Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label‐free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion‐induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion‐induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS‐Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label‐free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome‐wide scale.     

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.     

Glutathione adducts, not carbamylated lysines, are the major modification of lens α-crystallins from renal failure patients

-Crystallins from the water-soluble and the water-insoluble, guanidine-soluble portions of lenses from four renal failure patients and two normal donors of similar age were isolated and enzymatically digested into peptides. Molecular weights of the peptides, determined by fast atom bombardment mass spectrometry, indicated modifications specifically associated with renal failure. The only modifications observed in the-crystallins from renal failure patients, but not in the normal old lenses, were glutathione adducts to Cys 131 and Cys 142. These adducts were present in the lenses of all four renal failure patients, but not in the two normal old lenses. The four lenses from the renal failure patients were searched for evidence of carbamylation at lysyl or cysteinyl residues: carbamylation was not detected. Because the same mass spectrometric methods had previously demonstrated sufficient sensitivity and specificity to detect as little as 5% modification in the examination ofin vitro carbamylated bovine lenses, these results indicated that carbamylation is not a major modification of the lens-crystallins of renal failure patients.     

Age‐related cleavages of crystallins in human lens cortical fiber cells generate a plethora of endogenous peptides and high molecular weight complexes

Low molecular weight peptides derived from the breakdown of crystallins have been reported in adult human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilization of crystallins. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of four human lenses representing young, middle and old‐age human lenses. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C‐terminus with significantly higher frequency compared to other residues, suggesting that trypsin‐like proteolysis may be active in the lens cortical fiber cells. Selected reaction monitoring analysis of an endogenous αA‐crystallin peptide (αA_57‐65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA_57‐65 formation escalated significantly. Using 2D gel electrophoresis/nanoLC‐ESI‐MS/MS, 12 protein complexes of 40–150 kDa consisting of multiple crystallin components were characterized from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross‐linking, with these complexes potentially acting to stabilize degraded crystallins by sequestration into water soluble complexes. Proteins 2015; 83:1878–1886. © 2015 Wiley Periodicals, Inc.     

The amino acid sequence of the rabbit glycoprotein hormone alpha subunit

The amino acid sequence of the alpha subunit of rabbit (lagomorph) lutropin (lLH) has been determined. Overlapping peptides from trypsin and chymotrypsin digestions were isolated by reverse-phase high-pressure liquid chromatography (HPLC). Sequencing was by the dansyl-Edman procedure. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH alpha subunit is (asterisks denote carbohydrate attachment sites): This proposed sequence is highly homologous with the porcine, murine, ovine, and bovine glycoprotein hormone alpha subunit sequences. Two unusual proteolytic cleavages were observed: (1) a cleavage by trypsin between Asn-77 and Ala-78, and (2) a cleavage by chymotrypsin between Ala-45 and Arg-46. Similar enzymatic cleavages were previously reported for equine chorionic gonadotropin alpha subunit by Wardet al. and for these sites in the ovine LH alpha subunit by Liuet al. Chymotrypsin cleaved on the carboxyl side of methionine sulfone residues at positions 51 and 75.     

Cleavage of Rabbit Myelin Basic Protein by Pepsin

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe⁸⁷-Phe⁸⁸ bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu¹⁵¹-Phe¹⁵² bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe⁴⁴-Phe⁴⁵ and Leu¹⁰⁹-Ser¹¹⁰ bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH₂-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr¹⁴Leu¹⁵ bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Lens Crystallin Modifications and Cataract in Transgenic Mice Overexpressing Acylpeptide Hydrolase

The accumulation of crystallin fragments in vivo and their subsequent interaction with crystallins are responsible, in part, for protein aggregation in cataracts. Transgenic mice overexpressing acylpeptide hydrolase (APH) specifically in the lens were prepared to test the role of protease in the generation and accumulation of peptides. Cataract development was seen at various postnatal days in the majority of mice expressing active APH (wt-APH). Cataract onset and severity of the cataracts correlated with the APH protein levels. Lens opacity occurred when APH protein levels were >2.6% of the total lens protein and the specific activity, assayed using Ac-Ala-p-nitroanilide substrate, was >1 unit. Transgenic mice carrying inactive APH (mt-APH) did not develop cataract. Cataract development also correlated with N-terminal cleavage of the APH to generate a 57-kDa protein, along with an increased accumulation of low molecular weight (LMW) peptides, similar to those found in aging human and cataract lenses. Nontransgenic mouse lens proteins incubated with purified wt-APH in vitro resulted in a >20% increase in LMW peptides. Crystallin modifications and cleavage were quite dramatic in transgenic mouse lenses with mature cataract. Affected lenses showed capsule rupture at the posterior pole, with expulsion of the lens nucleus and degenerating fiber cells. Our study suggests that the cleaved APH fragment might exert catalytic activity against crystallins, resulting in the accumulation of distinct LMW peptides that promote protein aggregation in lenses expressing wt-APH. The APH transgenic model we developed will enable in vivo testing of the roles of crystallin fragments in protein aggregation.     

High-Efficiency and Robust Expression System for Stable Isotope-Labeled Peptides

Ubiquitin–peptide fusion protein system enables preparation of stable isotope labeled peptides through the expression of the protein in E. coli cells in labeled media (Kohno et al. (1998) J Biomol NMR 12:109–121). Advantages of the system over others include: very specific cleavage of the bond between ubiquitin and target peptide moieties by yeast ubiquitin hydrolase and low cost for the protease which can also be expressed in E. coli cells. The former point is particularly important since other frequently used proteases, such as factor Xa and thrombin, often show non-specific cleavages at sites unexpected from their nominal specificities. We improved the yield of the peptide by adapting the codon usage of ubiquitin gene for the expression in E. coli cells, by using RNase E-deficient host strains, and by modifying purification procedure. The yield of mastoparan-X was increased threefold by these modifications. We also succeeded in the preparation of labeled magainin 2, an antimicrobial peptide that could not be expressed at all by the previous method, by choosing host strains and culture media. The HSQC signals of the ¹⁵N-labeled magainin 2 in an aqueous solution were completely resolved in spite of the severe overlap of the 1D proton signals, confirming that the stable isotope labeling is quite useful for analysis of peptides.     

Important roles for epithelial cell peptides in hydra development

It has been convincingly shown that peptides play important roles in the regulation and maintenance of a variety of tissues and organs in living animals. However, little is known concerning the potential role of peptides as signaling molecules in developmental processes. In Hydra, there is circumstantial evidence that small diffusible molecules act as morphogens in the regulation of patterning processes. In order to view the entire spectrum of peptide signaling molecules, we initiated a project aiming at the systematic identification of peptide signaling molecules in Hydra. In this review, we describe three peptide signaling molecules and one family of peptides that function as signaling molecules in the processes of axial pattern formation and neuron differentiation in Hydra. These peptides are produced by epithelial cells and are therefore termed “epitheliopeptides”. We discuss the importance of epitheliopeptides in developmental processes within a subset of hydrozoans.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

Molecular modelling,synthesis and biological evaluation of peptide inhibitors as anti-angiogenic agent targeting neuropilin-1 for anticancer application

Vascular endothelial growth factor (VEGF) and its co-receptor neuropilin-1 (NRP-1) are important targets of many pro-angiogenic factors. In this study, nine peptides were synthesized and evaluated for their molecular interaction with NRP-1 and compared to our previous peptide ATWLPPR. Docking study showed that the investigated peptides shared the same binding region as shown by tuftsin known to bind selectively to NRP-1. Four pentapeptides (DKPPR, DKPRR, TKPPR and TKPRR) and a hexapeptide CDKPRR demonstrated good inhibitory activity against NRP-1. In contrast, peptides having arginine residue at sites other than the C-terminus exhibited low activity towards NRP-1 and this is confirmed by their inability to displace the VEGF₁₆₅ binding to NRP-1. Docking study also revealed that replacement of carboxyl to amide group at the C-terminal arginine of the peptide did not affect significantly the binding interaction to NRP-1. However, the molecular affinity study showed that these peptides have marked reduction in the activity against NRP-1. Pentapeptides having C-terminal arginine showed strong interaction and good inhibitory activity with NRP thus may be a good template for anti-angiogenic targeting agent.     

New caerin antibacterial peptides from the skin glands of the Australian tree frog Litoria xanthomera

The secretion of the skin glands of the ‘orange-thighed frog’ Litoria xanthomera contains seven peptides. One of these is the known hypotensive peptide caerulein. Two new peptides, caerin 1.6 [GLFSVLGAVAKHVLPHVVPVIAEKL(NH₂)], and caerin 1.7 [GLFKVLGSVAKHLLPHVAPVIAEKL(NH₂)] show antibacterial properties. Two other peptides lack the first two amino acid residues of caerins 1.6 and 1.7 and show no antibacterial activity. The identification of the peptides in Litoria xanthomera confirms that this species is related to Litoria caerula, Litoria gilleni and Litoria splendida but not as closely as those three species are related to each other. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Prediction of neuropeptide prohormone cleavages with application to RFamides

Genomic information is becoming available for an ever-wider range of animals with the genes for several well-characterized peptide families, such as the RFamides, detected in a surprisingly diverse set of these animals. While bioinformatic tools allow the prediction of the RFamide-related prohormones from genetic information, it is more difficult to accurately predict the final processed peptides because of the large number of processing steps required to convert a prohormone into mature bioactive peptides. Several statistical-based methods for predicting basic site cleavages in prohormones are described, and their ability to predict the basic site cleavages in a variety of RFamide-related peptides from vertebrates and invertebrates is reported. Specifically, the cleavages in the invertebrate FMRFamides, and the vertebrate NPFFa, RFRPa, and PrRPa peptide families are modeled. The three models compared here are based on known cleavage motifs, a logistic regression, and artificial neural networks. Improvements in the accuracy and precision of the cleavage estimates will lead to increased utilization of these models for predicting bioactive neuropeptides before experimental verification is available.     

Active site studies of human thrombin and bovine trypsin: peptide substrates.

Twenty-four oligopeptides modeled after the N-terminal portion of the α(A)-chain of human fibrinogen were synthesized and tested as substrates for human thrombin and bovine trypsin. The peptides contained either an Arg-Gly bond, or an Arg-Val bond, or both. Glycine and glutamic acid were substituted at various positions within the peptides, and from the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for each peptide with thrombin and with trypsin, the importance of residues on both sides of the Arg-Gly bond was evaluated. The trypsincatalyzed hydrolysis is faster than the thrombin-catalyzed hydrolysis of the peptides with glutamic acid at various distances N-terminal to the Arg-Gly bond. The ratio of the thrombic rate to the tryptic rate is fastest when glutamic acid is six residues away. Thrombin selectively cleaves the Arg-Gly bond in Ac-Val-Arg-Gly-Pro-Arg-Val-OH but cleaves both arginyl bonds rapidly in Ac-Val-Arg-Gly-Pro-Arg-Val-OMe. Trypsin selectively cleaves an Arg-Val bond in Ac-Arg-Gly-Pro-Arg-Val-OMe. The results are discussed in the light of thrombic cleavages of proteins. Most of these are seen to occur at highly polar sequences that frequently contain a proline residue.     

Immunosuppressive,antimicrobial and insecticidal activities of inhibitor cystine knot peptides produced by teratocytes of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae)

Teratocytes are specialized cells released by parasitoid wasps into their hosts. They are known for producing regulatory molecules that aid the development of immature parasitoids. We have recently reported the primary structures of cystine-rich peptides, including some containing inhibitor cystine knot (ICK) motifs, produced by teratocytes of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae). ICKs are known for their stability and diverse biological functions. In this study, we produced four putative ICK peptides from the teratocytes of C. flavipes using solid-phase peptide synthesis or recombinant expression in E. coli, and investigated their functions on host immune modulation as well their potential to impair the development of two lepidopterans after ingestion of the peptides. In addition, the peptides were assayed against pathogens and human cells. The peptides did not influence total hemocyte count but suppressed cellular immunity, detectable as a reduction of hemocyte encapsulation (CftICK-I, CftICK-II, CftICK-III) and spread indexes (CftICK-IV) in the host. None of the peptides influenced the activities of prophenoloxidase and phenoloxidase in the hemolymph of larval Diatraea saccharalis (Lepidoptera: Crambidae). CftICK-I and CftICK-II with previously unknown function showed antifungal activity against Candida albicans but were non-toxic to human cells. CftICK-I, CftICK-II, and CftICK-III increased larval mortality and reduced leaf consumption of D. saccharalis, a permissive host for C. flavipes. The CftICK-III also increased larval mortality and reduced leaf consumption of Spodoptera frugiperda (Lepidoptera: Noctuidae), a non-permissive host for C. flavipes. This study highlights biological functions and biotechnological potential of ICK peptides from the teratocytes of C. flavipes.     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

Voltage-gated sodium channels are targets for toxins from the venom of the spider Heriaeus melloteei

Three novel peptides were isolated from the venom of the spider Heriaeus melloteei (Thomisidae) and characterized. The peptides named Hm-1, 2 and 3 blocked voltage-gated Na⁺ channels at concentrations in the order of 100 nM. Activity of the purified peptides was investigated in Na⁺ channel isoforms of mammals and insects. Hm-1 and 2 appeared to act as pore blockers, whereas Hm-3 modulated the channel activation process. The toxins described exhibit minor similarity with other known peptides and may therefore constitute new groups of Na⁺ channel ligands.     

Folding thermodynamics of β‐hairpins studied by replica‐exchange molecular dynamics simulations

We study the differences in folding stability of β‐hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3‐1, and TrpZip4. By performing replica‐exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3‐1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β‐hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild‐type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state‐of‐the‐art all‐atom protein force fields. Proteins 2015; 83:1307–1315. © 2015 Wiley Periodicals, Inc.