S100B和S100A1可作為抗癌藥物之醫藥用蛋白｜國立清華大學博碩士論文庫

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研究生：	東郭旺 Dowarha, Deepu
論文名稱：	S100B和S100A1可作為抗癌藥物之醫藥用蛋白 S100B and S100A1 are the pharmaceutical proteins against cancer disease
指導教授：	余靖 Yu, Chin
口試委員:	蘇士哲 Su, Shih-Che 莊偉哲 Chuang, Woei-Jer 陳金榜 Chen, Chin-Pan 鄒瑞煌 Chou, Ruey-Hwang
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	139
中文關鍵詞：	醫藥用蛋白
外文關鍵詞：	Pharmaceutical proteins
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目前已知S100蛋白質家族具有許多細胞內和細胞外功能，例如鈣穩態，細胞增殖和分化，凋亡，轉錄，組織發育和修復。近年來，S100蛋白引起了研究界的關注，因為S100蛋白與多種導致腫瘤的過程有關，其表達模式的改變與多種人類癌症有關。RAGE（晚期糖基化終產物的受體）是S100蛋白的一般受體之一，S100蛋白也與癌症控制蛋白p53和MDM2（鼠雙分2）有關。
S100，RAGE，p53和MDM2的蛋白質與蛋白質交互作用研究將有助於我們理解它們在癌症控制和生長中的作用。S100蛋白及其靶分子之間形成的大分子復合物結構將能提供有用的數據，這些數據可為尋求抗癌特異性治療分子的藥物研究提供支持。為了理解這一點，我們研究了蛋白質-蛋白質相互作用研究，包括S100A11，S100B，RAGE，S100A1，p53和MDM2蛋白質。
在研究項目1（第二章）中，我們通過1H-15N HSQC-NMR（heteronuclear single-quantum correlation-NMR）滴定法研究了S100A11和S100B蛋白之間的相互作用。然後，我們利用HADDOCK程序構建了S100A11–S100B異二聚體複合物，然後將其與之前報導的S100A11–RAGE V域複合物疊加。疊圖數據證明，S100B可能阻礙S100A11與RAGE V域的結合界面區域。此外，WST-1（水溶性四唑-1）測定法可提供這些蛋白質在體外癌症模型中作用的功能性讀數。我們的研究證實，S100B拮抗劑的改良可以在S100和RAGE的人類疾病治療中扮演重要的角色。

在研究項目2（第三章）中，我們將工作重點放在可能干擾p53-MDM2相互作用的拮抗劑的開發上，因為據推測，刺激wild-type p53活性的有效策略可能需要打斷p53-MDM2相互作用，從而恢復具有wild-type p53的腫瘤中p53腫瘤抑制能力。目前已知，S100A1蛋白與MDM2和p53蛋白的N末端結構域有相互作用，我們對於當S100A1蛋白與MDM2和p53蛋白相互作用時， S100A1的界面區域的研究十分感興趣，此介面區物將作為結構的藥物設計方法的一部分，以發展出合適的拮抗劑干擾p53-MDM2交互作用。我們應用核磁共振光譜研究了S100A1與MDM2和p53的N末端結構域之間的結合界面。使用NMR和HADDOCK方法進行的數據分析顯示出S100A1片段（17個殘基）可能成功阻止p53-MDM2相互作用。為了檢驗癌細胞系中的假設，我們合成了源自S100A1蛋白的17個殘基的肽，並將其連接到可穿透細胞的HIV-TAT肽上，並將其命名為Peptide 1。來自HSQC-NMR competitive bingding實驗，WST-1分析，蛋白質印跡和細胞週期分析的合作數據支持了我們的假設，並表明peptide 1可以成功干擾p53-MDM2相互作用並激活正常的p53功能導致癌細胞的細胞週期停滯和凋亡細胞死亡。此證明了針對癌症生長，開發更多訂製藥物分子的可能性。
總體而言，通過我們的研究工作，我們更理解分子層級上與S100A11，S100B，RAGE，S100A1，p53和MDM2蛋白質有關的蛋白質-蛋白質和/或蛋白質-配體之間的交互作用，並為他們在訂製的抗癌藥物分子的開發與應用做出了貢獻。

S100 family of proteins are known to possess numerous intracellular and extracellular functions such as calcium homeostasis, cell proliferation and differentiation, apoptosis, transcription, tissue development, and repair. In recent times, S100 proteins have attracted the focus of the research community as S100 proteins are found to relate with various tumorigenic processes and their altered expression patterns are associated with multiple human cancers. One of the general receptors of S100 proteins is RAGE (Receptors for advanced glycation end products) and S100 proteins are also found to associate with the cancer controlling proteins p53 and MDM2 (Murine double minute 2). Protein-protein interaction studies involving the S100, RAGE, p53, and MDM2 will elucidate important aspects which will be helpful to understand their role in cancer control and/or progression. The structural insights into the macromolecular complex formation amongst S100 proteins with its target molecules will provide useful data which will be supportive in the pharmaceutical research for the quest of specific therapeutic molecule/s against cancer. To comprehend this, we studied the protein-protein interaction studies which included S100A11, S100B, RAGE, S100A1, p53, and MDM2 proteins.
In research project 1 (Chapter II), we studied the interactions amongst S100A11 and S100B proteins by employing the 1H–15N HSQC-NMR (heteronuclear single-quantum correlation-NMR) titrations. Then, we utilized the HADDOCK program to construct the S100A11–S100B heterodimer complex following which it was superimposed with the S100A11–RAGE V domain complex reported earlier. The overlay data exploration revealed that the S100B could obstruct the binding interface region of S100A11 and the RAGE V domain. Furthermore, WST-1 (water-soluble tetrazolium-1) assay provided a functional read-out of the effects of these proteins in an in vitro cancer model. Our study establishes that the improvement of a S100B antagonist could perform a vital part in the treatment of S100- and RAGE-dependent human diseases.
In research project 2 (Chapter III), we focused our work towards the development of antagonist that could interfere with the p53-MDM2 interaction, as it was postulated that the effective strategy to stimulate the activity of wild type p53 may require the breakdown of p53-MDM2 interaction, restoring the p53 tumor suppressor capability in tumors with wild-type p53. The S100A1 protein was reported to interact with the N-terminal domain of MDM2 and p53 protein and therefore attracted our interest to study the interface region of S100A1 when interacted with MDM2 and p53 protein as a part of structure-based drug design approach for the development of suitable antagonist interfering with the p53-MDM2 interaction. We applied NMR spectroscopy to study the binding interface amongst the S100A1 and N-terminal domain of MDM2 and p53. The data analysis using NMR and HADDOCK methods showed the possibility of S100A1 segment (17 residues) that could block the p53-MDM2 interaction successfully. To test the hypothesis in the cancer cell line, we synthesized the 17-residue peptide derived from the S100A1 protein and attached it to the cell-penetrating HIV-TAT peptide and named it as Peptide 1. The collaborative data from the HSQC-NMR competitive binding experiment, WST-1 assay, western blotting, and the cell cycle analysis supported our assumption and showed that the Peptide 1 could successfully interfere with the p53-MDM2 interaction and could activate the normal p53 functions, leading to cell cycle arrest and apoptotic cell death in cancer cells. This information shows the possibility for the development of more customized drug molecules against the cancer development.
Overall, through our research work we have contributed towards the understanding of protein-protein and/or protein-ligand interactions at a molecular level pertaining to the S100A11, S100B, RAGE, S100A1, p53, and MDM2 proteins, and their application in the development of customized drug molecule against cancer.

Table of contents
Abstract in Chinese………………………………………………………I
Abstract in English………………………………………………………III
Acknowledgement……………………………………………………………VI
Table of Contents…………………………………………………………IX
List of figures……………………………………………………………XIV
Abbreviations………………………………………………………………XXVI

Chapter - I
Introduction
1.1. Introduction ………………………………………………………………2
1.2. S100 family of Proteins…………………………………………………9
1.3. Receptor for advanced glycation end products (RAGE)……………11
1.4. Tumor protein 53 (TP53 or p53)…………………………………….…13
1.5. Murine double minute 2 (MDM2)…………………………………………15
1.6. Protein extraction and purification…………………………………18
1.7. Reverse-phase high-performance liquid chromatography (RP-HPLC).21
1.8. Protein analysis……………………………………………………………23
1.9. Protein-ligand interactions…  ………………………………………24
1.10. NMR spectroscopy to map protein-protein interaction in solution………………..............................................30
1.11. Chemical shift perturbation to demonstrate protein-ligand binding………………...............................................32
1.12. HADDOCK- a data driven molecular docking tool……………………………........................................37
1.13. WST-1 assay over MTT assay…………………………………………..41

Chapter - II
S100B as an antagonist to interfere with the interface area flanked by S100A11 and RAGE V domain
2.1. Introduction………………………………………………………………………44
2.2. Materials and Methods……………………………………………………46
2.2.1. Materials……………………………………………………………………….…46
2.2.2. Expression, Labeling, and Purification…………………………46
2.2.3. HSQC- NMR Titration Experiments…………………………………55
2.2.4. Molecular Docking……………………………………………………56
2.2.5. Cell Proliferation Assay……………………………………………57
2.3. Results and Discussion…………………………………………………57
2.3.1. The binding interface (S100A11/S100B) region in S100A11…………………….........................................57
2.3.2. The binding interface (S100A11/S100B) in S100B…………………………..…….................................60
2.3.3. The fundamental depiction of the S100A11-S100B complex……………………….......................................62
2.3.4. Dissociation constant………………………………………………65
2.3.5. WST-1 assay……………………………………………………………69
2.3.6. S100B inhibits the S100A11-RAGE V domain……………………70
2.4. Conclusion………………………………………………………………72

Chapter - III
S100A1 blocks the interaction between p53 and mdm2 and decreases cell proliferation activity
3.1. Introduction………………………………………………………………………75
3.2. Materials and methods……………………………………………………77
3.2.1 Materials………………………………………………………………………….77
3.2.2. Expression, Labeling, and Purification………………………….78
3.2.3. Peptide synthesis………………………………………………………95
3.2.4. HSQC- NMR titration experiments……………………………………95
3.2.5. Molecular docking……………………………………………………..96
3.2.6. HSQC- NMR competitive binding experiment………….……………96
3.2.7. Fluorescence experiment………………………………………………97
3.2.8. Circular dichroism (CD) spectroscopy…………………………….98
3.2.9. Cell proliferation assay…………………………………………….98
3.2.10. Western blotting……………………………………………………..98
3.2.11. Cell cycle analysis………………………………………………….99
3.3. Results and discussion………………………………………………….99
3.3.1. Interaction amongst labeled S100A1 and unlabeled MDM2………………………............................................99
3.3.2. Interaction amongst unlabeled S100A1 and labeled MDM2…………………….............................................101
3.3.3. Illustration of the S100A1−MDM2 complex……………………….103
3.3.4. Overlapping of S100A1-MDM2 complex with p53 (peptide)-MDM2 complex…………………………………………………………………………..106
3.3.5. S100A1 could interfere with the interaction amongst p53 and MDMX……..….....................................................107
3.3.6. Interaction amongst unlabeled S100A1 and labeled p53 (1-73)…………….….................................................108
3.3.7. Interaction amongst labeled S100A1 and unlabeled p53 (1-73) and comparison of S100A1-p53 (1-73) HSQC-NMR data with S100A1-MDM2 HSQC-NMR data……....................................................110
3.3.8. S100A1 derived peptide construction……………………………111
3.3.9. CD spectra……………………………………………………………113
3.3.10. Fluorescence experiment…………………………………………114
3.3.11. Competitive binding experiment by NMR………………………116
3.3.12. WST-1 assay…………………………………………………………118
3.3.13. Western Blotting………………………………………………….120
3.3.14. Cell cycle analysis………………………………………………121
3.4. Conclusion……………………………………………………………  122













List of figures
Chapter- I
Figure 1.1. Schematic illustration of bacterial cell E. coli……….2
Figure 1.2. Schematic depiction of eukaryotic cell…………………….3
Figure 1.3. The diagrammatic representation of flow of genetic information…..……….…...........................................4
Figure 1.4. The diagrammatic representation of secretary pathway of protein in a eukaryotic cell………………………………………………………………............….5
Figure 1.5. (a) Illustration of the basic amino acid structure and (b) the association of two amino acid subunits leads to the formation of peptide bond…………………..……….............................….7
Figure 1.6. Figure showing the four structural stages of a protein structure……….……..............................................8
Figure 1.7. Schematic and cartoon illustration of S100 protein basic structure. (a) L1 and L2 are the Ca2+ binding loop flanked by the α-helices HI to HIV. Hinge region, H, connects the HII and HIII.20 (b) The cartoon displaying apo-S10011 dimeric form with Ca2+ binding loops in red color and α-helices labelled in both chains of the apo-S100A11 protein…………………………………………………………………...…...10
Figure 1.8. Calcium induced conformational changes in calcium-free (Apo) and calcium-bound S100A11 protein………………..………...…..11
Figure 1.9. Schematic representation of RAGE protein…………………12
Figure 1.10. S100 Protein binding to RAGE triggers various RAGE mediated cellular signaling. Few S100 proteins, such as, S100B, S100P, S100A8/A9, S10012, and S100A14 binds to RAGE and activates NF-KB, MAPK, and PI-3K/AKT signaling pathways thereby regulating cellular processes including inflammation and cancer. S100A6 initiates the cell apoptosis by activating JNK pathway and restricts cell proliferation…………………………………………………...……………..13
Figure 1.11. Schematic diagram of p53 indicating various functional domains……...……................................................14
Figure 1.12. Graphic illustration of MDM2 indicating various functional domains……..........................................…16
Figure 1.13. Schematic diagram of the p53-mdm2 auto-regulatory feedback loop. P53 positively regulates MDM2 by controlling MDM2 gene expression and MDM2 negatively regulates p53 biological role by three possible means………………………………….…......................17
Figure 1.14. Schematic representation of steps involved in protein purification after its expressio…………………………………...…….21
Figure 1.15. Schematic representation of HPLC system basic components…………............................................……23
Figure 1.16. Schematic basic illustration of mass spectrometer components and data flow……………………………………………...…...24
Figure 1.17. The schematic representation of all the three binding models. (a) Protein and ligand are rigid and binds to each other as interface region is complementary to each other as a feature of lock and key model. (b) Ligand binding induces a conformational change in protein binding site for enhanced fitting as the protein binding interface is quite flexible and represents induced fit model. (c) In conformational selection model, ligand binds to the best fitting conformational state of the protein amongst a population of varied protein states……………………………………………………………………….…...27
Figure 1.18. Superimposition of the 2D spectra of labeled MDM2 (red) and in complex with Amx drug in 1:1 (blue) and 1:2 (yellow) ratio. Peak shift is observed as a result of ligand binding (Amx) to protein (MDM2) and is highlighted in dotted white color………......……...34
Figure 1.19. Illustration of fast and slow exchange system. Left panel shows the fast exchange, where peaks move from free (blue) to bound state (red) freely with some broadening at midway locations. Right panel shows the slow exchange, where the free peak (blue) intensity decreases during the course of titration and the bound peak (red) intensity increases……………………………………….……………….….36
Figure 1.20. The online HADDOCK server showing Easy interface mode…..…………..................................................40
Figure 1.21. The conversion of MTT to Formazan indicative of living cells………..…...................................................41
Figure 1.22. The conversion of WST-1 to Formazan indicative of living cells……….......................................................42

Chapter- II
Figure 2.1. Human S100A11 (wild type) clone construct. (a) The map of pET-20b (+) vector displaying various cloning sites. NdeI and/or XhoI restriction sites was used for the insertion of S100A11 protein sequence. (b) Amino acid sequence of S100A11 protein showing one letter amino acid code for each amino acid…………………………….47
Figure 2.2. Chromatogram displaying (a) S100A11 purification via HIC column followed by (b) SDS-PAGE………………….……………………..49
Figure 2.3. Elute fractions (E1 and E2), from HIC column purification, were mixed, concentrated and purified further via HPLC column. (a) HPLC chromatogram displaying pure S100A11 peak which is identified and verified for purity with (b) SDS-PAGE followed by (c) ESI-MS mass confirmation……………………………………......………..…………..50
Figure 2.4. Amino acid sequence of S100B protein showing one letter amino acid code for each amino acid……………………………………51
Figure 2.5. Chromatogram displaying S100B purification via Q column and the inset shows the SDS-PAGE indicating the E1 and E2 elute fractions contain S100B protein...........................................................52
Figure 2.6. Elute fractions (E1 and E2), from Q column purification, were mixed, concentrated and purified further via SEC column. Chromatogram illustrating S100B purification via SEC column and the inset shows the SDS-PAGE displaying the fractions 1 to 9 tested for S100B protein. Fractions 6 and 7 contained least impure S100B protein…………………………………………………………………………...53
Figure 2.7. Elute fractions (6 and 7), after SEC column purification, were mixed, concentrated and purified further via HPLC column. (a) HPLC chromatogram displaying pure S100B peak which is identified and verified for purity with (b) SDS-PAGE followed by (c) ESI-MS mass confirmation…………………………….………………........……….….54
Figure 2.8. Diagrammatic representation of steps involved in the dialysis process…….............................................56
Figure 2.9. Analysis of the binding interface (S100A11/S100B) reion in S100A11. (a) 1H-15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11 binding to unlabeled S100B (blue). Cross-peaks illustrating changes in intensities are shown in boxes. Horizontal lines connect the NH2 side chains. (b) Cross-peak intensity plot (I/I0) where (I) denotes cross peak intensity of S100A11-S100B complex and (I0) indicates the early intensity of free S100A11 versus a number of amino-acid residues (1-105) illustrated as a bar diagram. (c) A picture illustration of the S100A11 monomer with residues exhibiting decreases in cross-peak signals distinguished by the cyan color………………………………………….….........................59
Figure 2.10. Analysis of the binding interface (S100A11/S100B) region in S100B. (a) 1H-15N HSQC spectra overlay of free 15N S100B (red) and 15N S100B binding to unlabeled S100A11 (blue). Cross-peaks showing changes in intensities are shown in boxes. (b) Cartoon representation of S100B monomer with residues exhibiting decreases in cross-peak signals are shown in yellow…………………………………………………62
Figure 2.11. (a) Cartoon representing S100A11-S100B complex created with HADDOCK program. S100A11 is depicted in red and the S100B in blue color. Residues near the interaction sites are illustrated in cyan (from the S100A11 side) and yellow (from S100B side) respectively. (b) List of S100A11 and S100B residues as constraints for HADDOCK program……………………………..……………………………………………..63
Figure 2.12. The Ramachandran plot. The online PROCHECK program was used to test the structural stereochemistry of the complex shown in figure 2.11. The Ramachandran plot disclosed the occurrence of 82.1% residues in the maximum favoured regions, 13.4% in additionally allowed areas, 2.8% in allowed sector, and 1.7% in the disallowed zone………………………………………………………………………………..64
Figure 2.13. 1H- 15N HSQC spectra of free 15N S100A11 (red)………65
Figure 2.14. 1H- 15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11 homodimer + S100B homodimer mixture (blue)…………..66
Figure 2.15. 1H- 15N HSQC spectra overlay of free 15N S100A11 (red) and 15N S100A11- S100B heterodimer (green)……………………........67
Figure 2.16. Dissociation constant. The Kd calculated by the designated residues detected in 15N S100A11 HSQC titrations with S100B (yellow) and vice-versa (brown). The overall average Kd is about 2.65 μM…………………………………………………..…………...............68
Figure 2.17. WST-1 assay analysis. The SW480 cells were treated with 100 nM S100 A11 (dark blue), 100 nM S100B (light blue), and 100 nM S100A11-S100B heterodimer (green). The proportional cell counts after subsequent treatment with S100A11 and S100B are depicted as fold inductions with serum-free media alone as the control (red)………………………………………………………………..…….……..70
Figure 2.18. S100B interfering S100A11-RAGE V domain. (a) The complex model of S100A11-RAGE V domain constructed via HADDOCK. (b) Superimposition of the complex between S100A11 (red) and the RAGE V domain (green), with the complex amid S100A11 (red) and S100B (blue). (c) The magnified depiction of the clear and significant hindrance to S100A11-RAGE V domain associated with the S100B protein…………………………………………………………………………...72

Chapter- III
Figure 3.1. The picture illustration of pET28-p53 (1-73) vector…79
Figure 3.2. His tag fused p53 (1-73) protein (fusion protein) showing the amino acid sequence (a) before enzyme digestion with the site for thrombin cutting, and (b) sequence after enzyme digestion…………79
Figure 3.3. Chromatogram displaying p53-TAD (1-73) purification via NiNATA Superflow resin column and the inset shows the SDS-PAGE indicating various fractions. S represents the crude sample loaded onto the column; LB, WB, and EB indicate the elute fractions collected by employing lysis buffer, wash buffer, and the elution buffer. EB fraction contained the p53-Histidine tag fusion protein (10.13 kDa) which comes above the 15 kDa band before enzyme digestion…………81
Figure 3.4. Following enzyme digestion with Thrombin, digested fusion protein sample was loaded onto the Superdex 75 (SEC) column and the inset shows SDS-PAGE displaying the fractions (1 to 7) collected after enzyme digestion and cleaved p53 (1-73) protein (8.6 kDa) is observed (fraction, 5, 6, and 7) below the 15 kDa band, though fraction 7 has less protein concentration…………………………………………………………………...82
Figure 3.5. Following SEC purification, fraction consisting pure protein was subjected to the ESI-MS analysis for the confirmation of cleaved p53 (1-73) protein having calculated mass of 8617.53 kDa…………………………………………………………….......…………83
Figure 3.6. The picture illustration of pGEX6P2-MDM2 (17-125) vector…….………….............................................84
Figure 3.7. GST tag fused MDM2 (17-125) protein (fusion protein) showing the amino acid sequence (a) before enzyme digestion with the site for PreScission Protease enzyme cleavage, and (b) sequence after enzyme digestion………………………………..……....................85
Figure 3.8. Chromatogram displaying GST-MDM2 fusion protein purification via GST column and the inset shows the SDS-PAGE indicating the E1 and E2 elute fractions containing the fusion protein…………………………………………..……………..........…….86
Figure 3.9. Chromatogram displaying the purification of cleaved MDM2 protein via GST column after enzyme digestion. The inset shows the SDS-PAGE where, S represents the crude MDM2-GST tag fusion protein, F1 is the flow and E1 is the elute collected before enzyme digestion, AED represents the E1 sample incubated with the PreScission protease enzyme for the 16 hours’ enzyme digestion, F2 represents the cleaved MDM2 protein (12.9 kDa) collected in flow after enzyme digestion, E3 represents the mixture of any remained fusion protein (39.3 kDa) together with cleaved GST tag (26.4 kDa), and M indicates the protein marker…………………………………………………………………………….87
Figure 3.10. (a) HPLC chromatogram displaying cleaved MDM2 peak at number 4……......................................................88
Figure 3.10. (b) SDS-PAGE displaying the presence of cleaved MDM2 protein in fraction 4 corresponding to peak 4 of HPLC profile……88
Figure 3.10. (c) Cleaved MDM2 protein in fraction 4 corresponding to peak 4 of HPLC profile was confirmed by ESI-MS analysis……………89
Figure 3.11. Human S100A1 clone construct. (a) The map of pET-20b (+) vector displaying various cloning sites. NdeI and/or XhoI restriction sites was used for the insertion of S100A1 protein sequence. (b) Amino acid sequence of S100A1 protein showing one letter amino acid code for each amino acid…………………………………………….……….........90
Figure 3.12. Purification of S100A1 protein. (a) Chromatogram displaying S100A1 purification via Q-column. Elute 1 consisting S100A1 protein is collected, concentrated, and loaded on to the HIC column. (b) Chromatogram displaying S100A1 purification via HIC-column and the inset shows the SDS-PAGE indicating various fractions. S represents the crude sample, F1 and E1 indicates the flow through and elute fractions collected by employing Q- column. F2 and E2 indicates the flow through and elute fractions collected by employing HIC- column…………………………………………………………..…….........92
Figure 3.12. (c) E2 fraction collected by employing HIC-column is concentrated and loaded on to the SEC column. Fractions 4,5, and 6 contained the S100A1 protein mostly…………………………………...93
Figure 3.13. (a) HPLC chromatogram shows the S100A1 purification profile and the inset SDS-PAGE illustrates the pure S100A1 peak (peak 2) which is identified and verified for purity, followed by (b) ESI-MS mass confirmation……………………………………….................94
Figure 3.14. Exploration of the connected interface (S100A1/MDM2) section in S100A1. (a) The 1H−15N HSQC spectra overlay of free 15N S100A1 (red) and in complex with unlabelled MDM2 (blue). Cross-peaks displaying variation in their magnitude are highlighted in yellow. Residues were assigned as published earlier.226 (b) A picture illustration of the S100A1 protein with residues demonstrating a change in cross-peak signals portrayed by the red color…………101
Figure 3.15. Exploration of the connected interface (S100A1/MDM2) section in MDM2. (a) The 1H−15N HSQC spectra overlay of free 15N MDM2 (red) and in complex with unlabelled S100A1 (yellow). Cross-peaks illustrating variation in their magnitude is highlighted in blue. Residues were assigned as published earlier.229 (b) A picture showing the MDM2 protein with residues displaying change in cross-peak signs, which are characterized by the blue color……………………………..102
Figure 3.16. (a) Animation displaying S100A1−MDM2 complex generated via the HADDOCK program. S100A1 is painted in green and MDM2 in yellow color. Residues neighboring the interface regions are indicated in red (from the S100A1 side) and blue (from MDM2 side), respectively. (b) List of S100A1 and MDM2 residues as constraints for HADDOCK program.…………………...………………………...……………………..104
Figure 3.17. The Ramachandran Plot. The online PROCHECK program was used to test the structural stereochemistry of the complex shown in figure 3.16. The Ramachandran plot disclosed the occurrence of 93.2% residues in the maximum favored regions, 5.6% in additionally allowed areas, 1.2% in allowed sector, and 0.0% in the disallowed zone…………………………………………………………...………………….105
Figure 3.18. S100A1 interferes with the p53-MDM2 complex formation. (a) The complex model of the S100A1−MDM2 protein generated using the HADDOCK program. (b) The reported complex model of the p53 peptide−MDM2 domain obtained via the X-ray diffraction technique. (c) Overlay of the complex amid S100A1 (green) and the MDM2 domain (yellow), to the complex amid p53 peptide (blue) and MDM2 (yellow) showing S100A1 interference to p53-MDM2 complex………………………106
Figure 3.19. S100A1 can interfere with the interaction between p53 and MDM2/MDMX. (a) A reported complex model of p53 peptide and MDM2 N-terminal domain (PDB ID- 1YCR). (b) A reported complex model of p53 peptide and MDMX N-terminal domain (PDB ID- 3DAC). (c) A complex model of S100A1 and MDM2 N-terminal domain generated via the HADDOCK program. (d) Overlapping of the complex models indicated in a, b, and c showed possible interference of S100A1 between p53 peptide and MDM2/MDMX N-terminal domain…………………………………………………108
Figure 3.20. The 1H−15N HSQC spectra overlay of free 15N p53 (1-73) (red) and in complex with unlabelled S100A1 (yellow). Cross-peaks proving deviations in intensities are highlighted in yellow. Residues were assigned as published earlier……………………...............110
Figure 3.21. Exploration of the connected interface (S100A1/p53 (1-73)) section in S100A1. (a) The 1H-15N HSQC spectra overlay of free 15N S100A1 (red) and in complex with unlabelled p53 (1-73) (blue). Cross-peaks displaying variation in their magnitude are highlighted in yellow. (b) Table illustrating a list of the affected residues when 15N S100A1 interacts with the unlabeled p53 (1-73) and unlabeled MDM2 and also showing the common interacting residues……………………111
Figure 3.22. S100A1 complete sequence indicating highlighted region selected for peptide construction, attaching it to HIV-TAT peptide forming Peptide 1 and its scramble arrangement in Peptide 2……113
Figure 3.23. Far-UV CD spectra of (a) S100A1 protein and (b) Peptide 1……………......................................................114
Figure 3.24. The p53-MDM2 interaction was monitored via fluorescence spectroscopy. (a) Changes in the fluorescence emission spectra of p53 upon the addition of increasing concentration of MDM2 in the micro-molar range was observed. (b)  Relative intensity versus the MDM2 total concentration………………………………………..………….115
Figure 3.25. The p53-Peptide 1 interaction was monitored via fluorescence spectroscopy. (a) Changes in the fluorescence emission spectra of p53 upon the addition of increasing concentration of Peptide 1 in the micro-molar range was observed. (b)  Relative intensity versus the Peptide 1 total concentration………………………………………..……….........116
Figure 3.26. Competitive binding experiment. (a) The 1H-15N HSQC spectra of 15N MDM2 in complex with unlabeled Peptide 1 (red) in a 1:1 ratio. (b) The 1H-15N HSQC spectra of 15N MDM2 in complex with unlabeled p53 (blue) in a 1:1 ratio. To the same mixture, we added the Peptide 1 in a 1:1 ratio and obtained the spectrum as shown in (c). Then the spectra obtained in (b) and (c) is overlapped to the spectra indicated in (a) and the resulting overlapped spectra are shown in (d)……………………………………………….…….....................117
Figure 3.27. Analysis of WST-1 assay. The (a) MCF-7 and (b) AGS cells were treated with 5, 10, and 20 µM concentrations of Peptide 1 (blue) and Peptide 2 (green). The comparative cell counts after successive treatment with Peptides 1 and 2 are shown as fold inductions with the control (red) as serum-free media alone……………………………..…119
Figure 3.28. The MCF-7 and/or MDA-MB-468 cells were treated with or without 10 µM Peptide 1 for 48h. After the treatment, cells were collected and lysed. The protein levels of p53, its down-stream p21, and actin were examined by Western blotting. The Actin was used as an internal control………………………………………………………………121
Figure 3.29. MCF-7 cells were treated with 0, 10, or 20 µM of Peptide 1 or Peptide 2 for 48h. Subsequently, cells were collected, fixed with 70% ethanol, and stained with 10 µg/ml propidium iodide. The cell cycle distribution was analyzed by a flow cytometer…………………122
Figure 3.30. Table summarizing the list of interacting residues when (a) S100A1 interacts with p53 TAD and MDM2, (b) MDM2 interacts with S100A1 and p53 TAD, and (c) p53 TAD interacts with S100A1 and MDM2.123

                                

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