title: Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.
abstract: 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 pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A 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. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.|Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal.
authors.forename: Ana Rita
authors.initials: AR
authors.lastname: Leal
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.|Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Cruz
authors.affiliation: Actelion Pharmaceuticals Ltd, CH-4123 Allschwil, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bur
authors.affiliation: Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, 52425 Jülich, Germany.
authors.forename: Pitter F
authors.initials: PF
authors.lastname: Huesgen
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: Rosário
authors.initials: R
authors.lastname: Faro
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.|Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal.
authors.forename: Bruno
authors.initials: B
authors.lastname: Manadas
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702 Maryland, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.|Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Faro
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.|Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal.
authors.forename: Isaura
authors.initials: I
authors.lastname: Simões
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005075:Biological Evolution; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D019151:Peptide Library; D059748:Proteolysis; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010436:Pepstatins; D019151:Peptide Library; D020543:Proteome; D011994:Recombinant Proteins; D010434:Pepsin A; C031375:pepstatin