The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Rahman, Mizanur; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A.; Ahmed, S. Ashraf

doi:10.1074/jbc.m113.451286

Cited by 23 publications

(35 citation statements)

References 45 publications

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“…The inhibition appears to be more pronounced with the 66-mer substrate than with the full length SNAP25 substrate. In contrast to the inhibition by the 57-mer product, the reaction of LcA with the 17-mer substrate was not inhibited by either its N-terminal 11-mer or the C-terminal 6-mer or peptide products [24]. Only 5–10% inhibition of these larger substrate cleavage reactions at 10 µM peptide (Figure 6) suggest that the nonlinearity of the time course in Figure 5A may not be due to inhibition by the formed N-terminal product.…”

Section: Resultsmentioning

confidence: 65%

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

2014

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Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25) and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of kcat, Km, and catalytic efficiency kcat/Km. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.

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Section: Resultsmentioning

confidence: 65%

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

2014

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“…Šilhár et al (2013b) proposed that the additional 23 C-terminal residues in LC/A 448 increases the conformational flexibility of the active site, which reduces the binding affinity for SMIs. Although full-length LC/A 448 is clearly the more relevant target for BoNT/A inhibitor studies, it is less often used since the recombinant enzyme in solution has a tendency to form dimers and to undergo autocatalysis (Šilhár et al, 2013b; Mizanur et al, 2013). …”

Section: Discussionmentioning

confidence: 99%

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A

Jacobson¹,

Adler²,

Silvaggi

et al. 2017

Toxicon

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Botulinum neurotoxins (BoNTs) are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism. Their ease of production and extreme toxicity have caused these neurotoxins to be classified as Tier 1 bioterrorist threat agents and have led to a sustained effort to develop countermeasures to treat intoxication in case of a bioterrorist attack. While timely administration of an approved antitoxin is effective in reducing the severity of botulism, reversing intoxication requires different strategies. In the present study, we evaluated ABS 252 and other mercaptoacetamide small molecule active-site inhibitors of BoNT/A light chain using an integrated multi-assay approach. ABS 252 showed inhibitory activity in enzymatic, cell-based and muscle activity assays, and importantly, produced a marked delay in time-to-death in mice. The results suggest that a multi-assay approach is an effective strategy for discovery of potential BoNT therapeutic candidates.

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“…A study by Mizanur et al . () on the function of C terminus of the catalytic domain of type A botulinum neurotoxin also proved that the C‐terminal peptides competitively inhibit the normal catalytic activity of serotype A (LcA).…”

Section: C‐terminal Protease Domains Have Influence On Enzyme Activitymentioning

confidence: 89%

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications

Huang

Liú

et al. 2016

J Appl Microbiol

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Summary C‐terminal domains widely exist in the C‐terminal region of multidomain proteases. As a β‐sandwich domain in multidomain protease, the C‐terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C‐terminal protease domains are described. Furthermore, the application prospects of C‐terminal domains, including polycystic kidney disease, prepeptidase C‐terminal and collagen‐binding domain, in the area of medicine and biological artificial materials are also discussed.

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The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Cited by 23 publications

References 45 publications

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications

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