Canonical protein inhibitors of serine proteases

Background: Chymotrypsin C (CTRC) targets specific regulatory cleavage sites within trypsinogens and procarboxypeptidases. Results: The crystal structure of CTRC reveals the structural basis of substrate specificity. Conclusion: Long-range electrostatic and hydrophobic complementarity drives CTRC association with preferred substrates. Significance: The observations reveal the mechanistic basis for CTRC selectivity in digestive enzyme activation and degradation.

Section: Resultsmentioning

confidence: 99%

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation

Batra

Szabó

Caulfield

et al. 2013

“…These inhibitors, representing at least 18 different convergently evolved protein families (14,15), inhibit their cognate proteases via the "Laskowski mechanism," in which inhibitors act as highly specific, limited proteolysis substrates for target enzymes (14,16). They bind so as to position a specific peptide bond, the "reactive site," in the active site of the enzyme, ideally oriented for enzymatic cleavage (17), but due to a binding affinity many orders of magnitude stronger than that associated with ordinary substrate binding and to interactions at the protease-inhibitor interface that deter progress of the reaction and dissociation of the cleaved product peptides, enzymatic turnover proceeds extremely slowly.…”

mentioning

confidence: 99%

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

Salameh

Robinson

Navaneetham

et al. 2010

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-␤ peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg 15 -Ala 16 reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.Mesotrypsin is a human trypsin encoded by the PRSS3 gene found on chromosome 9p13 (1). Normal expression of PRSS3 is restricted to pancreas, brain, and, to a lesser extent, small intestine and colon (2-4); additionally, PRSS3 appears to be transcriptionally up-regulated with cancer progression in epithelial cancers, including lung (5), colon (6), and prostate. Mesotrypsin exhibits substantially different specificity from other trypsins toward protein substrates. It fails to activate pancreatic zymogens and also shows reduced capacity to degrade trypsinogens (7). Compared with other trypsins, it is significantly compromised in its ability to cleave protease-activated receptors (8 -10). Despite limited activity toward these classic trypsin substrates, mesotrypsin displays enhanced catalytic activity compared with other trypsins in the cleavage of certain specific protein substrates, most notably several canonical protease inhibitors (7, 11).The "canonical" inhibitors of serine proteases, named for a protease-binding loop of highly characteristic backbone conformation (12, 13), fulfill the paradoxical function of binding to a protease in a substrate-like manner yet acting as an inhibitor rather than an ordinary substrate. These inhibitors, representing at least 18 different convergently evolved protein families (14, 15), inhibit their cognate proteases via the "Laskowski mechanism," in which inhibitors act as highly specific, limited proteolysis substrates for target enzymes (14, 16). They bind so as to position a specific peptide bond, the "reactive si...

“…The inhibitory domains of these inhibitors, as exemplified by the archetypal example of bovine pancreatic trypsin inhibitor (BPTI), 3 are composed of about 58 amino acid residues that assume a very compact pearshaped fold stabilized by three intramolecular disulfide bonds (17). Like other serine protease inhibitors belonging to the canonical inhibitor class, they feature a protease binding loop that is preconfigured in a conserved canonical backbone conformation, highly complementary to the serine protease active site (18,19). These inhibitors follow the Laskowski mechanism of inhibition, wherein they bind very tightly to a target protease in a substrate-like fashion, positioning a specific "reactive site" bond for cleavage in the active site, but are cleaved many orders of magnitude more slowly than a normal substrate (19 -21).…”

Section: Mesotrypsin Is An Isoform Of Trypsin That Is Uniquely Resistmentioning

confidence: 99%

Sequence and Conformational Specificity in Substrate Recognition

Pendlebury

Wang

Henin

et al. 2014

Background: Mesotrypsin targets protease inhibitors as substrates; substrate recognition depends on sequence and conformational motifs. Results: Mesotrypsin cleaves three human Kunitz domains as specific substrates, but other similarly shaped substrates are cleaved less efficiently. Conclusion: Substrate conformation aids recognition by mesotrypsin but is not sufficient for efficient cleavage. Significance: Mesotrypsin cleavage of human Kunitz domains may contribute to cancer progression.