scholarly journals Substrate Requirements for Secondary Cleavage by HIV-1 Reverse Transcriptase RNase H

2002 ◽  
Vol 277 (32) ◽  
pp. 28400-28410 ◽  
Author(s):  
Michele Wisniewski ◽  
Yan Chen ◽  
Mini Balakrishnan ◽  
Chockalingam Palaniappan ◽  
Bernard P. Roques ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rnase H ◽  
Secondary Cleavage ◽  
Hiv 1

scholarly journals Partial Blocking of Mouse DSPP Processing by Substitution of Gly451–Asp452Bond Suggests the Presence of Secondary Cleavage Site(s)

2011 ◽  
Vol 53 (4) ◽  
pp. 307-312 ◽  
Author(s):  
Qinglin Zhu ◽  
Monica Prasad ◽  
Hui Kong ◽  
Yongbo Lu ◽  
Yao Sun ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Partial Blocking ◽  
Secondary Cleavage

scholarly journals Sorting of PC2 to the regulated secretory pathway in AtT20 cells

1998 ◽  
Vol 21 (2) ◽  
pp. 209-216 ◽  
Author(s):  
NA Taylor ◽  
G Jan ◽  
KT Scougall ◽  
K Docherty ◽  
KI Shennan
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Cleavage Sites ◽  
Large Deletions ◽  
C Terminus ◽  
Regulated Secretory Pathway ◽  
Primary Cleavage ◽  
Secondary Cleavage ◽  
Att20 Cell

PC2 and PC3 are neuroendocrine specific members of the eukaryotic subtilisin-like proprotein convertase (PC) family. Both are sorted via the regulated secretory pathway into secretory granules. In order to identify sequences in PC2 which are involved in targeting to the regulated secretory pathway we expressed a series of PC2 cDNAs containing mutations in the C terminal or propeptide domains in the mouse corticotrophic AtT20 cell line. Sorting of endogenous PC3 was used as a control. PC2 and PC3 were secreted with similar kinetics and sorted to secretory granules with similar efficiencies. Deletions of up to 50 amino acids from the C-terminus of proPC2 had no effect on secretion or sorting, but larger deletions completely prevented maturation or secretion. Two large deletions within the propeptide also prevented secretion. Smaller deletions between the primary and secondary cleavage sites, or of the primary cleavage site, reduced the amount of protein secreted but did not affect sorting to secretory granules. Replacement of the propeptide of PC2 with that of the endogenous PC3 also had no effect on secretion or sorting. The results indicate that targeting of proPC2 to the regulated secretory pathway is dependent on more than one region within the proPC2 molecule.

Secondary cleavage of a fluorescently labeled SNAP-25 substrate by Xeomin® drug product

Toxicon ◽  
2008 ◽  
Vol 51 ◽  
pp. 13 ◽  
Author(s):  
Hunt Terrence ◽  
Clarke Kenneth ◽  
Rupp David ◽  
Shimizu Gary ◽  
Tam Karen
Keyword(s):  
Drug Product ◽  
Secondary Cleavage

scholarly journals Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

2015 ◽  
Vol 197 (7) ◽  
pp. 1173-1184 ◽  
Author(s):  
Jerome Escano ◽  
Byron Stauffer ◽  
Jacob Brennan ◽  
Monica Bullock ◽  
Leif Smith
Keyword(s):  
Streptococcus Mutans ◽  
Posttranslational Modifications ◽  
Cleavage Site ◽  
Peptide Sequence ◽  
Leader Peptide ◽  
Peptide Antibiotics ◽  
The Core ◽  
Core Peptide ◽  
Mutacin 1140 ◽  
Secondary Cleavage

ABSTRACTLantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that is cleaved to yield the active antibacterial peptide. Significant advancements in molecular tools that promote the study of lantibiotic biosynthesis can be used inStreptococcus mutans. Herein, we further our understanding of leader peptide sequence and core peptide structural requirements for the biosynthesis and transport of the lantibiotic mutacin 1140. Our study on mutacin 1140 biosynthesis shows a dedicated secondary cleavage site within the leader peptide and the dependency of transport on core peptide posttranslational modifications (PTMs). The secondary cleavage site on the leader peptide is found at the −9 position, and secondary cleavage occurs before the core peptide is transported out of the cell. The coordinated cleavage at the −9 position was absent in alanTdeletion strain, suggesting that the core peptide interaction with the LanT transporter enables uniform cleavage at the −9 position. Following transport, the LanP protease was found to be tolerant to a wide variety of amino acid substitutions at the primary leader peptide cleavage site, with the exception of arginine at the −1 position. Several leader and core peptide mutations produced core peptide variants that had intermediate stages of PTM enzyme modifications, supporting the concept that PTM enzyme modifications, secondary cleavage, and transport are occurring in a highly coordinated fashion.IMPORTANCEMutacin 1140 belongs to the class I lantibiotic family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The biosynthesis of mutacin 1140 is a highly efficient process which does not lead to a discernible level of production of partially modified core peptide variants. The products isolated from an extensive mutagenesis study on the leader and core peptides of mutacin 1140 show that the posttranslational modifications (PTMs) on the core peptide occur under a highly coordinated dynamic process. PTMs are dictated by the distance of the core peptide modifiable residues from PTM enzyme active sites. The formation of lanthionine rings aids in the formation of successive PTMs, as was observed in a peptide variant lacking a C-terminal decarboxylation.

Fusion expression and purification of four disulfide-rich peptides reveals enterokinase secondary cleavage sites in animal toxins

Peptides ◽  
2013 ◽  
Vol 39 ◽  
pp. 145-151 ◽  
Author(s):  
Zongyun Chen ◽  
Song Han ◽  
Zhijian Cao ◽  
Yingliang Wu ◽  
Renxi Zhuo ◽  
...  
Keyword(s):  
Fusion Expression ◽  
Cleavage Sites ◽  
Expression And Purification ◽  
Secondary Cleavage

scholarly journals Specific conformational changes of plasminogen induced by chloride ions, 6-aminohexanoic acid and benzamidine, but not the overall openness of plasminogen regulate, production of biologically active angiostatins

2005 ◽  
Vol 392 (3) ◽  
pp. 703-712 ◽  
Author(s):  
Debra J. Warejcka ◽  
Sally S. Twining
Keyword(s):  
Conformational Changes ◽  
Umbilical Vein ◽  
Chloride Ions ◽  
Biologically Active ◽  
Buffer System ◽  
Cleavage Sites ◽  
Peptide Cleavage ◽  
Initial Cleavage ◽  
Umbilical Vein Endothelial Cells ◽  
Secondary Cleavage

The overall conformation of plasminogen depends upon the presence of anions and molecules such as AHA (6-aminohexanoic acid) and BZ (benzamidine). The purpose of the present study was to determine the effect of conformation on the initial and secondary cleavages of plasminogen to generate active angiostatins. Plasminogen was digested with the physiologically relevant neutrophil elastase in one of the four Tris/acetate buffers: buffer alone or buffer plus NaCl, AHA or BZ. The initial cleavage of Glu1-plasminogen was much slower in the tight NaCl-induced α-conformation, fastest in the intermediate BZ-induced β-conformation and intermediate both in the control and in the AHA-induced open γ-conformation. Although the buffer system determined the relative amounts of the initial cleavage products, the same four cleavage sites were utilized under all conditions. A fifth major initial cleavage within the protease domain was observed in the presence of BZ. N-terminal peptide cleavage required for angiostatin formation occurred as either the initial or the secondary cleavage. Angiostatins were generated fastest in the presence of BZ and slowest in the presence of NaCl. Both the initial and secondary cleavages were affected by the modifying agents, indicating that they influence the conformation of both Glu-plasminogen and the initial cleavage products. The angiostatins produced under the different conditions inhibited proliferation of human umbilical-vein endothelial cells. These results suggest that plasminogen conversion into active angiostatins is dependent more on the specific conformation changes induced by the various modifying reagents rather than on the overall openness of the molecule.

In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain

2013 ◽  
Vol 394 (8) ◽  
pp. 1069-1075 ◽  
Author(s):  
Christian Ursel ◽  
Uwe Fandrich ◽  
Anita Hoffmann ◽  
Torsten Sieg ◽  
Christian Ihling ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Cleavage Site ◽  
In Vitro Maturation ◽  
Dorsoventral Patterning ◽  
Cystine Knot ◽  
Toll Receptor ◽  
Almost All ◽  
Secondary Cleavage

Abstract Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

scholarly journals A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

RNA ◽  
2012 ◽  
Vol 18 (9) ◽  
pp. 1716-1724 ◽  
Author(s):  
B. Meineke ◽  
A. Kast ◽  
B. Schwer ◽  
F. Meinhardt ◽  
S. Shuman ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Secondary Cleavage

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Mariusz Olczak ◽  
Katarzyna Indyk ◽  
Teresa Olczak
Keyword(s):  
Proteolytic Activity ◽  
Active Site ◽  
Cleavage Site ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ph Optimum ◽  
Low Efficiency ◽  
Primary Cleavage ◽  
Secondary Cleavage

AbstractAzurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed inSf9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.

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