Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Abstract

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

Multivalent effect of peptide functionalized polymeric nanoparticles towards selective prostate cancer targeting

“… 43 Chymotrypsin protease is such an example, and it is known to specifically cleave at the C-terminal of aromatic amino acids such as tryptophan (W), tyrosine (Y) and phenylalanine (F). 44 In the sequence of the WQP peptide (WQPDTAHHWATLC), we expected a highly specific cleavage at the C-terminal end of tryptophan (W) at position 9 (starting from N-terminal). We confirmed the presence of the digested fragment having a molecular weight of 1177.23 Da by analysing the supernatant obtained after pelleting down the NPs and using UPLC-MS as shown in the schematic in Fig.…”

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin

“…Thrombin, like plasmin, is a serine protease of the trypsin family capable of hydrolyzing peptide bonds immediately following Arg or Lys residues. The enzyme is somewhat promiscuous and accepts a range of peptide substrates. − Previous work had identified a unique thrombin cutting site located between the N1 and the N2 domains of FnBPA. Here, we studied more generally how the binding regions of S. aureus MSCRAMMs are processed by this enzyme .…”

Comparative sequence analysis of vitamin K‐dependent coagulation factors

“…With some minor exceptions, position 284 is occupied by a basic residue (Figure 2), implicating evolutionary conservation of the autoproteolytic site. However, the propensity for auto‐proteolysis at position 284 likely varies across vertebrates, because amphibians, birds, and many mammals contain an acidic residue at position P2 (Figure 2), which should compromise cleavage by thrombin 36,37 . Interestingly, regardless of whether thrombin is generated after cleavage at R271 or R284, there appears to be strong selective pressure for a Thr (or less commonly Ser) as the first residue in the newly created N‐terminus (Figure 2), although the functional significance for such requirement is yet to be elucidated.…”