Design and characterization of helical peptides that inhibit the E6 protein of papillomavirus

The E6 protein from HPV type 16 binds proteins containing a seven-residue leucine-containing motif. Previous work demonstrated that peptides containing the consensus sequence are a mixture of alpha-helix and unstructured conformations. To design monomeric E6-binding peptides that are stable in aqueous solution, we used a protein grafting approach where the critical residues of the E6-binding motif of E6-associated protein, E6AP, LQELLGE, were incorporated into exposed helices of two stably folded peptide scaffolds. One series was built using the third zinc finger of the Sp1 protein, which contains a C-terminal helix. A second series was built using a Trp-cage scaffold, which contains an N-terminal helix. The chimeric peptides had very different activities in out-competing the E6-E6AP interaction. We characterized the peptides by circular dichroism spectroscopy and determined high-resolution structures by NMR methods. The E6-binding consensus motif was found to be helical in the high-quality structures, which had backbone root-mean-square deviations of less than 0.4 A. We have successfully grafted the E6-binding motif into two parent peptides to create ligands that have biological activity while preserving the stable, native fold of their scaffolds. The data also indicate that conformational change is common in E6-binding proteins during the formation of the complex with the viral E6 protein.     

Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides

We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified approximately 60,000-fold from rat brain cytosol through use of a chromatography step based on the enzyme's ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced approximately 50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.     

The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.     

Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine enzyme.

The glutamate dehydrogenase from a single human liver has been studied. The subunit size was found to be 55,200 +/- 1,500 by sedimentation equilibrium. The partial specific volume is 0.732 as calculated from the amino acid composition. The sequence was determined by isolation of peptides after cyanogen bromide (CNBr) cleavage; the fraction containing the largest peptides was hydrolyzed by trypsin after maleylation. Studies on these peptides accounted for 454 residues of the 505 residues that are presumably present in the protein. For the 51 residues that were not represented in isolated peptides, we have tentatively assumed that the sequence is the same as that of the bovine enzyme. Methionine and arginine residues in these peptides could be placed on the basis of the specificity of cleavage by CNBr or trypsin. In all, 349 residues were placed in sequence, and were aligned by homology with the corresponding peptides of the bovine and chicken enzymes. From the present information, there are 24 known differences in sequence between the human and bovine enzymes and 41 between the human and chicken enzymes. In addition, the human enzyme contains 4 additional residues at the NH2 terminus as compared to the bovine enzyme. In a peptide from the human enzyme, an additional residue, isoleucine 385, was detected by automated Edman degradation. Reinvestigation of the bovine sequence demonstrated that this residue is also present in the bovine enzyme (and presumably in the chicken enzyme also). Residue 384 of the bovine enzyme, previously reported as Glx has now been shown to be glutamine.     

A Dual‐Purpose Bromocoumarin Tag Enables Deep Profiling of the Cellular Cysteinome

Chemical proteomics enables comprehensive profiling of small molecules in complex proteomes. A critical component to understand the interactome of a small molecule is the precise location on a protein where the interaction takes place. Several approaches have been developed that take advantage of bio‐orthogonal chemistry and subsequent enrichment steps to isolate peptides modified by small molecules. These methods rely on target identification at the level of mass spectrometry making it difficult to interpret an experiment when modified peptides are not identified. Herein, an approach in which fluorescence‐triggered two‐dimensional chromatography enables the isolation of small molecule‐conjugated peptides prior to mass spectrometry analysis is described. In this study, a bromocoumarin moiety has been utilized that fluoresces and generates a distinct isotopic signature to locate and identify modified peptides. Profiling of a cellular cysteinome with the use of a bromocoumarin tag demonstrates that two‐dimensional fluorescence‐based chromatography separation can enable the identification of proteins containing reactive cysteine residues. Moreover, the method facilitates the interrogation of low abundance proteins with greater depth and sensitivity than a previously reported isotope‐targeted approach. Lastly, this workflow enables the identification of small‐molecule modified peptides from a protein‐of‐interest.     

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.     

Heparin-binding peptides from the type I repeats of thrombospondin. Structural requirements for heparin binding and promotion of melanoma cell adhesion and chemotaxis.

Synthetic peptides derived from the type I repeats of human platelet thrombospondin containing a consensus sequence Trp-Ser-Xaa-Trp bind to heparin, promote cell adhesion, and inhibit heparin-dependent interactions of melanoma cells with extracellular matrix components (Guo, N. H., Krutzsch, H. C., Nègre, E., Vogel, T., Blake, D. A., and Roberts, D. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3040-3044). In the present study, we further examined the structural requirements for activity of these peptides. The minimal active sequence for heparin or sulfatide binding based on inhibition studies is Trp-Ser-Pro-Trp, although an octapeptide is required for optimal activity. The 2 Trp residues and the Ser residue are essential. Peptides with more than 2 residues between the Trp residues are inactive. The Pro residue is essential for activity of the pentapeptide Trp-Ser-Pro-Trp-Ser, but some larger peptides with substitutions for the Pro residue are active. For direct high affinity binding to heparin, both the consensus sequence and a flanking sequence of basic amino acids are essential. Peptides containing the consensus sequence promote cell adhesion and act cooperatively with the adjacent basic amino acid sequence to promote cell spreading. Chemical modification of the Trp residues in the peptides with amino-terminal basic amino acids abolished both cell adhesion and heparin-binding. Peptides containing the consensus sequence and basic amino acids are chemotactic for A2058 human melanoma cells. The functional importance of this novel heparin and sulfatide-binding motif is suggested by its conservation in other members of the thrombospondin gene family, complement components, and in many members of the cytokine receptor and transforming growth factor beta superfamilies.     

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F₂Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F₂Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F₂Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F₂Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.     

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.     

Re-evaluation of amino acid sequence and structural consensus rules for cysteine-nitric oxide reactivity

Nitric oxide (NO), produced in different cell types through the conversion of L-arginine into L-citrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NO-reactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acid-base catalysed Cys beta93-nitrosylation and Cys beta393NO-denitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites -1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for Cys-NO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the -2 position has been observed in some NO-reactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri- or tetra-peptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691-696]. Here, we analyse the three-dimensional structure of several proteins containing NO-reactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the Cys-Sy atomic structural microenvironment rather than on the tri- or tetra-peptide sequence consensus motif.     

Determination of the sequence specificity of XIAP BIR domains by screening a combinatorial peptide library

Inhibitor of apoptosis (IAP) proteins regulate programmed cell death by inhibiting members of the caspase family of proteases. The X-chromosome-linked IAP (XIAP) contains three baculovirus IAP repeat (BIR) domains, which bind directly to the N-termini of target proteins including those of caspases-3, -7, and -9. In the present study, we defined the consensus sequences of the motifs that interact with the three BIR domains in an unbiased manner. A combinatorial peptide library containing four random residues at the N-terminus was constructed and screened using BIR domains as probes. We found that the BIR3 domain binds a highly specific motif containing an alanine or valine at the N-terminus (P1 position), an arginine or proline at the P3 position, and a hydrophobic residue (Phe, Ile, and Tyr) at the P4 position. The BIR2-binding motif is less stringent. Although it still requires an N-terminal alanine, it tolerates a wide variety of amino acids at P2-P4 positions. The BIR1 failed to bind to any peptides in the library. SPR analysis of individually synthesized peptides confirmed the library screening results. Database searches with the BIR2- and BIR3-binding consensus sequences revealed a large number of potential target proteins. The combinatorial library method should be readily applicable to other BIR domains or other types of protein modular domains.     

Proteomic profile of reversible protein oxidation using PROP, purification of reversibly oxidized proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells.     

Identification of the major site of in vitro PKA phosphorylation in the polycystin-1 C-terminal cytosolic domain.

Sequence analysis of the C-terminal cytosolic domain of human and mouse polycystin-1 has identified three RxS consensus protein kinase A (PKA) phosphorylation motifs. GST-fusion proteins containing the full-length and truncated C-terminal cytosolic domain of murine polycystin-1 were phosphorylated in vitro by the purified catalytic subunit of PKA. This identified a sequence of 25 amino acids, immediately downstream of a previously identified heterotrimeric G-protein activation sequence, as the major site of PKA phosphorylation. Phosphorylation of wild-type and alanine substituted synthetic peptides containing this motif demonstrated that alanine substitution of serine 4159 largely eliminated phosphorylation. Mutation of this residue in the fusion protein reduced phosphorylation by about 70%, whereas mutation of the other two conserved phosphorylation motifs had little effect. We conclude that serine 4159 is the major site of PKA phosphorylation in the C-terminal cytosolic domain of murine polycystin-1.     

Acceleration of alpha-synuclein aggregation by homologous peptides

alpha-Synuclein (alpha-Syn), amyloid beta-protein and prion protein are among the amyloidogenic proteins that are associated with the neurodegenerative diseases. These three proteins share a homologous region with a consensus sequence mainly consisting of glycine, alanine and valine residues (accordingly named as the GAV motif), which was proposed to be the critical core for the fibrillization and cytotoxicity. To understand the role of the GAV motif in protein amyloidogenesis, we studied the effects of the homologous peptides corresponding to the sequence of GAV motif region (residues 66-74) on alpha-Syn aggregation. The result shows that these peptides can promote fibrillization of wild-type alpha-Syn and induce that of the charge-incorporated mutants but not the GAV-deficient alpha-Syn mutant. The acceleration of alpha-Syn aggregation by the homologous peptides is under a sequence-specific manner. The interplay between the GAV peptide and the core regions in alpha-Syn may accelerate the aggregation process and stabilize the fibrils. This finding provides clues for developing peptide mimics that could promote transforming the toxic oligomers or protofibrils into the inert mature fibrils.     

Quantitative analysis of human cerebrospinal fluid proteins using a combination of cysteine tagging and amine-reactive isobaric labeling

Highly complex and dynamic protein mixtures are hardly comprehensively resolved by direct shotgun proteomic analysis. As many proteins of biological interest are of low abundance, numerous analytical methodologies have been developed to reduce sample complexity and go deeper into proteomes. The present work describes an analytical strategy to perform cysteinyl-peptide subset enrichment and relative quantification through successive cysteine and amine-isobaric tagging. A cysteine-reactive covalent capture tag (C3T) allowed derivatization of cysteines and specific isolation on a covalent capture (CC) resin. The 6-plex amine-reactive tandem mass tags (TMT) served for relative quantification of the targeted peptides. The strategy was first evaluated on a model protein mixture with increasing concentrations to assess the specificity of the enrichment and the quantitative performances of the workflow. It was then applied to human cerebrospinal fluid (CSF) from post-mortem and ante-mortem samples. These studies confirmed the specificity of the C3T and the CC technique to cysteine-containing peptides. The model protein mixture analysis showed high precision and accuracy of the quantification with coefficients of variation and mean absolute errors of less than 10% on average. The CSF experiments demonstrated the potential of the strategy to study complex biological samples and identify differential brain-related proteins. In addition, the quantification data were highly correlated with a classical TMT experiment (i.e., without C3T cysteine-tagging and enrichment steps). Altogether, these results legitimate the use of this quantitative C3T strategy to enrich and relatively quantify cysteine-containing peptides in complex mixtures.     

Characterization of fertilin beta-disintegrin binding specificity in sperm-egg adhesion

An essential step leading to fertilization is the binding of a sperm to the egg plasma membrane. Fertilin beta, a membrane bound protein on the extracellular surface of sperm, partially mediates this binding via the alpha6beta1 integrin. Fertilin beta is a member of the still expanding family of ADAM proteins (a disintegrin and metalloprotease) that are implicated in many cellular functions ranging from neurogenesis to myoblast fusion and cytokine processing. Fertilin beta contains a highly conserved motif (D/E)ECD in the disintegrin domain. This suggests that (D/E)ECD could be the consensus sequence for recognition of disintegrins by alpha6beta1 integrins. Previously, it has been demonstrated that small peptides containing different moieties of this consensus sequence are inhibitors of in vitro fertilization. In the present study, we sought to determine whether a four amino acid peptide sequence with two adjacent acidic residues improved inhibition, and investigated the importance for inhibition of a cysteine versus a cystine. A series of linear and cyclic peptides were synthesized, in which either one or both adjacent acidic residues in the sequence DECD were mutated to their corresponding amides (N or Q). To explore the required oxidation state of the cysteine in the (D/E)ECD sequence, it was protected as a mixed disulfide. Our results indicate that only one acidic residue is required for inhibition of fertilization and a reduced C is required.     

The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.     

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.     

An antibody-free enrichment approach enabled by reductive glutaraldehydation for monomethyllysine proteome analysis

Protein lysine monomethylation is an important post-translational modification participated in regulating many biological processes. There is growing interest in identifying these methylation events. However, the introduction of one methyl group on lysine residues has negligible effect on changing the physical and chemical properties of proteins or peptides, making enriching and identifying monomethylated lysine (Kme1) proteins or peptides extraordinarily challenging. In this study, we proposed an antibody-free chemical proteomics approach to capture Kme1 peptides from complex protein digest. By exploiting reductive glutaraldehydation, 5-aldehyde-pentanyl modified Kme1 residues and piperidine modified primary amines were generated at the same time. The peptides with aldehyde modified Kme1 residues were then enriched by solid-phase hydrazide chemistry. This chemical proteomics approach was validated by using several synthetic peptides. It was demonstrated that it can enrich and detect Kme1 peptide from peptide mixture containing 5000-fold more bovine serum albumin tryptic digest. Besides, we extended our approach to profile Kme1 using heavy methyl stable isotope labeling by amino acids in cell culture (hmSILAC) labeled Jurkat T cells and Hela cells. Totally, 29 Kme1 sites on 25 proteins were identified with high confidence and 11 Kme1 sites were identified in both two types cells. This is the first antibody-free chemical proteomics approach to enrich Kme1 peptides from complex protein digest, and it provides a potential avenue for the analysis of methylome.