Apoptotic RBM38, TUSC2 and ANO1 Proteins ใน Human Serum Proteome มีความสัมพันธ์กับภาวะ

วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 9

วารสารพิษวิทยาไทย Apoptotic RBM38, TUSC2 and ANO1 Proteins ใน Human Serum Proteome มีความสัมพันธ์กับภาวะ

THAI JOURNAL OF TOXICOLOGY โฮโมซิสเตอนี สูงในเลอื ด

Volume 33, Number 2, July-December 2018 ณฐั วุฒิ ลายน า้ เงิน1, สิทธิรักษ์ รอยตระกูล2, ตวงรัตน์ ต้ังเสถียรพนั ธ์ุ3, ปิยะมิตร ศรีธรา4, จินตนา ศิริวราศัย5* Research Articles 1หลักสูตรวิทยาศาสตรมหาบัณฑิต (โภชนศาสตร์) โครงการร่วมคณะแพทยศาสตร์โรงพยาบาลรามาธิบดีและสถาบัน Apoptotic RBM38, TUSC2 and ANO1 Proteins in Human Serum Proteome Are xx Associated with Hyperhomocysteinemia โภชนาการ มหาวิทยาลัยมหิดล 2 Nuttawut Lainumngen, Sittiruk Roytrakul, Tuangrat Tangstheanphan, Piyamitr Sritara, ห้องปฏิบัติการวิจัยโปรตีโอมิกส์ สถาบันจีโนม ศูนยพ์ นั ธุวศิ วกรรมและเทคโนโลยชี ีวภาพแห่งชาติ ปทุมธานี Jintana Sirivarasai ………….………………………………………………………………………… 3 ฝ่ายการแพทยแ์ ละอนามยั การไฟฟ้ าฝ่ายผลิตแห่งประเทศไทย นนทบุรี 4สาขาวิชาอายุรศาสตร์โรคหัวใจ ภาควิชาอายุรศาสตร์, 5กลุม่ สาขาวชิ าโภชนศาสตร์ คณะแพทยศาสตร์โรงพยาบาล Migration of Melamine from Melamine-formaldehyde Food Containers Available on xx

The Market รามาธิบดี มหาวิทยาลัยมหิดล Uma Boriboon, Sasithorn Homdumrongwong, Passarin Saysuwary ……………….…………. บทคัดย่อ In vitro Antioxidant Activities of Nipa Palm Vinegar xx ภาวะโฮโมซิสเตอีนสูงในเลือด เป็นหน่ึงในปัจจยั เสี่ยงต่อการเกิดโรคหลอดเลือดหวั ใจ เนื่องจาก Udomratana Vattanasit, Supabhorn Yimthiang, Wiyada Kwanhian……………………………. สามารถชกั นา ใหเ้ กิดภาวะเซลลข์ องกลา้ มเน้ือหลอดเลือดตายได้ (apoptosis) ในปัจจุบันการศึกษาทางด้าน Effects of Alcoholic Extracts of Sweet-Fruited Tamarind (Tamarindus indica L.) and xx โปรติโอมิกส์มีการนา มาใชก้ นั อยา่ งแพร่หลายในงานวิจัยทางด้านวิทยาศาสตร์และทางคลินิก งานวจิ ยั น้ีเป็น Banana (Musa sapientum L.) on H2O2-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells การศึกษาเบ้ืองตน้ ที่ใชเ้ ทคนิคทางโปรติโอมิกส์ โดยมีวตั ถุประสงคเ์ พื่อศึกษาความแตกตา่ งของโปรตีนที่ Jiraporn Rangphueng, Ratchanee Kongkachuichai, Sujira Mukda, Jintana Sirivarasai, แสดงออกที่เกี่ยวขอ้ งกบั การเกิดโรคหลอดเลือดหวั ใจในผทู้ ี่มีภาวะโฮโมซิสเตอีนสูงในเลือด 3 ระดับ ใช้ Pattarana Sae-chew, Supanart Srisala, Bunyada Jithontham and Rodjana Chunhabundit chemiluminescense immunoassay สาหรับ วัดระดับโฮโมซิสเตอีนในเลือด ศึกษาการแสดงออกของโปรตีน ในซีรัมด้วยเทคนิค gel electrophoresis และ LC/MS-MS วิเคราะห์ปริมาณโปรตีนด้วย DecyderMs software

Review Article เปรียบเทียบความแตกตา่ งของโปรตีนในรูปของ Venn diagram ด้วย Jvenn และจดั กลุ่มของโปรตีนด้วย Panther software ทา นายความสัมพนั ธ์ระหวา่ งโปรตีนและสารโมเลกุลอื่นโดย STITCH โปรแกรมเวอร์ชน่ั A Review of Thermal and pH Stability of Important Foodborne Pathogen Toxins and xx Their Applications 5.0 ผลการศึกษาพบวา่ มีโปรตีนเฉพาะ 8 ชนิดที่มีการพบในปริมาณสูง (up-regulated) ในกลุ่มคนที่มีภาวะ Woranon Leevongcharoen, Sasiphatlapa Thanpolphat, Tumnoon Charaslertrangsi ……….. โฮโมซิสเตอีนสูงในเลือด ไดแ้ ก่ MUC-19, LPAR1, MAPO2, GPALPP1, FECH, TUSC2, RBM38 และ

Thirdhand Smoke : Emerging, Existing, and Never Ending xx ANO1 ประเด็นสา คญั พบวา่ RBM38, TUSC2 และ ANO1 เป็นโปรตีนที่พบเฉพาะในกลุ่มที่มีโฮโมซิสเตอีน Udomratana Vattanasit, Sawanya Laohaprapanon and Prasert Makkaew…………………… ในเลือดสูงกวา่ 50 µmol/L ที่มีสัมพนั ธ์กบั โรคหลอดเลือดซ่ึงเกี่ยวขอ้ งกบั ความผิดปกติในขบวนการตายของ เซลล์ ข้อมูลเชิงลึกที่ไดจ้ ากการศึกษาน้ีจะช่วยเพ่ิมเติมความรู้ความเข้าใจในการเปลี่ยนแปลงของโปรตีน ภายหลังการแปลงรหัสที่มีความสัมพนั ธ์กบั ความผิดปกติของระดับโฮโมซิสเตอีน และความเสี่ยงของการ เกิดโรคหลอดเลือดหวั ใจได้ ค าส าคัญ : ภาวะโฮโมซิสเตอีนสูงในเลือด โปรตีโอมิกส์ เซลล์ตาย (apoptosis) *ผู้รับผดิ ชอบบทความ ผศ.ดร.จินตนา ศิริวราศัย กลุ่มสาขาวิชาโภชนศาสตร์ คณะแพทยศาสตร์โรงพยาบาลรามาธิบดี มหาวิทยาลัยมหิดล ถ. พระราม 6 กรุงเทพฯ 10400 E-mail: [email protected] วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 10

Apoptotic RBM38, TUSC2 and ANO1 Proteins in Human Serum Proteome Are Introduction cardiovascular diseases. Notably, they Associated with Hyperhomocysteinemia highlighted the novel association among Proteomics is one of the new era of Nuttawut Lainumngen1, Sittiruk Roytrakul2, Tuangrat Tangstheanphan3, Piyamitr genetic factor, homocysteine levels and Sritara4, Jintana Sirivarasai5* omics studies involving whole protein alteration of protein expression. However, expressions profiles in cells together with 1Master of Science Program in Nutrition, Faculty of Medicine Ramathibodi Hospital and Institute of Nutrition, previous proteomic studies of hyper- Mahidol University, Thailand quantify and identify the protein product 2 Genome Technology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), homocysteinemia in human biofluids of all genes. Proteomics is able to provide Pathum Thani, Thailand related homocysteine (e.g., serum, plasma, 3 Health Office, Electricity Generating Authority of Thailand, Nonthaburi, Thailand better understanding in molecular basis 4Department of Medicine 5Graduate Program in Nutrition, Faculty of Medicine Ramathibodi Hospital, Mahidol urine, and saliva) are very limited. To our insight of diseases and to identify University, Thailand knowledge, this is a first preliminary study biological marker, which may be a specific Abstract to investigate serum protein expression in indicator in different conditions. Previous Hyperhomocysteinemia (HHcy) is known as an independent risk factor for Thai men with hyperhomocysteinemia. studies of proteomics and homocysteine in cardiovascular diseases. It can induce apoptosis of vascular smooth muscle cells. Currently, Well-known risk factors of animal model reported some significant proteomic has been widely applied in scientific and clinical researches. This research is a cardiovascular disease include age, alteration of protein expression. A study of preliminary study that used proteomic technique for comparative protein expressions with genetic, lifestyle pattern and health proteome in rat off spring related to focusing on the molecular basis of CVD in HHcy condition. The aim of this study was to conditions such as hypertension, maternal food intake during pregnancy identify protein profiles between control (non-HHcy) group and three groups of HHcy. hyperlipidemia and hyperhomo- showed that protein expressions in Chemiluminescense immunoassay was used for determination of plasma homocysteine cysteinemia. Homocysteine is an amino homocysteine cycle was altered among rat levels. Protein expressions in human serum were determined and analyzed by gel acid, which results from methionine groups with different levels of electrophoresis and LC/MS-MS technique. Proteins were quantified by using DeCyderMS utilization in cells. The normal range of homocysteine intake1. Experimental analysis software. Jvenn was used for comparing protein lists with venn diagram. Function total plasma homocysteine in adult with studies revealed association between and class of protein were demonstrated by using PANTHER software. The interaction of fasting is usually in 5-15 µmol/L, and its protein expressions and genetic variations chemical and protein was predicted by using STITCH V.5.0. Eight unique proteins were up- level greater than 15 µmol/L is defined as which involved in homocysteine regulated in hyperhomocysteinemia groups, including MUC-19, LPAR1, MAPO2, hyperhomocysteinemia5. Elevated level of metabolism such as cystathionine β- GPALPP1, FECH, TUSC2, RBM38 and ANO1. Notably, RBM38, TUSC2, and ANO1 homocysteine can induce oxidative stress synthase (CBS)2 and paraoxonase1 found in subjects with plasma homocysteine levels more than 50 µmol/L were discovered and by disruption of anti-oxidant enzymes. (PON1) in induced mouse to linked to CVD risks via apoptotic signaling pathways. This study provided better insight into Simultaneously, it also initiates endothelial hyperhomocysteinemia condition3. There the interaction between expressed proteins profiles after post-translational modification cell injury through increased arterial was a report with alteration of which may resulted in induction of physiological alteration that may related to homocysteine- stiffness by decreased nitric oxide (NO) apolipoprotein A-I in patients carrying induced CVD. bioavailability6,7. Another mechanism of MTHFR C677T mutations in Italian Keywords: Hyperhomocysteinemia, Proteomics, Apoptosis hyperhomocysteinmia induced abnormal population4. A quantitative reduction of * Corresponding author molecular function involves apoptosis apolipoprotein A-I in mutant individuals Assist. Prof. Jintana Sirivarasai, Ph.D signaling pathway via endoplasmic with hyper-homocysteinemia was Graduate Program in Nutrition, Faculty of Medicine Ramathibodi Hospital, Mahidol University reticulum stress (ER stress). ER stress can E-mail: [email protected] associated with an increased risk of lead to alter function/ structure of proteins วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 12 and disturb lipid metabolism, which may male, aged 50-70 years. Subjects with characteristics, genetic background of Plasma homocysteine measurement: lead to cardiovascular diseases. Moreover, diseases related to abnormal homocysteine MTHFR gene and some components of Participants were asked for fasting before homocysteine play role in apoptosis via metabolism e.g., liver disease, renal metabolic syndrome. A control group with collecting blood sample. Plasma P53 mediated pathway or methyl disease, and cancer were excluded. normal level of Hcy were 2 cases (from 20 homocysteine level was measured by transferase inhibition. A large number of Subjects were categorized into non-hyper- subjects) with plasma Hcy level of <15 electrochemiluminescence immunoassay genes related to apoptotic were reported homocysteinemia group (non- µmol/L, BMI 18.5-22.9 kg/m2, LDL- technique with COBAS (C800, Roche.) such as Bax, Bcl-2, Casp 12, Casp 3, Cav HHcy/control group; plasma total cholesterol <130 mg/dL and blood Determination of protein concentration: 3, Cdk2, P38, P53 and Pkc with modulated homocysteine level ≤15 µmol/l) or pressure <130/85 mmHg. For Bradford assay was used for measurement by elevated level of homocysteine. In hyperhomocysteinemia group (HHcy/case hyperhomocysteinemia group, they were of serum protein concentrations. Briefly, addition, homocysteine was associated group; plasma total homocysteine level > subdivided as shown in the following; Bradford reagent was added to a well-plate with regulation of gene expressions, which 15 µmol/l). This study was approved by - Case group I (N=2 selected from 20 along with samples with triplicate involved in various metabolic diseases8. the Ethic Committee on Human Right cases): plasma Hcy level of 15-30 µmol/L, determination. The absorbance at 595 nm According to evidences from previous Related to Research Involving Human and no metabolic syndrome. was read after 5 min of incubation by studies, the strong interaction between Subjects, Faculty of Medicine - Case group II (N=3 selected from 20 Rayto Rt-2100c microplate reader. hyperhomocysteinemia and risk of Ramathibodi Hospital, Mahidol University cases): plasma Hcy level of 30-50 µmol/L, Amount of protein were calculated by cardiovascular diseases via apoptotic (MURA2017/116). 2 BMI > 22.9 kg/m , and two metabolic using a standard curve of protein standard signaling pathway has been shown. This syndrome components which was diluted accordingly to various study hypothesized that elevated Study design: Data of general - Case group III (N=3 selected from 20 concentration. homocysteine levels may associate with characteristics and clinical parameters, cases): plasma Hcy level of >50 µmol/L, alteration of apoptotic proteins expressions including body mass index (BMI), plasma BMI>22.9 kg/m2, two metabolic syndrome SDS-PAGE: The 10 µg proteins samples in human serum and possibly relate to homocysteine levels, low-density components and cardio-ankle vascular were separated by 12.5% SDS-PAGE increased risk of cardiovascular diseases. lipoprotein levels (LDL, high-density index (CAVI). Two cases showed CAVI through two layers of gel, including 12.5% Therefore, this research aims to investigate lipoprotein levels (HDL, HbA1c, and values more than 9.0 with indicative separating gel and 4% stacking gel. the different expression of human serum fasting blood glucose were completely diagnosis of suspected arteriosclerosis. Proteins samples were loaded and proteome between control and collected. Moreover, physical In this study, we have already performed by using Atto AE-6540 with hyperhomocysteinemia groups by using examinations and specific medical performed the pooled samples which can constant Amps power supply (40 mA) for proteomic technique. assessment were performed in this study. be used as a powerful strategy to approximately 1-2 hours. After According to NCEP ATP III criteria for compensate for limited amounts of electrophoresis, protein bands were Materials and Methods metabolic syndrome, cut point of blood samples or high biological variation. visualized by using silver nitrate solution Subjects: Human serums were obtained pressure were applied at greater than or Pooling of samples in proteomics and subsequently scanned with imaging from the Electricity Generating Authority equal to 130/85 mmHg10. Based on the experiments may help overcome resource densitometer as described in previous of Thailand study (EGAT cohort study) rational of this pilot study, we further 11 constraints when many individuals are study . in 2013, which has been described investigated subjects with intensively analyzed. previously9. This study recruited Thai matched age and other general วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 13 and disturb lipid metabolism, which may male, aged 50-70 years. Subjects with characteristics, genetic background of Plasma homocysteine measurement: lead to cardiovascular diseases. Moreover, diseases related to abnormal homocysteine MTHFR gene and some components of Participants were asked for fasting before homocysteine play role in apoptosis via metabolism e.g., liver disease, renal metabolic syndrome. A control group with collecting blood sample. Plasma P53 mediated pathway or methyl disease, and cancer were excluded. normal level of Hcy were 2 cases (from 20 homocysteine level was measured by transferase inhibition. A large number of Subjects were categorized into non-hyper- subjects) with plasma Hcy level of <15 electrochemiluminescence immunoassay genes related to apoptotic were reported homocysteinemia group (non- µmol/L, BMI 18.5-22.9 kg/m2, LDL- technique with COBAS (C800, Roche.) such as Bax, Bcl-2, Casp 12, Casp 3, Cav HHcy/control group; plasma total cholesterol <130 mg/dL and blood Determination of protein concentration: 3, Cdk2, P38, P53 and Pkc with modulated homocysteine level ≤15 µmol/l) or pressure <130/85 mmHg. For Bradford assay was used for measurement by elevated level of homocysteine. In hyperhomocysteinemia group (HHcy/case hyperhomocysteinemia group, they were of serum protein concentrations. Briefly, addition, homocysteine was associated group; plasma total homocysteine level > subdivided as shown in the following; Bradford reagent was added to a well-plate with regulation of gene expressions, which 15 µmol/l). This study was approved by - Case group I (N=2 selected from 20 along with samples with triplicate involved in various metabolic diseases8. the Ethic Committee on Human Right cases): plasma Hcy level of 15-30 µmol/L, determination. The absorbance at 595 nm According to evidences from previous Related to Research Involving Human and no metabolic syndrome. was read after 5 min of incubation by studies, the strong interaction between Subjects, Faculty of Medicine - Case group II (N=3 selected from 20 Rayto Rt-2100c microplate reader. hyperhomocysteinemia and risk of Ramathibodi Hospital, Mahidol University cases): plasma Hcy level of 30-50 µmol/L, Amount of protein were calculated by cardiovascular diseases via apoptotic (MURA2017/116). 2 BMI > 22.9 kg/m , and two metabolic using a standard curve of protein standard signaling pathway has been shown. This syndrome components which was diluted accordingly to various study hypothesized that elevated Study design: Data of general - Case group III (N=3 selected from 20 concentration. homocysteine levels may associate with characteristics and clinical parameters, cases): plasma Hcy level of >50 µmol/L, alteration of apoptotic proteins expressions including body mass index (BMI), plasma BMI>22.9 kg/m2, two metabolic syndrome SDS-PAGE: The 10 µg proteins samples in human serum and possibly relate to homocysteine levels, low-density components and cardio-ankle vascular were separated by 12.5% SDS-PAGE increased risk of cardiovascular diseases. lipoprotein levels (LDL, high-density index (CAVI). Two cases showed CAVI through two layers of gel, including 12.5% Therefore, this research aims to investigate lipoprotein levels (HDL, HbA1c, and values more than 9.0 with indicative separating gel and 4% stacking gel. the different expression of human serum fasting blood glucose were completely diagnosis of suspected arteriosclerosis. Proteins samples were loaded and proteome between control and collected. Moreover, physical In this study, we have already performed by using Atto AE-6540 with hyperhomocysteinemia groups by using examinations and specific medical performed the pooled samples which can constant Amps power supply (40 mA) for proteomic technique. assessment were performed in this study. be used as a powerful strategy to approximately 1-2 hours. After According to NCEP ATP III criteria for compensate for limited amounts of electrophoresis, protein bands were Materials and Methods metabolic syndrome, cut point of blood samples or high biological variation. visualized by using silver nitrate solution Subjects: Human serums were obtained pressure were applied at greater than or Pooling of samples in proteomics and subsequently scanned with imaging from the Electricity Generating Authority equal to 130/85 mmHg10. Based on the experiments may help overcome resource densitometer as described in previous of Thailand study (EGAT cohort study) rational of this pilot study, we further 11 constraints when many individuals are study . in 2013, which has been described investigated subjects with intensively analyzed. previously9. This study recruited Thai matched age and other general วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 14

In-solution digestion: The samples were PepMap100, C18, 300 μm i.d.×5 mm), peptide charge state (1+, 2+ and 3+) and database of known and predicted subjected to the in-solution digestion and using full loop injection. The samples were max missed cleavages (3). Protein levels interaction between chemical and proteins. purified to remove buffer and salts. separated on analytical column (PepSwift were presented in log2 value. Interaction sources in STITCH are derived Briefly, an aliquot of 2 µl (4 µg of protein) Monolithic Nano Column, 100 μm × 5 cm Jvenn diagram was applied for from five main sources, including genomic were added into low-binding tubes. Protein i.d.). A linear gradient for elution method counting and comparing lists of proteins in context predictions, high-throughput lab samples were reduced by addition of 5 µl was used to elute peptides into the mass each group15. It is an integrative tool for experiments, (conserved) co-expression, of 10 mM dithiothreitol (DTT) in 10 mM spectrometer, mobile phases A and B at a computing and identifying the number of automated text mining and previous ammonium bicarbonate ( Ambic) and 5 constant flow rate of 1 µl/min which specific or intersection proteins among knowledge databases. mM DTT (final concentration). Then, the mobile phase A was 0.1% formic acid in groups. Jvenn displays the data as venn Results samples were spun down at 10,000 g for 1 water and mobile B was 0.1 % formic acid diagram and two statistic chart for minute and incubated at 56°C for 1 hour. in 80 % acetonitrile. The gradient evaluating homogeneity of the lists size SDS-PAGE analysis: Serum protein Protein samples were alkylated with 10 µl condition was performed as follows; the and comparing the compactness of concentrations were determined by using of iodoacetamide ( IAA) and incubated at mobile phase gradients were conditioned multiple venn diagram. Moreover, proteins Bradford assay. Sodium dodecyl sulfate room temperature in the dark for 1 hour. as follows: equilibrated period, 0-4.0 min: were classified according to its function, polyacrylamide gel electrophoresis (SDS- Next, 10 µl of trypsin solution ( 2 ng/µl) 15% B, separation period, 4.01-14.0 min: which related to protein class and PAGE) analysis was performed for was added into each sample tubes and 15– 60% B, washing period, 14.01-16.00 biological process at the level of the cell or separating proteins of 10 different groups. incubated at 37°C overnight. The digested min: 95% B and re-equilibration period organism by using Protein ANalysis Protein (10 µg) were loaded and visualized samples were dried by SpeedVac. The 16.01-20.00 min, 15% B. THrough Evolutionary Relationships or with silver staining as shown in Figure 1. dried samples were re-dissolved by the PANTHER classification system Differential protein expressions according Protein identification and database addition of 42 µl formic acid and (available at http://www.pantherdb.org)16. to various molecular weights (kDa) of analysis: Proteins were quantified by centrifuged at 10,000 rpm for 10 minutes. The PANTHER classifications are the each sample were observed. The using DeCyder MS Differential Analaysis The peptides were provided in vials for result of bioinformatics algorithms. Lastly, differences in the protein bands with software (DeCyderMS, GE Healthcare)12, liquid chromatography-tandem mass the identified proteins were submitted to molecular mass 17-30 kDa were observed 13. Consequently, the data were submitted spectrometry (LC-MS/MS) analysis. The Search Tool for Interacting Chemicals between pooled control group and to the Mascot software (Matrix Science, (STITCH) (http://stitch.embl.de) for hyperhomocysteinemia groups. Total LC-MS/MS: The purified peptides were London, UK14 and searched against the predicting interactions between proteins proteins in serum samples were digested analyzed by Ultimate 3000 Nano/Capillary NCBI database. Database interrogation and small molecules17. STITCH is a and analyzed by LC-MS/MS. LC System (Dionex Ltd., UK) coupled to a was; taxonomy (Homo sapiens); enzyme Hybrid quadrupole Q-Tof impact II™ (trypsin); variable modifications (Bruker Daltonics GmbH, Germany) (carbamidomethyl, oxidation of equipped with a Nano captive spray ion methionine residues); mass values source. The 500 nl of the purified (monoisotopic); protein mass bioactive peptide was subjected to the (unrestricted); peptide mass tolerance (1.2 trapping column (Thermo Scientific, Da); fragment mass tolerance (±0.6 Da), วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 15

Figure 1. SDS-PAGE protein profiles of human serum presented sample in the pooled of (B) control and case groups.

Protein classification: A total of 625 biological process (Figure 2A). In differentially expressed proteins were addition, these proteins were further classified by Panther program and categorized by the class of protein that presented into the categories of biological mainly distributed in nucleic acid binding process and protein class as shown in (18.9%), hydrolases (13.9%), enzyme Figure 2. Most proteins involved in the modulators (8.8%) and transcription cellular process (27.6%) and metabolic factors (8.4%) (Figure 2B). process (23%) when classified by the

Figure 2. Classification of total proteins by their functions in the categories of (A) biological process and (B) protein class.

Figure 1. SDS-PAGE protein profiles of human serum presented sample in the pooled of (B) control and case groups.

Figure 2. Classification of total proteins by their functions in the categories of (A) biological process and (B) protein class.

Interaction of unique proteins and displays the chart. The jvenn statistics homocysteine in different groups: exhibited that the different homocysteine Detection of unique proteins in control groups generate gene list with different group (normal homocysteine levels) and sizes (minimum 649 from case group III – hyperhomocysteinemia group were maximum 685 from case group II) and presented as Jvenn diagrams ( Figure 3) . most of these were shared between groups. Jvenn computes the intersection count The overlapping protein of all four number for each element and subsequently datasets was found to be 625, which was วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 18 located in central area of venn diagram. were summarized in Table 1. By using Table 1: Unique proteins were identified by UniProt in each group and the relative fold The specific list of protein to only one STITCH 5.0 for further analysis, only 5 changed of protein expressions were compared with control group

Mass Fold dataset will be considered as the unique out of 9 proteins are reported that they are Group No. gi number Protein names Function (Da) changed protein. Name and functions of the unique indirectly related to homocysteine. Non- 1. gi|160380653 Double homeobox 885.4 DNA-binding, regulation - hyperhomocysteinemia protein A (DUXA) of transcription proteins were subsequently identified by Homocysteine interact with anoctamin 1, (control) Case group I 1. gi|1035664363 Mucin 19 1391.6 Mucin family members are 18.1 UniProt. For control group, double ferrochelatase, tumor suppressor candidate (MUC19) glycoproteins that have tandem repeats which are homeobox A was the only 1 unique protein 2, and RNA binding motif protein 38. The extensively O-glycosy- lated. The structural detected. Group of mild results showed that these proteins features of mucin proteins are responsible for the gel- hyperhomocysteinemia (case group I) expressions were induced in high like properties of mucus Case group II 1. gi|26454626 Lysophosphatidic 3184.3 G-protein coupled 18.6 exhibited two specific proteins. Five and homocysteine group (group III, > 50 acid receptor 1 receptor, lipid binding (LPAR1) six unique proteins were found in case µmol/L, with high BMI and CAVI > 9). 2. gi|74746565 O6-methylguanine- 1749.6 Apoptosis 10.4 induced apoptosis 2 group II and III, respectively. Of total 17 Homocysteine can also interact with (MAPO2 ) Case group III 1. gi|7920153 GPALPP motifs- 2003.0 Unknown 17.6 gi numbers of uniques from venn diagram, lysophosphatidic acid receptor 1. This containing protein 1 (GPALPP1) only 9 unique proteins could be identified protein level is upregulated in the group of 2. gi|62897941 Ferrochelatase 1003.7 Heme biosynthetic process 10.5 (FECH) by UniProt. The identification of protein moderate hyperhomocysteinemia (group 3. gi|7531123 Tumor suppressor 654.3 Cell cycle, inflammatory 9.9 candidate 2 (TUSC2) response markers in human serum with different II, 30-50 µmol/L). 4. gi|215273895 RNA-binding motif 1512.6 RNA-binding, cell cycle, 9.2 protein 38 (RBM38) cell differentiation levels of homocysteine and their functions 5. gi|158259637 Anoctamin-1 881.7 Intracellular calcium 7.4 (ANO1) activated chloride channel activity

Discussion Based on these results of protein identifications in different groups, one Hyperhomocysteinemia is an unique protein (Mucin 19) from serum of independent risk factor for cardiovascular subjects with mild level of homocysteine diseases. The promoting effect of (15-30 µmol/L) was markedly induced. homocysteine on vascular dysfunction has Mucin 19 (MUC19) is a glycoproteins been considered as one of the important localized to salivary mucous cells. pathological bases of atherosclerosis. Alterations of salivary expression of However, the various mechanism induced MUC19 affecting the oral microbiota may by homocysteine remains unclear. The in turn influence the intestinal microbiota present research used proteomic and immune homeostasis, leading to CVD techniques to initially analyze the protein risk18. Homocysteine may contribute to changes in this clinical event. In this study, pyroptosis (caspase 1-dependent Figure 3. Venn diagram presented the unique proteins detected in four groups (control group, case we identify the alteration of serum group I, II and III). The specific lists of gene included 4, 2, 5 and 6 for control, case group I, II and III, programmed cell death) and changes gut proteome in subjects with hyper- respectively. The vertical chart displayed the number of elements in each list for checking the microbiome. homogeneity of the lists size. The horizontal chart showed the number of specific or shared elements. homocysteinemia (Table 1). วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 19 located in central area of venn diagram. were summarized in Table 1. By using Table 1: Unique proteins were identified by UniProt in each group and the relative fold The specific list of protein to only one STITCH 5.0 for further analysis, only 5 changed of protein expressions were compared with control group

In other point of view, suggested greater homocysteine level and enzymes, such as FECH can lead to to increase the expression of plasminogen lysophosphatidic acid receptor 1 (LPAR1) greater risks of CVD. cellular damage through altering activator inhibitor-1 (PAI-1) and to and O6-methylguanine induce apoptosis 2 In group of very high homocysteine membrane permeability and increasing enhance ex vivo blood coagulation on an ( MAPO2) were unique proteins that up- levels (> 50 µmol/L), up-regulation of five oxidative stress and is associated with endothelial monolayer25. Findings from regulated in moderate hyper- unique proteins were observed, including abnormal cardiovascular functions23. this study also showed that another protein homocysteinemia group (30-50 µmol/L). GPALPP motifs-containing protein 1 Notably, proteins involved in the related to Hcy was Anoctamin-1 ( ANO1). The impact of HHcy over different cell (GPALPP1 or KIAA1704), ferrochelatase apoptosis pathway were observed to be This protein involves in Ca2+-activated Cl- signaling pathways including G-protein (FECH), tumor suppressor candidate 2 upregulated by high Hcy expression. channels (CaCCs) and plays role in coupled receptor (GPCR) and consequent (TUSC2, RNA-binding protein 38 Tumor suppressor candidate 2 (TUSC2), numerous physiological functions. CaCCs cellular outcomes are increasingly realized. (RBM38) and anoctamin-1 (ANO1). also known as FUS1, is a novel tumor are mediated by intracellular calcium26. GPCRs are very important in modulation GPPALPP1 gene encodes the protein suppressor gene candidate that has been Activation of ANO1 promotes apoptosis of of cell structure, cell microenvironment, in human cell as lipopolysaccharide- identified in the 3p21.3 chromosomal pulmonary endothelial cells via increased and cell responses to various stimuli. specific response protein 7 (LSR7). Up- region. Activation of FUS1 in normal cells mtROS and p38 phosphorylation, leading Receptor of lysophosphatidic acid regulation of this protein was observed in in response to apoptotic stimuli or stress to apoptosis27. Previous studies confirmed (LPAR1) is one of the GPCRs and play U937 cells when treated with nicotin. can lead to trigger cytochrome c (Cyt C) ANO1 activity and expression in smooth role in LPA pathways related to CVD However, the function of this protein was release from the inner membrane of muscles cell from various arteries risks. LPA acts as phospholipid messenger not yet well understood but likely related mitochondria to the cy-tosol, selectively veins28,29. There were supported that with multi-functions and mainly produced to inflammation, immune responses22. For and directly interacts with Apaf-1. After vasorelaxation of murine and human by activated platelets. Therefore, blood ferrochelatase (FECH), it is an enzyme this stage, it can facilitate downstream arteries were induced by ANO1 inhibition. LPA level has been targeted to be an ideal which involves in heme biosynthesis. Apaf-1-mediated apoptosome assembly, Moreover, the expression of ANO1 was molecular marker for reflecting platelet Heme is an essential prosthetic group for caspase activation, and apoptosis significantly up-regulated in various activation state19. In addition, hemoproteins involved in numerous induction24. Another protein group is hypertension models30, 31. homocysteine causes a multitude of cardiovascular processes, including oxygen RNA-binding proteins (RBPs) which are In this study, we further analyze adverse effects that disrupt vascular cell transport (hemoglobin), oxygen storage master regulators of RNA biogenesis and up-regulated proteins related to increased homeostasis via activating apoptosis by (myoglobin), oxygen metabolism metabolism. This study found alteration of homocysteine levels with the specific ROS release and ER stress20. For O6- (oxidases), antioxidation (peroxidases, one kind of RBPs which is RNA-binding program STITCH version 5.0. This methylguanine-induced apoptosis 2 catalases), and electron transport motif protein 38 (RBM38). It is a target of software provides global interactions (MAPO2), it has involved in the induction (cytochromes). Another important issue is the p53 family and modulates p53 between proteins and small molecules ae 21 of cellular apoptosis . Upstream DNA heme function in the liver which is the expression via mRNA translation. An well as an integral part of biological damage pathway with increased apoptotic synthesis of cytochrome P450 enzymes. experimental study demonstrated that processes in living organisms. In Figure 4, proteins is involved in triggering apoptosis This enzyme system are required for liver overexpression of p53 in endothelial cells our results from STITCH 5.0 demonstrated vascular smooth muscle cell and this detoxification under different conditions. It was found to reduce the expression of a variety of the differentially expressed consequence can hasten plaque rupture. is also recognized that heme accumulation nitric oxide synthase and thrombomodulin, proteins in hyperhomocysteinemia Increased both of two proteins level caused by up-regulation of important condition. LPAR1, FECH, TUSC2, วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 21

RBM38 and ANO1 proteins indirectly extremely high homocysteine levels that process in all cells. Heme modulates Conclusion interacted with homocysteine via apoptotic involved in apoptotic signaling. The enzyme activity of cystathionine-β- To our knowledge, this is the first pathways. The possible process linking association between ANO1 and FAS synthase (CBS) in response to redox preliminary study that applied proteomic HHcy and apoptosis is endoplasmic associated via death domain (FADD) was change. CBS is known as pyridoxal techniques for studying protein markers reticulum (ER) stress. Previous study reported. FADD is an adaptor molecule phosphate-dependent enzyme that also and identified expression profiles in serum indicated that homocysteine induces ER interacting with many kinds of death contains heme b as a cofactor. CBS is the with hyperhomocysteinemia condition in stress–mediated activation of caspase-3 in receptors and induces apoptosis by key enzyme in transulfuration pathway for Thai men population. In this study, 8 endothelial progenitor cells from patients caspase-836,37. Noticeably, high levels of clearance of excessive homocysteine levels unique proteins were up-regulated in with coronary heart disease. In other FADD and caspase-8 were associated with to form cystathionine and promotes human serum with hyperhomocysteinemia words, enhanced ER stress-mediated increased incidence of coronary events38. glutathione synthesis46. Glutathione is the condition, including MUC-19, LPAR1, activation of caspase-3 in endothelial For RBM38, it has been implicated in cell important antioxidant, which play critical MAPO2, FECH, GPALPP1, TUSC2, progenitor cells might be involved in cycle regulation and differentiation. The role for defense mechanism in every cell RBM38 and ANO1. Interestingly unique hyperhomocysteinemia-associated vascular diverse biological functions of RBM 38 are from HHcy induced oxidative stress. proteins in very high homocystetine level, pathology32. A large number of published probably due to its role as an RNA-binding Interplay of homocysteine induced TUSC2, RBM38 and ANO1 interacted researches were used to support overall protein to regulate multiple targets. For apoptosis-proteomics outcomes may be with homocysteine via apoptotic pathways data from this proteomic study. LPA could example, RBM38 regulates mRNA applicable in facilitating the assessing that may indirectly associated to vascular facilitate endothelial cell death by stability of mdm239 and p2140. In addition, potential novel molecular targets to reduce pathology. modulating the redox environment, which RBM38 can regulate p53 translation by cardiovascular risk related with elevated dependd on LPAR1-mediated modulation modulating the binding of eIF4E, a Hcy levels in various human populations. of cell redox state33. For TUSC2, it has translation initial factor, to the p53 In addition, it can lead to elucidate new Conflicts of Interest been shown to induce G1 cell cycle arrest mRNA41, 42. The importance of p53 is its mechanisms through which protein The authors declare that there are no and promote apoptosis24. A study in small ability as a transcription factor to regulate a functions can be regulated by the redox conflicts of interests regarding the cell lung cancer (SCLC) exhibited that series of downstream target genes, such as status with the use of various strategies publication of this paper. TUSC2 induces cell death through p53 induces p21 (WAF1/CIP1) for cell (such as dietary, lifestyle modification or mechanism related to caspase-dependent cycle regulation43. MAPK can regulate targeted drugs). However, the limitation of apoptosis34. Interestingly, TUSC2 apoptosis through specific phosphorylation this study is a small sample size in each expression was negatively regulated by of downstream mediators of apoptosis, group for proteomic analysis. Further several microRNAs, especially miRNA- including the tumor suppressor p53, thus proteomic studies in large population are

378. Previous study revealed that miRNA- linking cellular stress signaling and needed to validate protein markers and

378 binded to 3′-UTR of the TUSC2 regulation of p53 activity44, 45. The last clarify role of different protein expressions transcript and repressed its translation, finding of unique protein related to related to hyperhomocysteinemia in human resulting in enhances cell survival and homocysteine in group of extremely serum. reduce caspase-3 activity35. For ANO1, it hyperhomocysteine levels is FECH. It is was another protein presented in group of the enzyme of the heme biosynthetic วารสารพิษวิทยาไทย 2561 ; 33(2) : 9-28 23

378. Previous study revealed that miRNA- linking cellular stress signaling and needed to validate protein markers and

marker in liver of rat offspring. Mol Cell homocysteinemia interactions with LDL, Proteomics 2015; 14(11):2901-9. macrophage function, paraoxonase 1, and 2. DiBello PM, Dayal S, Kavetu S, Zhang exercise. Ann N.Y. Acad Sci 2016; D, Kinter M, Lentz SR, et al. The 1363(1):138-54. nutrigenetics of hyperhomocysteinemia: 8. Sharma P, Senthilkumar RD, quantitative proteomics reveals differences Brahmachari V, Sundaramoorthy E, in the methionine cycle enzymes of gene- Mahajan A, Sharma A, et al. Mining indyced versus diet-induced hyper- literature for a comprehensive pathway homocysteinemia. Mol Cell Proteomics analysis: a case study for retrieval of

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homeostasis. Acta Biochim Pol 2014; Vanavanan S, Yamwong S, et al. Cohort Figure 4. An interaction of homocysteine and unique proteins related to apoptotic pathways (including LPAR1, FECH, TUSC2, RBM38 and ANO1 by using STITCH 5.0. ; LPAR1, 61(4):815-23. Profile: The electricity generating lysophosphatidic acid receptor 1; FECH, ferrochelatase; TUSC2, tumor suppressor candidate 2; 4. Greco M, Chiriaco F, Boccio PD, authority of Thailand study. Int J RBM38, RNA binding motif protein 38; ANO1, anoctamin1; FADD, fas-associated via death domain; CASP3, caspase 3; CASP4, caspase 4; CASP7, caspase 7; CASP8, caspase 8; MAPK8, mitogen- Tagliaferro L, Acierno R, Menegazzi P, et Epidemiol 2012; 41(2):359-65. activated protein kinase 8; EP300, E1A binding protein p300; CDKN1A, cyclin-dependent kinase al. A proteomic approach for the 10. National Cholesterol Education inhibitor 1A; MDM2, mdm2; BRCA1, breast cancer; KAT2B, K(lysine) acetyltransferase 2B; SIRT1, sirtuin 1; Haem, heme b; TP53, tumor protein p53, CDKN2A, cyclin-dependent kinase inhibitor 2A; characterization of C677T mutation of the Program ( NCEP) . Third report of the ATM, ataxia telangiectasia mutated). **, *** represented unique proteins from case group II and case group III, respectively. human gene methylenetetrahydrofolate national cholesterol education program reductase. Proteomics 2006; 6(19):5350- (NCEP) expert panel on detection,

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สำ 昱กคุณภาำและคุาำปละดภากอดำาำ ค ปาิทอำศำสต ์คำ ลแทอ์ ถ. ติาำ昱昱ท์ ด.เปืดง จ. 昱昱ทบณ ี 11000

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