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Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant.

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  • معلومة اضافية
    • المصدر:
      Publisher: MDPI Country of Publication: Switzerland NLM ID: 101530765 Publication Model: Electronic Cited Medium: Internet ISSN: 2072-6651 (Electronic) Linking ISSN: 20726651 NLM ISO Abbreviation: Toxins (Basel) Subsets: MEDLINE
    • بيانات النشر:
      Original Publication: Basel : MDPI
    • الموضوع:
      Botulinum Toxins, Type A/*chemical synthesis ; Botulinum Toxins, Type A/*metabolism ; Protein Engineering/*methods ; Qb-SNARE Proteins/*chemical synthesis ; Qb-SNARE Proteins/*metabolism ; Qc-SNARE Proteins/*chemical synthesis ; Qc-SNARE Proteins/*metabolism; Amino Acid Sequence ; Animals ; Botulinum Toxins, Type A/genetics ; Botulinum Toxins, Type A/pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; HEK293 Cells ; Humans ; Neurons/drug effects ; Neurons/metabolism ; Protein Structure, Secondary ; Qb-SNARE Proteins/genetics ; Qc-SNARE Proteins/genetics ; Rats ; Rats, Sprague-Dawley
    • نبذة مختصرة :
      Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc. The expansion of its application to these medical conditions is prevented because the major non-neuronal SNAP-25 isoform responsible for exocytosis, SNAP-23, is, in humans, virtually resistant to BoNT/A. Based on previous structural data and mutagenesis studies of SNAP-23 we optimized substrate binding pockets of the enzymatic domain for interaction with SNAP-23. Systematic mutagenesis and rational design yielded the mutations E148Y, K166F, S254A, and G305D, each of which individually increased the activity of LC/A against SNAP-23 between 3- to 23-fold. The assembled quadruple mutant showed approximately 2000-fold increased catalytic activity against human SNAP-23 in in vitro cleavage assays. A comparable increase in activity was recorded for the full-length BoNT/A quadruple mutant tested in cultivated primary neurons transduced with a fluorescently tagged-SNAP-23 encoding gene. Equipped with a suitable targeting domain this quadruple mutant promises to complete successfully tests in cells of the immune system.
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    • Grant Information:
      none International Ipsen
    • Contributed Indexing:
      Keywords: SNAP-23; SNAP-25; botulinum toxin; screening method; substrate specificity; zinc protease
    • الرقم المعرف:
      0 (Qb-SNARE Proteins)
      0 (Qc-SNARE Proteins)
      0 (SNAP23 protein, human)
      EC 3.4.24.69 (Botulinum Toxins, Type A)
    • الموضوع:
      Date Created: 20201223 Date Completed: 20210623 Latest Revision: 20210623
    • الموضوع:
      20231215
    • الرقم المعرف:
      PMC7766560
    • الرقم المعرف:
      10.3390/toxins12120804
    • الرقم المعرف:
      33352834