Cleavage Specificity of Human Rhinovirus-2 2A Protease for Peptide Substrates

1997 ◽  
Vol 235 (3) ◽  
pp. 562-566 ◽  
Author(s):  
Q.May Wang ◽  
Wolfgang Sommergruber ◽  
Robert B. Johnson
Keyword(s):  
Human Rhinovirus ◽  
Peptide Substrates ◽  
Cleavage Specificity ◽  
2A Protease

scholarly journals Enzymatic Characterization of Refolded Human Rhinovirus Type 14 2A Protease Expressed in Escherichia coli

1998 ◽  
Vol 72 (2) ◽  
pp. 1683-1687 ◽  
Author(s):  
Q. May Wang ◽  
Robert B. Johnson ◽  
Gregory A. Cox ◽  
Elcira C. Villarreal ◽  
Lisa M. Churgay ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Cdna Sequence ◽  
Colorimetric Assay ◽  
Human Rhinovirus ◽  
Bacterial Cells ◽  
Enzymatic Characterization ◽  
Cleavage Specificity ◽  
Microplate Reader ◽  
2A Protease

ABSTRACT Reported here is the production of recombinant human rhinovirus 14 (HRV14) 2A protease from bacterial cells transformed with a heat-inducible plasmid containing the HRV14 2A cDNA sequence. Overexpressed 2A protein partitioned into the inclusion bodies was solubilized in urea and then refolded in the presence of Zn2+. Transition metals were required for the restoration of 2A protease activity as a structural component, but appeared to be inhibitory if added exogenously once the enzyme was refolded. Based on the cleavage specificity studies, a colorimetric assay was developed for the highly purified HRV14 2A protease. A peptide with the sequence RKGDIKSY–p-nitroanilide was found to be cleaved by the 2A protease with a k cat/Km ratio of ∼335 M−1s−1, which allows its activity to be measured continuously with a spectrophotometer or a microplate reader.

scholarly journals Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

1992 ◽  
Vol 267 (31) ◽  
pp. 22639-22644
Author(s):  
W Sommergruber ◽  
H Ahorn ◽  
A Zöphel ◽  
I Maurer-Fogy ◽  
F Fessl ◽  
...  
Keyword(s):  
Synthetic Peptide ◽  
Human Rhinovirus ◽  
Peptide Substrates ◽  
Cleavage Specificity

Development ofin VitroPeptide Substrates for Human Rhinovirus-14 2A Protease

1998 ◽  
Vol 356 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Q.May Wang ◽  
Robert B. Johnson ◽  
Wolfgang Sommergruber ◽  
Timothy A. Shepherd
Keyword(s):  
Human Rhinovirus ◽  
2A Protease

scholarly journals Cleavage Specificity ofMycobacterium tuberculosisClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

2015 ◽  
Vol 290 (17) ◽  
pp. 11008-11020 ◽  
Author(s):  
Tatos Akopian ◽  
Olga Kandror ◽  
Christopher Tsu ◽  
Jack H. Lai ◽  
Wengen Wu ◽  
...  
Keyword(s):  
Bacterial Activity ◽  
Peptide Substrates ◽  
Cleavage Specificity

scholarly journals Full length and protease domain activity of chikungunya virus nsP2 differ from other alphavirus nsP2 proteases in recognition of small peptide substrates

2015 ◽  
Vol 35 (3) ◽  
Author(s):  
Chonticha Saisawang ◽  
Pornpan Sillapee ◽  
Kwanhathai Sinsirimongkol ◽  
Sukathida Ubol ◽  
Duncan R. Smith ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Virus Replication ◽  
Chikungunya Virus ◽  
Full Length ◽  
Protease Domain ◽  
Small Peptide ◽  
Peptide Substrates ◽  
Cleavage Specificity ◽  
Structural Polypeptide

The protease role of alphavirus nsP2 is critical for virus replication as only the virus protease processes the viral non-structural polypeptide. We show chikungunya nsP2 protease possesses different substrate specificity to the canonical alphavirus nsP2 polyprotein cleavage specificity.

scholarly journals Arabidopsis thaliana phytaspase: identification and peculiar properties

2018 ◽  
Vol 45 (2) ◽  
pp. 171 ◽  
Author(s):  
Nina V. Chichkova ◽  
Raisa A. Galiullina ◽  
Larisa V. Mochalova ◽  
Svetlana V. Trusova ◽  
Zulfazli M. Sobri ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Synthetic Peptide ◽  
Single Gene ◽  
Amino Acid Residues ◽  
Reaction Conditions ◽  
Peptide Substrates ◽  
Cleavage Specificity ◽  
Cellular Environment ◽  
Detection And Identification ◽  
Additional Amino Acid

Phytaspases are plant cell death-related proteases of the subtilisin-like protease family that possess an unusual aspartate cleavage specificity. Although phytaspase activity is widespread in plants, phytaspase of Arabidopsis thaliana (L.) Heynh. has escaped detection and identification thus far. Here, we show that a single gene (At4 g10540) out of 56 A. thaliana subtilisin-like protease genes encodes a phytaspase. The recombinant phytaspase was overproduced in Nicotiana benthamiana Domin leaves, isolated, and its substrate specificity and properties were characterised. At pH 5.5, at physiological mildly acidic reaction conditions, the Arabidopsis phytaspase was shown to be strictly Asp-specific. The strongly preferred cleavage motifs of the enzyme out of a panel of synthetic peptide substrates were YVAD and IETD, while the VEID-based substrate preferred by the tobacco and rice phytaspases was almost completely resistant to hydrolysis. At neutral pH, however, the Arabidopsis phytaspase could hydrolyse peptide substrates after two additional amino acid residues, His and Phe, in addition to Asp. This observation may indicate that the repertoire of Arabidopsis phytaspase targets could possibly be regulated by the conditions of the cellular environment. Similar to tobacco and rice phytaspases, the Arabidopsis enzyme was shown to accumulate in the apoplast of epidermal leaf cells. However, in stomatal cells Arabidopsis phytaspase was observed inside the cells, possibly co-localising with vacuole. Our study thus demonstrates that the Arabidopsis phytaspase possesses both important similarities with and distinctions from the already known phytaspases, and is likely to be the most divergent member of the phytaspase family.

scholarly journals Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

2016 ◽  
Vol 90 (24) ◽  
pp. 11032-11042 ◽  
Author(s):  
Erin Walker ◽  
Lora Jensen ◽  
Sarah Croft ◽  
Kejun Wei ◽  
Alex J. Fulcher ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Human Rhinovirus ◽  
Subcellular Targeting ◽  
Nuclear Targeting ◽  
3C Protease ◽  
2A Protease ◽  
Targeting Ability ◽  
Green Fluorescent

ABSTRACTThe human rhinovirus (HRV) 3C and 2A proteases (3Cproand 2Apro, respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3Cprois present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2Aproactivity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3Cpro, 3D, and mutant derivatives thereof, we show that 3Cprois located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3Cproactivity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2Aprofusion proteins, we demonstrate formally that 2Aproactivity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3Cprois insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2Aproactivity and not 3Cproactivity, which is observed only later in infection. The results thus define the temporal activities of 2Aproand 3CD/3Cproactivities in HRV serotype16 infection.IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

scholarly journals Ex Vivo and In Vivo Inhibition of Human Rhinovirus Replication by a New Pseudosubstrate of Viral 2A Protease

2011 ◽  
Vol 86 (2) ◽  
pp. 691-704 ◽  
Author(s):  
N. Falah ◽  
S. Violot ◽  
D. Decimo ◽  
F. Berri ◽  
M.-L. Foucault-Grunenwald ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Rhinovirus ◽  
2A Protease

Further Characterization of Earthworm Serine Proteases: Cleavage Specificity Against Peptide Substrates and on Autolysis

1999 ◽  
Vol 63 (11) ◽  
pp. 2031-2033 ◽  
Author(s):  
Nobuyoshi NAKAJIMA ◽  
Manabu SUGIMOTO ◽  
Kohji ISHIHARA ◽  
Kaoru NAKAMURA ◽  
Hiroki HAMADA
Keyword(s):  
Serine Proteases ◽  
Peptide Substrates ◽  
Cleavage Specificity
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