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<document id="61DBDBFA9B87B1815AD21560B36AAC8D" ID-DOI="10.1016/j.phytochem.2020.112466" ID-ISSN="1873-3700" ID-Zenodo-Dep="8293508" IM.bibliography_approvedBy="jonas" IM.illustrations_approvedBy="jonas" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="jonas" IM.taxonomicNames_approvedBy="jonas" IM.treatments_approvedBy="jonas" checkinTime="1693252666754" checkinUser="felipe" docAuthor="Zheng, Sheng, Zhu, Ning, Shi, Cheng &amp; Zheng, Heng" docDate="2020" docId="BB74B629FFE1FFCCE651FB9904586981" docLanguage="en" docName="Phytochemistry.179.112466.pdf" docOrigin="Phytochemistry (112466) 179" docSource="http://dx.doi.org/10.1016/j.phytochem.2020.112466" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.6:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="6" docTitle="Ganoderma sinense J. D. Zhao, L. W. Hsu &amp; X. Q. Zhang" docType="treatment" docVersion="4" lastPageNumber="2" masterDocId="474DCE51FFE0FFCEE635FF8907356D52" masterDocTitle="Genomic data mining approaches for the discovery of anticancer peptides from Ganoderma sinense" masterLastPageNumber="8" masterPageNumber="1" pageNumber="2" updateTime="1693484751065" updateUser="ExternalLinkService">
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<mods:title id="7E4C500AD6BDD0F2F7ACC59FBA564B95">Genomic data mining approaches for the discovery of anticancer peptides from Ganoderma sinense</mods:title>
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<mods:namePart id="8F16A42303EE2ABE4B2D4A1761DCAFC6">Zheng, Sheng</mods:namePart>
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<mods:namePart id="179B6947D5619CD3B33013C1047A5A6C">Shi, Cheng</mods:namePart>
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<paragraph id="3362073FFFE1FFCFE651FB99054C6971" blockId="1.[100,633,1040,1059]" box="[100,633,1040,1059]" pageId="1" pageNumber="2">
<heading id="682AB053FFE1FFCFE651FB99054C6971" bold="true" box="[100,633,1040,1059]" fontSize="36" level="1" pageId="1" pageNumber="2" reason="1">
<emphasis id="01A9DB2DFFE1FFCFE651FB99054C6971" bold="true" box="[100,633,1040,1059]" italics="true" pageId="1" pageNumber="2">
2.1. Prediction of anticancer activity of 
<taxonomicName id="F4DD7CBCFFE1FFCFE7E7FB9905196971" ID-CoL="6K8NP" ID-ENA="36075" authority="J. D. Zhao, L. W. Hsu &amp; X. Q. Zhang" authorityName="J. D. Zhao, L. W. Hsu &amp; X. Q. Zhang" box="[466,556,1040,1059]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">G. sinense</taxonomicName>
proteins
</emphasis>
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</paragraph>
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<paragraph id="3362073FFFE1FFCFE6B1FBC103716C61" blockId="1.[100,770,1096,1478]" lastBlockId="1.[818,1487,148,307]" pageId="1" pageNumber="2">
Genomic and proteomic data on 
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<emphasis id="01A9DB2DFFE1FFCFE783FBC1053B6909" bold="true" box="[438,526,1096,1115]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
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were obtained from NCBI genomic database, and include over 15,000 predicted protein sequences (
<bibRefCitation id="574C7ACEFFE1FFCFE659FB09063769C1" author="Zhu, Y. &amp; Xu, J. &amp; Sun, C. &amp; Zhou, S. &amp; Xu, H. &amp; Nelson, D. R. &amp; Qian, J. &amp; Song, J. &amp; Luo, H. &amp; Xiang, L. &amp; Li, Y. &amp; Xu, Z. &amp; Ji, A. &amp; Wang, L. &amp; Lu, S. &amp; Hayward, A. &amp; Sun, W. &amp; Li, X. &amp; Schwartz, D. C. &amp; Wang, Y. &amp; Chen, S." box="[108,258,1152,1171]" pageId="1" pageNumber="2" pagination="11087" refId="ref6831" refString="Zhu, Y., Xu, J., Sun, C., Zhou, S., Xu, H., Nelson, D. R., Qian, J., Song, J., Luo, H., Xiang, L., Li, Y., Xu, Z., Ji, A., Wang, L., Lu, S., Hayward, A., Sun, W., Li, X., Schwartz, D. C., Wang, Y., Chen, S., 2015. Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense. Sci. Rep. 5, 11087." type="journal article" year="2015">Zhu et al., 2015</bibRefCitation>
). The length distribution of these proteins is shown in 
<figureCitation id="ABE61BBAFFE1FFCFE651FB1507AF69FD" box="[100,154,1180,1199]" captionStart="Fig" captionStartId="1.[170,200,1964,1981]" captionTargetBox="[107,766,1539,1935]" captionTargetId="figure-1113@1.[106,767,1538,1936]" captionTargetPageId="1" captionText="Fig. 1. Protein length distribution in the G. sinense proteome." figureDoi="http://doi.org/10.5281/zenodo.8293510" httpUri="https://zenodo.org/record/8293510/files/figure.png" pageId="1" pageNumber="2">Fig. 1</figureCitation>
. Most of the translated proteins have more than 200 amino acid residues, and only 5.8% proteins (899 sequences) are shorter than 100 amino acids in length. First, we attempted to identify ACPs from the proteome of 
<taxonomicName id="F4DD7CBCFFE1FFCFE6EAFB66060C6850" box="[223,313,1263,1282]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE6EAFB66060C6850" bold="true" box="[223,313,1263,1282]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
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; thus, proteins shorter than 100 amino acids in length were screened by mACPpred. None of the proteins translated from the 
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<emphasis id="01A9DB2DFFE1FFCFE68FFAAE06276868" bold="true" box="[186,274,1319,1338]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
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genome was predicted to have anticancer activity. It is possible that bioactive peptides are in fact present but are inactive in their parent proteins, exhibiting activity only after being cleaved by enzymes (Manzanares et al., 2015). Considering that 
<taxonomicName id="F4DD7CBCFFE1FFCFE46CFAF205DB68DC" box="[601,750,1403,1422]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="undetermined">
<emphasis id="01A9DB2DFFE1FFCFE46CFAF205F468DC" bold="true" box="[601,705,1403,1422]" italics="true" pageId="1" pageNumber="2">Ganoderma</emphasis>
spp.
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is consumed as a health-care food and medicine, trypsin was selected as a model enzyme to hydrolyze the proteins. The proteins were blasted with a dataset containing 760 known ACPs to investigate whether they contain ACP sequences (
<bibRefCitation id="574C7ACEFFE1FFCFE223FF3903836D91" author="Chen, W. &amp; Ding, H. &amp; Feng, P. &amp; Lin, H. &amp; Chou, K. C." box="[1046,1206,176,195]" pageId="1" pageNumber="2" pagination="16895 - 16909" refId="ref4878" refString="Chen, W., Ding, H., Feng, P., Lin, H., Chou, K. C., 2016. iACP: a sequence-based tool for identifying anticancer peptides. Oncotarget 7, 16895 - 16909." type="journal article" year="2016">Chen et al., 2016</bibRefCitation>
; 
<bibRefCitation id="574C7ACEFFE1FFCFE2F4FF3902606D91" author="Wei, L. &amp; Zhou, C. &amp; Chen, H. &amp; Song, J. &amp; Su, R." box="[1217,1365,176,195]" pageId="1" pageNumber="2" pagination="4007 - 4016" refId="ref6479" refString="Wei, L., Zhou, C., Chen, H., Song, J., Su, R., 2018. ACPred-FL: a sequence-based predictor based on effective feature representation to improve the prediction of anti-cancer peptides. Bioinformatics 34, 4007 - 4016." type="journal article" year="2018">Wei et al., 2018</bibRefCitation>
; 
<bibRefCitation id="574C7ACEFFE1FFCFE354FF3904576D8D" author="Tyagi, A. &amp; Kapoor, P. &amp; Kumar, R. &amp; Chaudhary, K. &amp; Gautam, A. &amp; Raghava, G. P." pageId="1" pageNumber="2" pagination="2984" refId="ref6330" refString="Tyagi, A., Kapoor, P., Kumar, R., Chaudhary, K., Gautam, A., Raghava, G. P., 2013. In silico models for designing and discovering novel anticancer peptides. Sci. Rep. 3, 2984." type="journal article" year="2013">Tyagi et al., 2013</bibRefCitation>
). The proteins containing more than four ACP sequences were then 
<emphasis id="01A9DB2DFFE1FFCFE507FF6E04436DA8" bold="true" box="[818,886,231,250]" italics="true" pageId="1" pageNumber="2">in silico</emphasis>
digested by trypsin, and the resulting fragments were screened by mACPpred to identify possible ACPs. A flowchart of the overall procedure is shown in 
<figureCitation id="ABE61BBAFFE1FFCFE23DFE9603716C61" box="[1032,1092,287,307]" captionStart="Fig" captionStartId="2.[100,130,827,844]" captionTargetBox="[173,1429,160,788]" captionTargetId="figure-376@2.[154,1433,148,800]" captionTargetPageId="2" captionText="Fig. 2. Experimental flowchart. (A) Blast search and alignment of G. sinense proteins with known anticancer peptides. (B) An example of a Hit sequence. (C) Hit sequences were cleaved by in silico enzyme digestion of trypsin. (D) The digested fragments were screened by mACPpred, an SVM-based algorithm, to identify putative ACPs. (E) The resulting peptides were then subjected to sequence comparison to known ACPs." figureDoi="http://doi.org/10.5281/zenodo.8293512" httpUri="https://zenodo.org/record/8293512/files/figure.png" pageId="1" pageNumber="2">Fig. 2.</figureCitation>
</paragraph>
<paragraph id="3362073FFFE1FFCFE507FEDE03C76C38" blockId="1.[818,1266,343,363]" box="[818,1266,343,363]" pageId="1" pageNumber="2">
<emphasis id="01A9DB2DFFE1FFCFE507FEDE03C76C38" bold="true" box="[818,1266,343,363]" italics="true" pageId="1" pageNumber="2">2.2. Prediction of ACP-containing parent proteins</emphasis>
</paragraph>
<paragraph id="3362073FFFE1FFCFE564FE0604486FD0" blockId="1.[818,1487,399,642]" pageId="1" pageNumber="2">
The sequences of 
<taxonomicName id="F4DD7CBCFFE1FFCFE230FE06038C6CF0" box="[1029,1209,399,418]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="7" phylum="Basidiomycota" rank="subSpecies" species="sinense" subSpecies="proteins">
<emphasis id="01A9DB2DFFE1FFCFE230FE0603576CF0" bold="true" box="[1029,1122,399,418]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
proteins
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comprising fewer than 100 amino acids were blasted against a dataset of known ACPs. The sequence alignment results are shown in 
<figureCitation id="ABE61BBAFFE1FFCFE267FE4E03AB6C88" box="[1106,1182,455,474]" captionStart="Fig" captionStartId="2.[156,186,1379,1396]" captionTargetBox="[107,766,954,1350]" captionTargetId="figure-444@2.[106,767,953,1351]" captionTargetPageId="2" captionText="Fig. 3. The number of parent proteins containing putative ACPs." figureDoi="http://doi.org/10.5281/zenodo.8293514" httpUri="https://zenodo.org/record/8293514/files/figure.png" pageId="1" pageNumber="2">Fig. 3. A</figureCitation>
total of 477 sequences possessed at least one possible ACP sequence. Some of these sequences from the 
<taxonomicName id="F4DD7CBCFFE1FFCFE507FE7604BA6F40" box="[818,911,511,530]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE507FE7604BA6F40" bold="true" box="[818,911,511,530]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
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proteome (parent proteins) were similar to one or more anticancer peptide sequences. Some of the parent proteins possess multiple anticancer peptides; 26 parent proteins contained more than 6 putative ACP sequences, 4 of which contain more than 12 possible ACPs (
<figureCitation id="ABE61BBAFFE1FFCFE50FFDE7045A6FD0" box="[826,879,622,642]" captionStart="Fig" captionStartId="2.[156,186,1379,1396]" captionTargetBox="[107,766,954,1350]" captionTargetId="figure-444@2.[106,767,953,1351]" captionTargetPageId="2" captionText="Fig. 3. The number of parent proteins containing putative ACPs." figureDoi="http://doi.org/10.5281/zenodo.8293514" httpUri="https://zenodo.org/record/8293514/files/figure.png" pageId="1" pageNumber="2">Fig. 3</figureCitation>
).
</paragraph>
<paragraph id="3362073FFFE1FFCFE507FD2E028D6FEB" blockId="1.[818,1464,678,698]" box="[818,1464,678,698]" pageId="1" pageNumber="2">
<emphasis id="01A9DB2DFFE1FFCFE507FD2E028D6FEB" bold="true" box="[818,1464,678,698]" italics="true" pageId="1" pageNumber="2">2.3. In silico enzyme digestion and ACP screening of digested fragments</emphasis>
</paragraph>
<paragraph id="3362073FFFE1FFCFE564FD5603396E83" blockId="1.[818,1488,734,1200]" pageId="1" pageNumber="2">
Most of the currently discovered active peptides are hydrolysates of proteins. Many of the products play regulatory roles and exhibit a range of activities, including antimicrobial, anticancer, antihypertensive and immunomodulatory activities (
<bibRefCitation id="574C7ACEFFE1FFCFE26CFCBB02216E17" author="Yousefi, H. &amp; Yuan, J. &amp; Keshavarz-Fathi, M. &amp; Murphy, J. F. &amp; Rezaei, N." box="[1113,1300,818,837]" pageId="1" pageNumber="2" pagination="1001 - 1015" refId="ref6667" refString="Yousefi, H., Yuan, J., Keshavarz-Fathi, M., Murphy, J. F., Rezaei, N., 2017. Immunotherapy of cancers comes of age. Expet Rev. Clin. Immunol. 13 (10), 1001 - 1015." type="journal article" year="2017">Yousefi et al., 2017</bibRefCitation>
; Manzanares et al., 2015). Trypsin was used as a model enzyme to hydrolyze parent proteins from 
<taxonomicName id="F4DD7CBCFFE1FFCFE551FCE304896E2F" box="[868,956,874,893]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE551FCE304896E2F" bold="true" box="[868,956,874,893]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
. The parent proteins containing more than four putative ACP sequences (a total of 65 proteins) were subjected to 
<emphasis id="01A9DB2DFFE1FFCFE370FC0F02BD6ECA" bold="true" box="[1349,1416,901,921]" italics="true" pageId="1" pageNumber="2">in silico</emphasis>
trypsin digestion. The fragments generated by trypsin cleavage are listed in the supplement (
<tableCitation id="7E5F3284FFE1FFCFE598FC3704CB6E83" box="[941,1022,958,977]" captionStart="Table 1" captionStartId="3.[140,190,154,170]" captionTargetPageId="3" captionText="Table 1 Peptide fragment prediction results." httpUri="http://table.plazi.org/id/67A257B7FFE3FFCDE6B9FF1306886D96" pageId="1" pageNumber="2" tableUuid="67A257B7FFE3FFCDE6B9FF1306886D96">Table S1</tableCitation>
).
</paragraph>
<paragraph id="3362073FFFE1FFCFE564FC5003D169E2" blockId="1.[818,1488,734,1200]" pageId="1" pageNumber="2">
After trypsin cleavage, a total of 307 peptides were produced. The length distribution is shown in 
<figureCitation id="ABE61BBAFFE1FFCFE261FC7C03BC695A" box="[1108,1161,1013,1032]" captionStart="Fig" captionStartId="2.[100,130,1876,1893]" captionTargetBox="[107,766,1453,1847]" captionTargetId="figure-458@2.[106,767,1452,1848]" captionTargetPageId="2" captionText="Fig. 4. Length distributions of cleaved peptides. The parent proteins were cleaved by in silico enzyme digestion with trypsin. The length distributions of peptides between 7 and 19 amino acids in length are shown." figureDoi="http://doi.org/10.5281/zenodo.8293516" httpUri="https://zenodo.org/record/8293516/files/figure.png" pageId="1" pageNumber="2">Fig. 4</figureCitation>
mACPpred was used to predict the anticancer activity of these cleaved peptides. The results are shown in 
<tableCitation id="7E5F3284FFE1FFCFE507FBA404A46912" box="[818,913,1069,1089]" captionStart="Table 1" captionStartId="3.[140,190,154,170]" captionTargetPageId="3" captionText="Table 1 Peptide fragment prediction results." httpUri="http://table.plazi.org/id/67A257B7FFE3FFCDE6B9FF1306886D96" pageId="1" pageNumber="2" tableUuid="67A257B7FFE3FFCDE6B9FF1306886D96">Table 1. A</tableCitation>
total of 34 trypsin-cleaved 
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<emphasis id="01A9DB2DFFE1FFCFE2A6FBA403DE6912" bold="true" box="[1171,1259,1069,1088]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
proteins
</taxonomicName>
were predicted to produce peptides with anticancer activity. In addition, some parent proteins were predicted to generate multiple fragments with anticancer activity. These ACPs might serve as active components of orally administered 
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<emphasis id="01A9DB2DFFE1FFCFE586FB14033969E2" bold="true" box="[947,1036,1181,1200]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
as an anticancer drug.
</paragraph>
<paragraph id="3362073FFFE1FFCFE507FB5C03BB69BA" blockId="1.[818,1166,1237,1256]" box="[818,1166,1237,1256]" pageId="1" pageNumber="2">
<emphasis id="01A9DB2DFFE1FFCFE507FB5C03BB69BA" bold="true" box="[818,1166,1237,1256]" italics="true" pageId="1" pageNumber="2">2.4. Verification of the screened ACPs</emphasis>
</paragraph>
<paragraph id="3362073FFFE1FFCFE564FA84030C68DD" blockId="1.[818,1488,1293,1814]" pageId="1" pageNumber="2">
To verify the anticancer activity of the mACPpred-screened ACPs, the peptides were blasted against known peptides with anticancer activity. Fifteen cleavage peptides had similar sequences to those of known anticancer peptides. These cleavage peptides belong to 14 parent proteins of 
<taxonomicName id="F4DD7CBCFFE1FFCFE548FAF504E368DD" box="[893,982,1404,1423]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE548FAF504E368DD" bold="true" box="[893,982,1404,1423]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
(
<tableCitation id="7E5F3284FFE1FFCFE5D0FAF5031968DD" box="[997,1068,1404,1423]" captionStart="Table 2" captionStartId="5.[100,150,150,166]" captionTargetPageId="5" captionText="Table 2 Sequence alignment results of trypsin-digested peptides with known ACPs and the properties of these peptides." httpUri="http://table.plazi.org/id/67A257B7FFE5FFCBE651FF1F03216D93" pageId="1" pageNumber="2" tableUuid="67A257B7FFE5FFCBE651FF1F03216D93">Table 2</tableCitation>
).
</paragraph>
<paragraph id="3362073FFFE1FFCFE564FA1102976A44" blockId="1.[818,1488,1293,1814]" pageId="1" pageNumber="2">
According to the results, the trypsin cleavage fragments of protein No. 11 (with proteins numbered according to their order in the proteome) and No. 20 produced the same sequences as known anticancer peptides (Identity =100%). Protein No. 2 yielded two peptide hits, with one of the peptides being identical in sequence to a known anticancer peptide. The results suggested that after oral administration of 
<taxonomicName id="F4DD7CBCFFE1FFCFE507F9B604BE6B00" box="[818,907,1599,1618]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE507F9B604BE6B00" bold="true" box="[818,907,1599,1618]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
, the parent proteins of 
<taxonomicName id="F4DD7CBCFFE1FFCFE25EF9B603F66B00" box="[1131,1219,1599,1618]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE25EF9B603F66B00" bold="true" box="[1131,1219,1599,1618]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
may be digested by trypsin, resulting in the release of ACPs. Jeong et al. reported that the anticancer peptide lunasin, a 43-amino acid peptide naturally present in soybean, was bioactive and presented in liver, kidney and blood after feeding rye to rats (
<bibRefCitation id="574C7ACEFFE1FFCFE548F926031C6B90" author="Jeong, H. J. &amp; Lee, J. R. &amp; Jeong, J. B. &amp; Park, J. H. &amp; Cheong, Y. K. &amp; de Lumen, B. O." box="[893,1065,1711,1730]" pageId="1" pageNumber="2" pagination="680 - 686" refId="ref5217" refString="Jeong, H. J., Lee, J. R., Jeong, J. B., Park, J. H., Cheong, Y. K., de Lumen, B. O., 2009. The cancer preventive seed peptide lunasin from rye is bioavailable and bioactive. Nutr. Canc. 61 (5), 680 - 686." type="journal article" year="2009">Jeong et al., 2009</bibRefCitation>
). The bioavailability of ACPs resulting from the digestion of 
<taxonomicName id="F4DD7CBCFFE1FFCFE5E2F94203BF6B8C" box="[983,1162,1739,1758]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="7" phylum="Basidiomycota" rank="subSpecies" species="sinense" subSpecies="proteins">
<emphasis id="01A9DB2DFFE1FFCFE5E2F94203016B8C" bold="true" box="[983,1076,1739,1758]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
proteins
</taxonomicName>
needs further study. Our results indicated that future research should perform in vitro digestion to aid the discovery of bioactive peptides from 
<taxonomicName id="F4DD7CBCFFE1FFCFE286F88A02396A44" box="[1203,1292,1795,1814]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE286F88A02396A44" bold="true" box="[1203,1292,1795,1814]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
or other TCMs.
</paragraph>
<paragraph id="3362073FFFE1FFCFE507F8B204B06A3B" blockId="1.[818,1435,1851,1897]" pageId="1" pageNumber="2">
<emphasis id="01A9DB2DFFE1FFCFE507F8B204B06A3B" bold="true" italics="true" pageId="1" pageNumber="2">2.5. Structural simulation of anticancer peptides based on molecular dynamics</emphasis>
</paragraph>
<paragraph id="3362073FFFE1FFCCE564F80604586981" blockId="1.[818,1487,1934,1982]" lastBlockId="2.[818,1488,936,1235]" lastPageId="2" lastPageNumber="3" pageId="1" pageNumber="2">
<taxonomicName id="F4DD7CBCFFE1FFCFE564F806049B6AF0" box="[849,942,1935,1954]" class="Agaricomycetes" family="Ganodermataceae" genus="Ganoderma" kingdom="Fungi" order="Polyporales" pageId="1" pageNumber="2" phylum="Basidiomycota" rank="species" species="sinense">
<emphasis id="01A9DB2DFFE1FFCFE564F806049B6AF0" bold="true" box="[849,942,1935,1954]" italics="true" pageId="1" pageNumber="2">G. sinense</emphasis>
</taxonomicName>
protein Nos. 14 and 15 produced fragments similar to multiple known anticancer peptides. This result indicated that these peptides have a high probability of being unreported ACPs. Molecular dynamics were used to construct structure models of these peptides to gain insight into their possible anticancer mechanisms; these models may aid the design of novel anticancer peptides. The energy changes of these peptides after 100 ns of molecular dynamics simulation are shown in 
<figureCitation id="ABE61BBAFFE2FFCCE57AFBBD04BC6915" box="[847,905,1076,1095]" captionStart="Fig" captionStartId="5.[100,130,1096,1113]" captionTargetBox="[190,681,803,1067]" captionTargetId="figure-691@5.[188,683,801,1069]" captionTargetPageId="5" captionText="Fig. 5. Energy change in 100-ns molecular dynamic simulation of putative ACPs P14 and P15 (trypsin-digested fragments from parent proteins No. 14 and 15 in the G. sinense proteome). Molecular dynamic simulation was performed by AMBER14." figureDoi="http://doi.org/10.5281/zenodo.8293518" httpUri="https://zenodo.org/record/8293518/files/figure.png" pageId="2" pageNumber="3">Fig. 5</figureCitation>
. During the equilibration phase, a brief energy change is experienced, followed by an energy minimize approach to stabilization. Therefore, it can be inferred that these peptides were in a relatively stable state after a period of stretching. We extracted the lowest energy conformation from 20 to 100 ns for analysis. The results are shown in 
<figureCitation id="ABE61BBAFFE2FFCCE507FB3604586981" box="[818,877,1215,1235]" captionStart="Fig" captionStartId="5.[818,848,1233,1250]" captionTargetBox="[906,1398,802,1203]" captionTargetId="figure-731@5.[905,1400,801,1205]" captionTargetPageId="5" captionText="Fig. 6. Three-dimensional structures of P14 and P15, shown in cartoon representation; red represents α-helix, yellow represents β-sheet, and blue represents turn structure. Structures were extracted from the trajectory of molecular dynamic simulation from 20 ns to 100 ns. The lowest energy conformation is presented. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)" figureDoi="http://doi.org/10.5281/zenodo.8293520" httpUri="https://zenodo.org/record/8293520/files/figure.png" pageId="2" pageNumber="3">Fig. 6.</figureCitation>
</paragraph>
</subSubSection>
</treatment>
</document>