Peptide delivery to tissues via reversibly linked protein transduction sequences

The development of peptide-based therapeutics has suffered from challenges associated with delivery to intact tissue. In skin, an array of protein targets resides only tens of micrometers below the surface; however, because of difficulties in traversing the cutaneous barrier, the potentialfor peptide-based therapeutics remains unrealized. We have developed a general approach for topical peptide delivery into skin using releasable protein transduction sequences to enable peptide transport across tissue boundaries. Upon entry into the cell, the disulfide linkage between the peptide transduction sequences and peptide cargo is cleaved, permitting the dissociation of the highly charged peptide transduction sequences from the active peptide. A protype cargo peptide, the hemagglutinin (HA) epitope, was conjugated to a hepta-arginine protein transduction sequence via a releasable disulfide linkage. This construct penetrated the skin to deep dermis within 1 h after topical application. Consistent with the dissociation of the protein transduction and cargo sequences, absorbed protein transduction sequences and HA peptides displayed differential intracellular localization. Reversible protein transduction sequence linkage thus represents a noninvasive platform for tissue delivery of intact peptides with no requirement for viral vectors or parenteral injection and may be of broad utility in molecular therapy.     

Virtual interaction profiles of proteins.

We have developed a new method for the prediction of peptide sequences that bind to a protein, given a three-dimensional structure of the protein in complex with a peptide. By applying a recently developed sequence prediction algorithm and a novel ensemble averaging calculation, we generate a diverse collection of peptide sequences that are predicted to have significant affinity for the protein. Using output from the simulations, we create position-specific scoring matrices, or virtual interaction profiles (VIPs). Comparison of VIPs for a collection of binding motifs to sequences determined experimentally indicates that the prediction algorithm is accurate and applicable to a diverse range of structures. With these VIPs, one can scan protein sequence databases rapidly to seek binding partners of potential biological significance. Overall, this method can significantly enhance the information contained within a protein- peptide crystal structure, and enrich the data obtained by experimental selection methods such as phage display.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Sequence requirements for synthetic peptide-mediated translocation to the nucleus.

The abilities of 18 synthetic peptides to target a carrier protein to the nucleus following microinjection into the cytoplasm of HeLa cells were determined. Eight of the sequences chosen for synthesis were based on published nuclear targeting regions as determined by gene fusion and deletion experiments. Six of these sequences were found to be effective when mimicked by a synthetic peptide and conjugated to a carrier protein. One additional peptide was based on a region of lamin L1, a nuclear protein from Xenopus laevis, in which the nuclear targeting region had not been previously investigated. This peptide was also able to target a carrier protein to the nucleus. Eight other peptides which resemble the known targeting signals had little or no nuclear targeting ability. Peptides which were able to target a carrier protein to the nucleus did so within 45 min of injection into the cytoplasm. Two peptides with little or no apparent nuclear targeting ability after 45 min were examined for longer times as well. No increase in nuclear accumulation was observed between 45 min and 4 h after cytoplasmic injection. Comparison of the sequences which were effective at nuclear targeting with those that were not revealed a possible consensus sequence for peptide-mediated nuclear transport.     

The reconstruction of a fragment of primordial genetic information from modern proteins: PGI-FI (AHAP).

In a continuation of earlier studies the connection between a reconstructed ancestral histone IV peptide and various sequences from non-histone proteins was investigated. This peptide, AHAP, was found to be related to partial peptides from the human encephalitogenic protein, immunoglobulin L-chains, fibrinopeptide A, glyceraldehyde-3-phosphate-dehydrogenase, TMV coat protein and several other proteins and protein families; for comparison, nodal sequences were employed wherever possible. The widespread occurrence of genetic information expressed in AHAP caused us to rename the peptide PGI-FI (primordial genetic information-fragment 1) since this peptide is apparently part of a very primitive and ancient genetic information, and may be called "protogene".     

BioPD: a web-based information center for bioactive peptides

Bioactive peptide database (BioPD) is a web-based knowledge base that contains more than 1100 protein sequences from human, mouse and rat, which are putative or are known to be bioactive peptides. In addition to peptide sequences and the annotation, the database also contains gene sequences with annotation, protein interaction and disease data related to the peptides. Each entry has as many references as possible to support the information represented. BioPD consists of six parts: PROTEIN, GENE, DISEASE, LINKS, INTERACTION, and REFERENCE. The database is searchable through keyword, gene and protein name, receptor name, etc. The links to PDB, InterPro, Pfam, OMIM, etc. are provided in each entry. Thus BioPD is formed as an information center for the bioactive peptide and serves as a gateway for exploration of bioactive peptides. The database can be accessed at http://biopd.bjmu.edu.cn.     

Getting more from less: algorithms for rapid protein identification with multiple short peptide sequences

We describe two novel sequence similarity search algorithms, FASTS and FASTF, that use multiple short peptide sequences to identify homologous sequences in protein or DNA databases. FASTS searches with peptide sequences of unknown order, as obtained by mass spectrometry-based sequencing, evaluating all possible arrangements of the peptides. FASTF searches with mixed peptide sequences, as generated by Edman sequencing of unseparated mixtures of peptides. FASTF deconvolutes the mixture, using a greedy heuristic that allows rapid identification of high scoring alignments while reducing the total number of explored alternatives. Both algorithms use the heuristic FASTA comparison strategy to accelerate the search but use alignment probability, rather than similarity score, as the criterion for alignment optimality. Statistical estimates are calculated using an empirical correction to a theoretical probability. These calculated estimates were accurate within a factor of 10 for FASTS and 1000 for FASTF on our test dataset. FASTS requires only 15-20 total residues in three or four peptides to robustly identify homologues sharing 50% or greater protein sequence identity. FASTF requires about 25% more sequence data than FASTS for equivalent sensitivity, but additional sequence data are usually available from mixed Edman experiments. Thus, both algorithms can identify homologues that diverged 100 to 500 million years ago, allowing proteomic identification from organisms whose genomes have not been sequenced.     

pepgrep: A Tool for Peptide MS/MS Pattern Matching

Typically, detection of protein sequences in collision-induced dissociation (CID) tandem MS (MS2) dataset is performed by mapping identified peptide ions back to protein sequence by using the protein database search (PDS) engine. Finding a particular peptide sequence of interest in CID MS2 records very often requires manual evaluation of the spectrum, regardless of whether the peptide-associated MS2 scan is identified by PDS algorithm or not. We have developed a compact cross-platform database-free command-line utility, pepgrep, which helps to find an MS2 fingerprint for a selected peptide sequence by pattern-matching of modelled MS2 data using Peptide-to-MS2 scoring algorithm. pepgrep can incorporate dozens of mass offsets corresponding to a variety of post-translational modifications (PTMs) into the algorithm. Decoy peptide sequences are used with the tested peptide sequence to reduce false-positive results. The engine is capable of screening an MS2 data file at a high rate when using a cluster computing environment. The matched MS2 spectrum can be displayed by using built-in graphical application programming interface (API) or optionally recorded to file. Using this algorithm, we were able to find extra peptide sequences in studied CID spectra that were missed by PDS identification. Also we found pepgrep especially useful for examining a CID of small fractions of peptides resulting from, for example, affinity purification techniques. The peptide sequences in such samples are less likely to be positively identified by using routine protein-centric algorithm implemented in PDS. The software is freely available at http://bsproteomics.essex.ac.uk:8080/data/download/pepgrep-1.4.tgz.     

Novel Zn(2+)-chelating peptides selected from a fimbria-displayed random peptide library.

The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the FimH adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of approximately 40 million individual clones, was screened for peptide sequences that conferred on recombinant cells the ability to bind Zn(2+). By serial selection, sequences that exhibited various degrees of binding affinity and specificity toward Zn(2+) were enriched. None of the isolated sequences showed similarity to known Zn(2+)-binding proteins, indicating that completely novel Zn(2+)-binding peptide sequences had been isolated. By changing the protein scaffold system, we demonstrated that the Zn(2+)-binding seems to be uniquely mediated by the peptide insert and to be independent of the sequence of the carrier protein. These findings might be applied in the design of biomatrices for bioremediation purposes or in the development of sensors for detection of heavy metals.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

How antigenic are antigenic peptides?

Most of a protein surface is potentially antigenic, consisting of numerous overlapping domains each complementary to antibody-combining sites. These domains may include peptide sequences that are demonstrably antigenic but only when antibodies from the appropriate host individuals and species are used. Methods for locating antigenic peptide sequences are described in which hydrophilic polyamide supports are used for peptide synthesis, then solid-phase radioimmunoassay with antisera and protein A. Most antigenic domains, however, comprise amino acid side chains contributed by two or more nearby polypeptide chains. Such domains can be identified by comparing the cross-reactivities of groups of very closely related proteins towards monoclonal antibodies raised to one of them. Such studies, using myoglobins, have identified a number of residues not previously shown to be antigenic and have provided a guide for the choice of synthetic peptides which are likely to carry several immunodominant side chains. One such peptide corresponding to residues (72–89) of beef myoglobin has been shown, using CD and antibodies to the parent protein, to have interesting conformational and antigenic properties. The peptide (25–55) is also antigenic.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Proteome analysis of the leukocytes from the American alligator (Alligator mississippiensis) using mass spectrometry

Mass spectrometry was used in conjunction with gel electrophoresis and liquid chromatography, to determine peptide sequences from American alligator (Alligator mississippiensis) leukocytes and to identify similar proteins based on homology. The goal of the study was to generate an initial database of proteins related to the alligator immune system. We have adopted a typical proteomics approach for this study. Proteins from leukocyte extracts were separated using two-dimensional gel electrophoresis and the major bands were excised, digested and analyzed by on-line nano-LC MS/MS to generate peptide sequences. The sequences generated were used to identify proteins and characterize their functions. The protein identity and characterization of the protein function were based on matching two or more peptides to the same protein by searching against the NCBI database using MASCOT and Basic Local Alignment Search Tool (BLAST). For those proteins with only one peptide matching, the phylum of the matched protein was considered. Forty-three proteins were identified that exhibit sequence similarities to proteins from other vertebrates. Proteins related to the cytoskeletal system were the most abundant proteins identified. These proteins are known to regulate cell mobility and phagocytosis. Several other peptides were matched to proteins that potentially have immune-related function.     

P-Mod: an algorithm and software to map modifications to peptide sequences using tandem MS data

The discovery of unanticipated protein modifications is one of the most challenging problems in proteomics. Whereas widely used algorithms such as Sequest and Mascot enable mapping of modifications when the mass and amino acid specificity are known, unexpected modifications cannot be identified with these tools. We have developed an algorithm and software called P-Mod, which enables discovery and sequence mapping of modifications to target proteins known to be represented in the analysis or identified by Sequest. P-Mod matches MS/MS spectra to peptide sequences in a search list. For spectra of modified peptides, P-Mod calculates mass differences between search peptide sequences and MS/MS precursors and localizes the mass shift to a sequence position in the peptide. Because modifications are detected as mass shifts, P-Mod does not require the user to guess at masses or sequence locations of modifications. P-Mod uses extreme value statistics to assign p value estimates to sequence-to-spectrum matches. The reported p values are scaled to account for the number of comparisons, so that error rates do not increase with the expanded search lists that result from incorporating potential peptide modifications. Combination of P-Mod searches from multiple LC-MS/MS analyses and multiple samples revealed previously unreported BSA modifications, including a novel decarboxymethylation or D-->G substitution at position 579 of the protein. P-Mod can serve a unique role in the identification of protein modifications both from exogenous and endogenous sources and may be useful for identifying modified protein forms as biomarkers for toxicity and disease processes.     

Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations.

Synthetic peptides corresponding to sequences in the human liver gap junction protein were chemically synthesized and used for generation of peptide antisera to defined sequences in the protein. The antibodies were affinity purified and characterized by demonstrating that they specifically recognized both their corresponding synthetic peptide (as indicated by dot blot analysis) and the native 32-kd gap junction protein (by immunoblotting). The specificity of a subset of the different site-specific antibodies was subsequently confirmed by demonstration of their binding to specific gap junction fragments produced by treatment with a lysine-specific endoproteinase. Immunoelectron microscopy was used to localize the specific peptide antibody epitopes to either the cytoplasmic or extracellular surfaces of the gap junction. Results indicate a transmembrane orientation for the protein with the amino and carboxyl termini located on the cytoplasmic side of the membrane. Based on these data, a model is proposed for the transmembrane folding of the gap junction protein.     

Identification of structured peptide segments in folding proteins.

Prediction of the structures of long polypeptides and small proteins has been carried out using conformational energy calculations. These calculations can be applied to large proteins if structured regions of their sequences can be identified. Three different approaches to identifying such sequences are presented. First, sequences of five or more contiguous hydrophobic residues tend to nucleate alpha-helices. Second, peptide sequences from parent proteins that have the same biological activities as the parent proteins are highly structured. Third, structured synthetic peptide segments from proteins inhibit the folding of the parent proteins by competing with the corresponding segment of the protein chain for associating with complementary regions. Examples of each of these approaches are presented.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.