Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining.

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.

Cloning and Genomic Characterization of a Natural Insecticidal Peptide LaIT1 with Unique DDH Structural Fold.

Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Transcriptome analysis of venom glands from a single fishing spider Dolomedes mizhoanus.

The spider venom is a large pharmacological repertoire composed of different types of bioactive peptide toxins. Despite the importance of spider toxins in capturing terrestrial prey and defending themselves against predators, we know little about the venom components from the spider acting on the fish. Here we constructed a cDNA library of a pair of venomous glands from a single fish-hunting spider Dolomedes mizhoanus. A total of 356 high-quality expressed sequence tags (ESTs) were obtained from the venom gland cDNA library and analyzed. These transcripts were further classified into 45 clusters (19 contigs and 26 singletons), most of which encoded cystine knot toxins (CKTs) and non-CKTs. The ESTs coding for 53 novel CKT precursors were abundant transcripts in the venom glands of the spider D. mizhoanus, accounting for 76% of the total ESTs, the precursors of which were grouped into six families based on the sequence identity and the phylogenetic analysis. In addition, the non-CKTs deduced from 21% of the total ESTs were annotated by Gene Ontology terms and eukaryotic orthologous groups. Fifty-five CKT precursors deduced from 273 ESTs are the largest dataset for a single spider specimen to date. The results may contribute to discovering novel potential drug leads from spider venoms and a better understanding of the evolutionary relationship of the spider toxin.

CCN2/CTGF regulates neovessel formation via targeting structurally conserved cystine knot motifs in multiple angiogenic regulators.

Blood vessels are formed during development and tissue repair through a plethora of modifiers that coordinate efficient vessel assembly in various cellular settings. Here we used the yeast 2-hybrid approach and demonstrated a broad affinity of connective tissue growth factor (CCN2/CTGF) to C-terminal cystine knot motifs present in key angiogenic regulators Slit3, von Willebrand factor, platelet-derived growth factor-B, and VEGF-A. Biochemical characterization and histological analysis showed close association of CCN2/CTGF with these regulators in murine angiogenesis models: normal retinal development, oxygen-induced retinopathy (OIR), and Lewis lung carcinomas. CCN2/CTGF and Slit3 proteins worked in concert to promote in vitro angiogenesis and downstream Cdc42 activation. A fragment corresponding to the first three modules of CCN2/CTGF retained this broad binding ability and gained a dominant-negative function. Intravitreal injection of this mutant caused a significant reduction in vascular obliteration and retinal neovascularization vs. saline injection in the OIR model. Knocking down CCN2/CTGF expression by short-hairpin RNA or ectopic expression of this mutant greatly decreased tumorigenesis and angiogenesis. These results provided mechanistic insight into the angiogenic action of CCN2/CTGF and demonstrated the therapeutic potential of dominant-negative CCN2/CTGF mutants for antiangiogenesis.

Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery.

Cyclotides are a unique and growing family of backbone cyclized peptides that also contain a cystine knot motif built from six conserved cysteine residues. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Aside from the conserved residues forming the cystine knot, cyclotides have been shown to have high variability in their sequences. Consisting of over 160 known members, cyclotides have many biological activities, ranging from anti-HIV, antimicrobial, hemolytic, and uterotonic capabilities; additionally, some cyclotides have been shown to have cell penetrating properties. Originally discovered and isolated from plants, cyclotides can also be produced synthetically and recombinantly. The high sequence variability, stability, and cell penetrating properties of cyclotides make them potential scaffolds to be used to graft known active peptides or engineer peptide-based drug design. The present review reports recent findings in the biological diversity and therapeutic potential of natural and engineered cyclotides.

Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein.

BACKGROUND: The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops. METHODOLOGY/PRINCIPAL FINDINGS: Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α(v)ß(3) and α(v)ß(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)ß(1) and α(iib)ß(3). In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)ß(3) and α(v)ß(5) integrins. A (64)Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys. CONCLUSIONS/SIGNIFICANCE: We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.

Engineered cystine-knot miniproteins for diagnostic applications.

Owing to their extraordinary thermal and biological stability, cystine-knot miniproteins provide an attractive scaffold for the development of peptide-based diagnostics. One of the major advantages of this scaffold lies in the fact that the disulfide-constrained structural core can be functionalized by decoration with bioactive-loop residues. Methods have been developed to generate miniproteins with prescribed binding characteristics to a broad spectrum of different target proteins. They combine structural, biophysical and functional features that are beneficial for applications in molecular diagnostics in vivo (i.e., high affinity and selectivity, small size, high biological stability and fast renal clearance). Promising candidates for tumor imaging have been generated recently and evaluated in animal models, and more applications are expected in the near future.

Wnt modulators in the biotech pipeline.

The purpose of this review is to provide a better understanding for the LRP co-receptor-mediated Wnt pathway signaling. Using proteomics, we have also subdivided the LRP receptor family into six sub-families, encompassing the twelve family members. This review includes a discussion of proteins containing a cystine-knot protein motif (i.e., Sclerostin, Dan, Sostdc1, Vwf, Norrin, Pdgf, Mucin) and discusses how this motif plays a role in mediating Wnt signaling through interactions with LRP.

Knottin cyclization: impact on structure and dynamics.

BACKGROUND: Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack. RESULTS: In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding. CONCLUSION: In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.

The Bmp pathway in skull vault development.

The Bmp pathway is of critical importance in the development of the skull vault. Analysis of gain and loss of function phenotypes of Bmp pathway effectors, particularly Msx genes, has shown that the Bmp pathway functions in the growth of both mesodermal and neural crest-derived calvarial bones. It is required for the development of the frontal and parietal bones during the interval between the initial osteogenic mesenchymal condensations at E12.5 to the apposition of the paired frontal and parietal bones at E18.5. During postnatal development, forced expression of the Bmp inhibitor, noggin, maintains the patency of sutures, consistent with a role for the Bmp pathway in regulating suture development. The availability of conditional mutants of Bmp ligands, receptors and downstream effectors will make possible an increasingly high resolution analysis of precisely how the Bmp functions in these processes and how aberrations in its activity can contribute to pathological conditions such as familial parietal foramina and craniosynostosis.

Evolutionary conservation of bursicon in the animal kingdom.

Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species.