995308dacea3cdc28b4650af3a3a1294 pharmaceutics-15-01777.pdf 62b1d3c17436b8f1acf6072800f8c45e0299330d pharmaceutics-15-01777.pdf edca732beb8097a5042c2437f4701fb516eb23acd76af5e09e1cefd4322127a7 pharmaceutics-15-01777.pdf Title: The C-Terminus of Panusin, a Lobster -Defensin, Is Crucial for Optimal Antimicrobial Activity and Serum Stability Subject: -defensins are one of the most abundant and studied families of antimicrobial peptides (AMPs). Because of their selective toxicity to bacterial membranes and a broad spectrum of microbicidal action, -defensins are regarded as potential therapeutic agents. This work focuses on a -defensin-like AMP from the spiny lobster Panulirus argus (hereafter referred to as panusin or PaD). This AMP is structurally related to mammalian defensins via the presence of an domain stabilized by disulfide bonds. Previous studies of PaD suggest that its C-terminus (Ct_PaD) contains the main structural determinants of antibacterial activity. To confirm this hypothesis, we made synthetic versions of PaD and Ct_PaD to determine the influence of the C-terminus on antimicrobial activity, cytotoxicity, proteolytic stability, and 3D structure. After successful solid-phase synthesis and folding, antibacterial assays of both peptides showed truncated Ct_PaD to be more active than native PaD, confirming the role of the C-terminus in activity and suggesting that cationic residues in that region enhance binding to negatively charged membranes. On the other hand, neither PaD nor Ct_PaD were hemolytic or cytotoxic in human cells. Proteolysis in human serum was also studied, showing high (>24 h) t1/2 values for PaD and lower but still considerable for Ct_PaD, indicating that the missing native disulfide bond in Ct_PaD alters protease resistance, albeit not decisively. NMR-2D experiments in water agree with the results obtained by circular dichroism (CD), where in SDS micelles, CD showed both peptides adopting an increasingly ordered structure in a hydrophobic environment, in tune with their ability to perturb bacterial membrane systems. In conclusion, while the -defensin features of PaD are confirmed as advantageous in terms of antimicrobial activity, toxicity, and protease stability, the results of the present work suggest that these same features are preserved, even enhanced, in the structurally simpler Ct_PaD, which must therefore be viewed as a valuable lead for the development of novel anti-infectives. Keywords: panusin; -defensins; antimicrobial peptides Author: Roberto Bello-Madruga, Javier Valle, M. Ángeles Jiménez, Marc Torrent, Vivian Montero-Alejo and David Andreu Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jun 20 14:05:11 2023 CEST ModDate: Tue Jun 20 14:13:46 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2669088 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EQJVKE+EURB10 Type 1 Builtin yes yes no 10 0 AIPYBR+VnURWPalladioL-Bold Type 1 Custom yes yes no 14 0 QFGBPA+VnURWPalladioL Type 1 Custom yes yes no 19 0 DIQYEN+EURM10 Type 1 Builtin yes yes no 23 0 DRORJQ+URWPalladioL-Bold Type 1 Custom yes yes no 27 0 HMEISL+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 RGTNAY+URWPalladioL-Ital Type 1 Custom yes yes no 36 0 WYKRWL+EURM7 Type 1 Builtin yes yes no 40 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi 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GMNPIF+PalatinoLinotype TrueType MacRoman yes yes no 304 0 GMNOHG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 322 0 GMNOHH+PalatinoLinotype CID TrueType Identity-H yes yes yes 325 0 GMNOHI+PalatinoLinotype TrueType WinAnsi yes yes no 331 0 GMNOIK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 334 0 GMNPIF+PalatinoLinotype TrueType MacRoman yes yes no 337 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-10 02:33:16 CET RepresentationInformation: pharmaceutics-15-01777.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-02-09 18:15:42 CET Size: 2669088 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 555 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Materials Destination: subsection.2.1 Item: Title: Peptide Synthesis Destination: subsection.2.2 Item: Title: RP-HPLC and LC-MS Destination: subsection.2.3 Item: Title: Anaerobic Oxidative Folding Destination: subsection.2.4 Item: Title: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Destination: subsection.2.5 Item: Title: Hemolytic Activity Destination: subsection.2.6 Item: Title: Cytotoxicity in Mammalian Cells Destination: subsection.2.7 Item: Title: Serum Stability Destination: subsection.2.8 Item: Title: NMR Spectroscopy Destination: subsection.2.9 Item: Title: Circular Dichroism Spectroscopy (CD) Destination: subsection.2.10 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Peptide Design and Synthesis Destination: subsection.3.1 Item: Title: Synthesis and Oxidative Folding of PaD and Ct_PaD Destination: subsection.3.2 Item: Title: Antimicrobial Activity Destination: subsection.3.3 Item: Title: Hemolytic Activity and Cell Viability Destination: subsection.3.4 Item: Title: Serum Stability Destination: subsection.3.5 Item: Title: NMR Structural Study Destination: subsection.3.6 Item: Title: Analysis of Secondary Structure by Circular Dichroism Destination: subsection.3.7 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.A. Info: Title: The C-Terminus of Panusin, a Lobster -Defensin, Is Crucial for Optimal Antimicrobial Activity and Serum Stability Author: Roberto Bello-Madruga, Javier Valle, M. Ángeles Jiménez, Marc Torrent, Vivian Montero-Alejo and David Andreu Subject: -defensins are one of the most abundant and studied families of antimicrobial peptides (AMPs). Because of their selective toxicity to bacterial membranes and a broad spectrum of microbicidal action, -defensins are regarded as potential therapeutic agents. This work focuses on a -defensin-like AMP from the spiny lobster Panulirus argus (hereafter referred to as panusin or PaD). This AMP is structurally related to mammalian defensins via the presence of an domain stabilized by disulfide bonds. Previous studies of PaD suggest that its C-terminus (Ct_PaD) contains the main structural determinants of antibacterial activity. To confirm this hypothesis, we made synthetic versions of PaD and Ct_PaD to determine the influence of the C-terminus on antimicrobial activity, cytotoxicity, proteolytic stability, and 3D structure. After successful solid-phase synthesis and folding, antibacterial assays of both peptides showed truncated Ct_PaD to be more active than native PaD, confirming the role of the C-terminus in activity and suggesting that cationic residues in that region enhance binding to negatively charged membranes. On the other hand, neither PaD nor Ct_PaD were hemolytic or cytotoxic in human cells. Proteolysis in human serum was also studied, showing high (>24 h) t1/2 values for PaD and lower but still considerable for Ct_PaD, indicating that the missing native disulfide bond in Ct_PaD alters protease resistance, albeit not decisively. NMR-2D experiments in water agree with the results obtained by circular dichroism (CD), where in SDS micelles, CD showed both peptides adopting an increasingly ordered structure in a hydrophobic environment, in tune with their ability to perturb bacterial membrane systems. In conclusion, while the -defensin features of PaD are confirmed as advantageous in terms of antimicrobial activity, toxicity, and protease stability, the results of the present work suggest that these same features are preserved, even enhanced, in the structurally simpler Ct_PaD, which must therefore be viewed as a valuable lead for the development of novel anti-infectives. 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