effective to date. Therefore, there are still gaps in understanding the mechanisms underlying TGF-β1-associated EMT and peritoneal fibrosis. We conducted network-based integrated analysis of transcriptomic and proteomic data to systemically characterize the molecular signature of TGF-β1-stimulated human peritoneal mesothelial cells (HPMCs). To increase the power of the data, multiple expression datasets of TGF-β1-stimulated human cells were employed, and extended based on a human functional gene network. Dense network sub-modules enriched with differentially expressed genes by TGF-β1 stimulation were prioritized and genes of interest were selected for functional analysis in HPMCs. Through integrated analysis, ECM constituents and oxidative stress-related genes were shown to be the top-ranked genes as expected. Among top-ranked sub-modules, TNFAIP6, ZC3H12A, and NNT were validated in HPMCs to be involved in regulation of E-cadherin, ZO-1, fibronectin, and αSMA expression. The present data shows the validity of network-based integrated analysis in discovery of novel players in TGF-β1-induced EMT in peritoneal mesothelial cells, which may serve as new prognostic markers and therapeutic targets for peritoneal dialysis patients.

Heat shock proteins are induced by activation of heat shock factor 1 (HSF1) in response to heat shock and protect against heat stress. However, the molecular mechanisms underlying the downstream signal of heat shock have not been fully elucidated. We found that similarly to canonical Hsps, Arc/Arg3.1 is also markedly induced by heat shock and by other cellular stress inducers, including diamide, sodium arsenite and H2O2 in various cells. We noted that heat stress–induced Arc/Arg3.1 protein is short lived, with a half-life of

Secretagogin (SCGN), a Ca2+-binding protein having six EF-hands, is selectively expressed in pancreatic β-cells and neuroendocrine cells. Previous studies suggested that SCGN enhances insulin secretion by functioning as a Ca2+-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.

In general, Q&A system retrieves passages by matching terms of a question in order to find an answer to the question. However it is difficult for Q&A system to find a correct answer because too many passages are retrieved and matching using terms is not enough to rank them according to their relevancy to a question. To alleviate this problem, we introduce a topic for a sentence, and adopt it for ranking in Q&A system. We define a set of person-related topic class and a clue expression which can indicate a topic of a sentence. A topic classification system proposed in this paper can determine a target topic for an input sentence by using clue expressions, which are manually collected from a corpus. We explain an architecture of the topic classification system and evaluate the performance of the components of this system.

&beta, /&gamma, 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 &beta, crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of &beta, &Beta, 2-crystallin and P24S/S31G of &gamma, D-crystallin, were found mutations occurred in the largest loop linking the distant &beta, 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 &beta, crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

Characterization of protein structural changes in response to protein modifications, ligand or chemical binding, or protein-protein interactions is essential for understanding protein function and its regulation. Amide hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is one of the most favorable tools for characterizing the protein dynamics and changes of protein conformation. However, currently the analysis of HDX-MS data is not up to its full power as it still requires manual validation by mass spectrometry experts. Especially, with the advent of high throughput technologies, the data size grows everyday and an automated tool is essential for the analysis. Here, we introduce a fully automated software, referred to as ‘deMix’, for the HDX-MS data analysis. deMix deals directly with the deuterated isotopic distributions, but not considering their centroid masses and is designed to be robust over random noises. In addition, unlike the existing approaches that can only determine a single state from an isotopic distribution, deMix can also detect a bimodal deuterated distribution, arising from EX1 behavior or heterogeneous peptides in conformational isomer proteins. Furthermore, deMix comes with visualization software to facilitate validation and representation of the analysis results.

Nm23-H1/NDPK-A is a tumor metastasis suppressor having NDP kinase (NDPK) activity. Nm23-H1 is positively associated with prolonged disease-free survival and good prognosis of cancer patients. Approaches to increasing the cellular levels of Nm23-H1 therefore have significance in the therapy of metastatic cancers. We found a small molecule, (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, that activates Nm23, hereafter called NMac1. NMac1 directly binds to Nm23-H1 and increases its NDPK activity. Employing various NMac1 derivatives and hydrogen/deuterium mass spectrometry (HDX-MS), we identified the pharmacophore and mode of action of NMac1. We found that NMac1 binds to the C-terminal of Nm23-H1 and induces the NDPK activation through its allosteric conformational changes. NMac1-treated MDA-MB-231 breast cancer cells showed dramatic changes in morphology and actin-cytoskeletal organization following inhibition of Rac1 activation. NMac1 also suppressed invasion and migration in vitro, and metastasis in vivo, in a breast cancer mouse model. NMac1 as an activator of NDPK has potential as an anti-metastatic agent.

// Hyun Jung Kim 1 , Venkataraman Magesh 1 , Jae-Jin Lee 1 , Sun Kim 1 , Ulla G. Knaus 2 and Kong-Joo Lee 1 1 Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Korea 2 Conway Institute, University College Dublin, Dublin, Ireland Correspondence to: Kong-Joo Lee, email: // Keywords : UCH-L1, ubiquitination, hydrogen peroxide, NOX4, invasion Received : December 19, 2014 Accepted : March 20, 2015 Published : April 15, 2015 Abstract This study explored the role of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in the production of ROS and tumor invasion. UCH-L1 was found to increase cellular ROS levels and promote cell invasion. Silencing UCH-L1, as well as inhibition of H 2 O 2 generation by catalase or by DPI, a NOX inhibitor, suppressed the migration potential of B16F10 cells, indicating that UCH-L1 promotes cell migration by up-regulating H 2 O 2 generation. Silencing NOX4, which generates H 2 O 2 , with siRNA eliminated the effect of UCH-L1 on cell migration. On the other hand, NOX4 overexpressed in HeLa cells happens to be ubiquitinated, and NOX4 following deubiquitination by UCH-L1, restored H 2 O 2 -generating activity. These in vitro findings are consistent with the results obtained in vivo with catalase (-/-) C57BL/6J mice. When H 2 O 2 and UCH-L1 levels were independently varied in these animals, the former by infecting with H 2 O 2 -scavenging adenovirus-catalase, and the latter by overexpressing or silencing UCH-L1, pulmonary metastasis of B16F10 cells overexpressing UCH-L1 increased significantly in catalase (-/-) mice. In contrast, invasion did not increase when UCH-L1 was silenced in the B16F10 cells. These findings indicate that H 2 O 2 levels regulated by UCH-L1 are necessary for cell invasion to occur and demonstrate that UCH-L1 promotes cell invasion by up-regulating H 2 O 2 via deubiquitination of NOX4.

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.