Trypsin Cleavage Site Research Articles

Sequential Digestion with Trypsin and Elastase in Cross-Linking Mass Spectrometry.

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein–protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4–12 complex allowed us to identify cross-links in previously inaccessible regions.

Pseudotrypsin: A Little-Known Trypsin Proteoform.

Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.

Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment.

Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.

2S protein Ara h 7.0201 has unique epitopes compared to other Ara h 7 isoforms and is comparable to 2S proteins Ara h 2 and 6 in basophil degranulation capacity.

Screening for specific IgE against 2S albumin proteins Ara h 2 and 6 has good positive predictive value in diagnosing peanut allergy. From the third 2S member Ara h 7, 3 isoforms have been identified. Their allergenicity has not been elucidated. This study investigated the allergenicity of Ara h 7 isoforms compared to Ara h 2 and 6. Sensitization of 15 DBPCFC-confirmed peanut-allergic patients to recombinant Ara h 2.0201, Ara h 6.01 and isoforms of recombinant Ara h 7 was determined by IgE immunoblotting strips. A basophil activation test (BAT) was performed in 9 patients to determine IgE-cross-linking capacities of the allergens. Sensitivity to the allergens was tested in 5 patients who were sensitized to at least 1 Ara h 7 isoform, by a concentration range in the BAT. 3D prediction models and sequence alignments were used to visualize differences between isoforms and to predict allergenic epitope regions. Sensitization to Ara h 7.0201 was most frequent (80%) and showed to be equally potent as Ara h 2.0201 and 6.01 in inducing basophil degranulation. Sensitization to Ara h 7.0201 together with Ara h 2.0201 and/or 6.01 was observed, indicating the presence of unique epitopes compared to the other 2 isoforms. Differences between the 3 Ara h 7 isoforms were observed in C-terminal cysteine residues, pepsin and trypsin cleavage sites and 3 single amino acid substitutions. The majority of peanut-allergic patients are sensitized to isoform Ara h 7.0201, which is functionally as active as Ara h 2.0201 and 6.01. Unique epitopes are most likely located in the C-terminus or an allergenic loop region which is a known allergenic epitope region for Ara h 2.0201 and 6.01. Due to its unique epitopes and allergenicity, it is an interesting candidate to improve the diagnostic accuracy for peanut allergy.

Detection of Proteome Diversity Resulted from Alternative Splicing is Limited by Trypsin Cleavage Specificity

Alternative splicing dramatically increases transcriptome complexity but its contribution to proteome diversity remains controversial. Exon-exon junction spanning peptides provide direct evidence for the translation of specific splice isoforms and are critical for delineating protein isoform complexity. Here we found that junction-spanning peptides are underrepresented in publicly available mass spectrometry-based shotgun proteomics data sets. Further analysis showed that evolutionarily conserved preferential nucleotide usage at exon boundaries increases the occurrence of lysine- and arginine-coding triplets at the end of exons. Because both lysine and arginine residues are cleavage sites of trypsin, the nearly exclusive use of trypsin as the protein digestion enzyme in shotgun proteomic analyses hinders the detection of junction-spanning peptides. To study the impact of enzyme selection on splice junction detectability, we performed in-silico digestion of the human proteome using six proteases. The six enzymes created a total of 161,125 detectable junctions, and only 1,029 were common across all enzyme digestions. Chymotrypsin digestion provided the largest number of detectable junctions. Our experimental results further showed that combination of a chymotrypsin-based human proteome analysis with a trypsin-based analysis increased detection of junction-spanning peptides by 37% over the trypsin-only analysis and identified over a thousand junctions that were undetectable in fully tryptic digests. Our study demonstrates that detection of proteome diversity resulted from alternative splicing is limited by trypsin cleavage specificity, and that complementary digestion schemes will be essential to comprehensively analyze the translation of alternative splicing isoforms.

Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution

The role of membrane proteins in cellular mechanisms strongly depends on their dynamics; herein we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motion of the β-barrel as a collective rocking of low amplitude and of hundreds of nano-seconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique Trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, indicating that this loop may be highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.

Targeting an Oncolytic Influenza A Virus to Tumor Tissue by Elastase

Oncolytic viruses are currently established as a novel type of immunotherapy. The challenge is to safely target oncolytic viruses to tumors. Previously, we have generated influenza A viruses (IAVs) containing deletions in the viral interferon antagonist. Those deletions have attenuated the virus in normal tissue but allowed replication in tumor cells. IAV entry is mediated by hemagglutinin (HA), which needs to be activated by a serine protease, for example, through trypsin. To further target the IAV to tumors, we have changed the trypsin cleavage site to an elastase cleavage site. We chose this cleavage site because elastase is expressed in the tumor microenvironment. Moreover, the exchange of the cleavage site previously has been shown to attenuate viral growth in lungs. Newly generated elastase-activated influenza viruses (AE viruses) grew to similar titers in tumor cells as the trypsin-activated counterparts (AT viruses). Intratumoral injection of AE viruses into syngeneic B16f1 melanoma-derived tumors in mice reduced tumor growth similar to AT viruses and had a better therapeutic effect in heterologous human PANC-1-derived tumors. Therefore, the introduction of the attenuation marker “elastase cleavage site” in viral HA allows for safe, effective oncolytic virus therapy.

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist.

Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution.

The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the β-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.

Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes

Trypsin modulating oostatic factor, a decapaptide isolated from the ovaries of A. aegypti, is the physiological factor that terminates the trypsin biosynthesis after the blood meal. Earlier results obtained from feeding mosquito larvae and injecting female mosquitoes with TMOF show that trypsin biosynthesis and egg development are inhibited, indicating that TMOF traverses the gut epithelial cells and modulates trypsin biosynthesis, making it a potential larvacidal peptide hormone.Therefore, TMOF and TMOF green fluorescent protein (GFP) fusion protein with a trypsin cleavage site, allowing TMOF release in the larval gut, were expressed in S. cerevisiae cells that were transformed using homologous recombination at ura3-52 with an engineered plasmid (pYDB2) carrying tmfA and gfp-tmfA and a strong galactose promoter (PGAL1). Southern blot analyses showed that each cell incorporated a single tmfA or gfp-tmfA. Western blot analyses of cells that were fermented up to 48h showed that the engineered S. cerevisiae cells synthesized both TMOF and GFP-TMOF and heat treatment did not affect the recombinant proteins. Engineered S. cerevisiae (3×108cells) that were fermented for 4h produced (2.1±0.2μg±S.E.M) of TMOF. Feeding the engineered cells producing TMOF and GFP-TMOF to larval mosquito caused high mortalities (66±12% and 83±8%, respectively). S. cerevisiae cells transfected with pYEX-BX carrying gfp-tmfA and (DPAR)4 or transformed by homologous recombination of pYDB2-gfp-tmfA carrying a heat shock promoter (PHP) were ineffective. Engineered heat treated yeast cells are consumed by mosquito larvae, and could be used to control mosquitoes.

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status.

Many neurodegenerative diseases arestill lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Oftenpotential new biomarkers do not make itinto the clinicdue to limitationsin their development andhigh costs.However,targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkersfor clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small moleculesin clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4). In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation. The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibratethe MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments

Integral membrane proteins are generally under-represented in routine proteomic analyses, mostly because of their relatively low abundance, hydrophobicity and lack of trypsin-cleavage sites. To increase the coverage of membrane proteomes, various strategies have been developed, targeting mostly the extra-membrane segments of membrane proteins. We focused our attention to the rather overlooked hydrophobic transmembrane segments. Such peptides can be isolated after carbonate stripping and protease “shaving” of membranes isolated by simple centrifugation procedure. The treated membranes with embedded hydrophobic peptides can then be solubilized in organic solvents, re-digested with CNBr, delipidated and subjected to LC-MS/MS analysis. We modified the original “hppK” method, and applied it for the analysis of human lymphoma cells. We identified 1224 proteins of which two-thirds were IMPs with 1–16 transmembrane segments. This method allowed us to identify 13 “missing proteins” — proteins with no previous evidence on protein level. Biological significanceIntegral membrane proteins execute numerous essential functions and represent substantial part of eukaryotic proteomes. Our knowledge of their function and expression is, however, limited. Novel approaches extending our knowledge of membrane proteome are therefore highly desired. As we demonstrate here, a non-conventional method which targets rather overlooked hydrophobic transmembrane segments of integral membrane proteins has wide potential to provide the missing information on the membrane proteome. We show that it can deliver identification and potentially also quantification of hundreds of integral membrane proteins including the so called “missing proteins”.

β-Lactoglobulin as nanotransporter – Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide

The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys121 turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.

Determination of trypsin activity using a gold electrode modified with a nanocover composed of graphene oxide and thionine

We report on an electrochemical method for the determination of the activity of trypsin. A multi-functional substrate peptide (HHHAKSSATGGC-HS) is designed and immobilized on a gold electrode. The three His residues in the N-terminal are able to recruit thionine-loaded graphene oxide (GO/thionine), a nanocover adopted for signal amplification. Once the peptide is cleaved under enzymatic catalysis by trypsin (cleavage site: Lys residue), the His residues leave the electrode, and the GO/thionine cannot cover the peptide-modified electrode anymore. Thus, the changes of the electrochemical signal of thionine, typically acquired at a voltage of -0.35 V, can be used to determine the activity of trypsin. A detection range of 1 × 10−4 to 1 U, with a detection limit of 3.3 × 10−5 U, can be achieved, which is better than some currently available methods. In addition, the method is highly specific, facile, and has the potential for the detection of trypsin-like proteases.

Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity.

By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.

Natural flanking sequences for peptides included in a quantification concatamer internal standard.

Quantification by targeted proteomics has largely depended on mass spectrometry and isotope-labeled internal standards. In addition to traditionally used recombinant proteins or synthetic peptides, concatenated peptides (QconCATs) were introduced as a conceptually new source of internal standard. In the present study, we focused on assessing the length of natural flanking sequences, which surround each peptide included in QconCAT and provide for identical rates of analyte and standard digestion by trypsin. We have expressed, purified, and characterized a set of seven (15)N-labeled QconCATs that cover seven tryptic peptides from human clusterin with a length of natural flanking sequences ranging from none (+0) to six amino acid residues (+6) for each tryptic peptide. Individual QconCATs were mixed with recombinant human clusterin at a 1:1 molar ratio and digested, and the actual ratios for each combination of peptide/flanking sequence were measured with a multiple reaction monitoring assay. Data analysis suggested that natural flanking sequences shorter than +6 residues can cause a quantitative error because the random appearance of other amino acid residues in close proximity to trypsin cleavage sites has unpredictable consequences for the digestion rates of QconCATs.

In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and angiotensin-converting enzyme inhibitory capacity

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A1, A2, B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence 106H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the 122Ser to 122Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of digestion (60min of pepsin + 120min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that genetic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion.

Protein painting reveals solvent-excluded drug targets hidden within native protein-protein interfaces.

Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.

Structure-Function Relationship of SW-AT-1, a Serpin-Type Protease Inhibitor in Silkworm

Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10−5 M−1s−1, for rSW-AT-1 and chymotrpsin is 2.85×10−6 M−1s−1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed.