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Recombinant shewasin D and shewasin A specificity preferences profiled by proteomic identification of protease cleavage sites (PICS)

ABSTRACT: We determined the specificity preferences of both recombinant shewasin D and shewasin A (pepsin-like aspartic proteases from S. denitrificans and S. amazonensis, respectively) using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1 thereby confirming their phylogenetic proximity. We further observed subtle differences in sequence preferences at several positions, suggesting variations in their subsite binding pockets.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Blood Cell, Monocyte, Blood

DISEASE(S): Acute Leukemia

SUBMITTER: Rui Cruz

LAB HEAD: Isaura Simões

PROVIDER: PXD003078 | Pride | 2016-04-05

REPOSITORIES: pride

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			Action	DRS
	BM_1_10uL.mgf	Mgf
	BM_1_10uL.mzXML	Mzxml
	BM_1_10uL.wiff	Wiff
	BM_1_10uL.wiff.scan	Wiff
	BM_1_1uL.mgf	Mgf

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Publications

Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.

Leal Ana Rita AR Cruz Rui R Bur Daniel D Huesgen Pitter F PF Faro Rosário R Manadas Bruno B Wlodawer Alexander A Faro Carlos C Simões Isaura I

Scientific reports 20160331

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the peps ...[more]

PMID: 27029611

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Project description:We performed an analysis of the substrate specificity preferences of native and synthetic cardosin B using the Proteomic Identification of protease Cleavage Sites methodology and some differences regarding preference and stringency of the subsite binding pockets were observed. Results clearly suggest that synthetic cardosin B is more stringent than native cardosin B explaining the observed suitability of the synthetic protein as biotechnological tool in cheese industry.

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Active Site Specificity Profiling of the Matrix Metalloproteinase Family: Proteomic Identification of 4,300 Cleavage Sites by MMPs 1, 2, 3, 7, 8, 9, 12, 13, and 14.

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The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B

Project description:The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

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TALE transcription factors binding and transcriptional effect [ChIP-Seq]

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