Adeno-Associated Virus VP1u Exhibits Protease Activity

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.

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NDM-4 Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05961-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 2184-2186 ◽

Cited By ~ 87

Author(s):

Patrice Nordmann ◽

Anne E. Boulanger ◽

Laurent Poirel

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Substitution ◽

Active Sites ◽

Hydrolytic Activity ◽

Single Amino Acid Substitution ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Content Type

ABSTRACTA clinicalEscherichia coliisolate resistant to all β-lactams, including carbapenems, expressed a novel metallo-β-lactamase (MBL), NDM-4, differing from NDM-1 by a single amino acid substitution (Met154Leu). NDM-4 possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to that of NDM-1. This amino acid substitution was not located in the known active sites of NDM-1, indicating that remote amino acid substitutions might also play a role in the extended activity of this MBL.

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Effect of amino acid substitution at the P3 and P4 subsites of fusion proteins on Kex2 protease activity

Biotechnology and Applied Biochemistry ◽

10.1042/ba20000012 ◽

2000 ◽

Vol 32 (1) ◽

pp. 53 ◽

Cited By ~ 6

Author(s):

Yuji Suzuki ◽

Nobue Ikeda ◽

Eri Kataoka ◽

Kazuhiro Ohsuye

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Fusion Proteins ◽

Protease Activity ◽

Kex2 Protease

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Amino acid substitution scoring matrices specific to intrinsically disordered regions in proteins

Scientific Reports ◽

10.1038/s41598-019-52532-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Rakesh Trivedi ◽

Hampapathalu Adimurthy Nagarajaram

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Compositional Bias ◽

Amino Acid Residues ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Scoring Matrices ◽

Disordered Regions

Abstract An amino acid substitution scoring matrix encapsulates the rates at which various amino acid residues in proteins are substituted by other amino acid residues, over time. Database search methods make use of substitution scoring matrices to identify sequences with homologous relationships. However, widely used substitution scoring matrices, such as BLOSUM series, have been developed using aligned blocks that are mostly devoid of disordered regions in proteins. Hence, these substitution-scoring matrices are mostly inappropriate for homology searches involving proteins enriched with disordered regions as the disordered regions have distinct amino acid compositional bias, and therefore expected to have undergone amino acid substitutions that are distinct from those in the ordered regions. We, therefore, developed a novel series of substitution scoring matrices referred to as EDSSMat by exclusively considering the substitution frequencies of amino acids in the disordered regions of the eukaryotic proteins. The newly developed matrices were tested for their ability to detect homologs of proteins enriched with disordered regions by means of SSEARCH tool. The results unequivocally demonstrate that EDSSMat matrices detect more number of homologs than the widely used BLOSUM, PAM and other standard matrices, indicating their utility value for homology searches of intrinsically disordered proteins.

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Multiclonal Expansion and High Prevalence of β-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00851-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 9

Author(s):

Hiroyuki Honda ◽

Toyotaka Sato ◽

Masaaki Shinagawa ◽

Yukari Fukushima ◽

Chie Nakajima ◽

...

Keyword(s):

Amino Acid ◽

Haemophilus Influenzae ◽

Amino Acid Substitution ◽

University Hospital ◽

Fluoroquinolone Resistance ◽

Content Type ◽

Group Iii ◽

High Prevalence ◽

High Level ◽

Sequence Types

ABSTRACT β-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of β-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were β-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

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Hubsm: A Novel Amino Acid Substitution Matrix for Comparing Hub Proteins

International Journal of Advanced Research in Computer Science and Software Engineering ◽

10.23956/ijarcsse.v7i8.53 ◽

2017 ◽

Vol 7 (8) ◽

pp. 212

Author(s):

Renganayaki G. ◽

Achuthsankar S. Nair

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Low Complexity ◽

Database Search ◽

Substitution Matrix ◽

Compositional Bias ◽

Sequence Alignments ◽

Amino Acid Substitution Matrix ◽

Alignment Algorithms ◽

Hub Proteins

Sequence alignment algorithms and database search methods use BLOSUM and PAM substitution matrices constructed from general proteins. These de facto matrices are not optimal to align sequences accurately, for the proteins with markedly different compositional bias in the amino acid. In this work, a new amino acid substitution matrix is calculated for the disorder and low complexity rich region of Hub proteins, based on residue characteristics. Insights into the amino acid background frequencies and the substitution scores obtained from the Hubsm unveils the residue substitution patterns which differs from commonly used scoring matrices .When comparing the Hub protein sequences for detecting homologs, the use of this Hubsm matrix yields better results than PAM and BLOSUM matrices. Usage of Hubsm matrix can be optimal in database search and for the construction of more accurate sequence alignments of Hub proteins.

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