9dc641ff792996aa8eca61759e2b7047 256464.pdf 68d4f9fad482680a78316a499efd37c8190e2292 256464.pdf 06074c0f74f49b7acf4db54e959a70b48d5443adce7415eadd00b6f38069f263 256464.pdf Title: Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin Subject: This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together in vivo at neuromuscular junctions and in motor neurons. Keywords: clostridium neurotoxins; neurotrophin receptors; carboxyl-terminal domain of tetanus neurotoxin; brain-derived neurotrophic factor Author: Ana Candalija, Thomas Scior, Hans-Richard Rackwitz, Jordan E. Ruiz-Castelan, Ygnacio Martinez-Laguna and José Aguilera Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Jul 4 10:48:31 2021 CEST ModDate: Sun Jul 4 11:12:21 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 23 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5141475 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MWHIBA+VnURWPalladioL Type 1 Custom yes yes no 10 0 QFIDSE+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 CNFWUB+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 LIFPNT+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 PUSVWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 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Item: Title: The Established Receptor Mechanism Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: The Inconsistent Dual-Receptor Mechanism Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: The Proposed Receptor Mechanism Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Hc-TeNT Interaction with Target TrkB Receptor Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Hc-TeNT Competes with BDNF for Binding and Internalization in Cultured Neurons Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Mutations in Predicted Binding Site Affect Binding and Internalization Ability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Computed Results at the Predicted Binding Site Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: The Peptide Design to Evaluate the Protein-Protein Interface Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Synthesis of the Cyclic Peptide and Negative Control Sequences Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Expression and Purification of Hc-TeNT Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Cloning and Expression of Hc-Mut with a Triple Mutation (Y266A-K311A-E343A) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Cerebellar Granule Neurons (CGNs) Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Dot-Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: In Vivo Hc-TeNT Intramuscular Injections Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Immunohystochemistry and Image Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Hc-TeNT Endocytic Assay and Uptake Competition Assays in CGNs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Binding Competition Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Flow Cytometry in Hc-TeNT Binding and Uptake Assays in CGNs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: CGNs Immunocytofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Molecular Modeling and Bioinformatics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Info: Title: Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin Author: Ana Candalija, Thomas Scior, Hans-Richard Rackwitz, Jordan E. Ruiz-Castelan, Ygnacio Martinez-Laguna and José Aguilera Subject: This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together in vivo at neuromuscular junctions and in motor neurons. Keywords: clostridium neurotoxins; neurotrophin receptors; carboxyl-terminal domain of tetanus neurotoxin; brain-derived neurotrophic factor Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sun Jul 04 10:48:31 CEST 2021 ModDate: Sun Jul 04 11:12:21 CEST 2021 ID: 0x25080a016f3fe8d54c200f979d34e68e, 0x25080a016f3fe8d54c200f979d34e68e Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LEVXPH+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: LEVXPH+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: CNFWUB+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: CNFWUB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LIFPNT+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: LIFPNT+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 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652, 553, 663 Annotation: Subtype: Link Rect: 225, 629, 262, 640 Annotation: Subtype: Link Rect: 245, 607, 283, 617 Annotation: Subtype: Link Rect: 494, 583, 529, 594 Annotation: Subtype: Link Rect: 331, 560, 368, 571 Annotation: Subtype: Link Rect: 519, 548, 552, 559 Annotation: Subtype: Link Rect: 203, 514, 240, 525 Annotation: Subtype: Link Rect: 272, 491, 310, 502 Annotation: Subtype: Link Rect: 316, 491, 352, 502 Annotation: Subtype: Link Rect: 95, 457, 133, 467 Annotation: Subtype: Link Rect: 139, 457, 175, 467 Annotation: Subtype: Link Rect: 501, 433, 537, 444 Annotation: Subtype: Link Rect: 478, 422, 516, 433 Annotation: Subtype: Link Rect: 197, 399, 234, 410 Annotation: Subtype: Link Rect: 127, 377, 164, 387 Annotation: Subtype: Link Rect: 486, 353, 523, 364 Annotation: Subtype: Link Rect: 309, 307, 347, 318 Annotation: Subtype: Link Rect: 397, 284, 434, 295 Annotation: Subtype: Link Rect: 430, 261, 468, 272 Annotation: Subtype: Link Rect: 474, 261, 510, 272 Checksum: cac347fb Type: CRC32 Checksum: 9dc641ff792996aa8eca61759e2b7047 Type: MD5 Checksum: 68d4f9fad482680a78316a499efd37c8190e2292 Type: SHA-1