Targeting the tumor vasculature with engineered cystine-knot miniproteins.

The extra domain B splice variant (EDB) of human fibronectin selectively expressed in the tumor vasculature is an attractive target for cancer imaging and therapy. Here, we describe the generation and characterization of EDB-specific optical imaging probes. By screening combinatorial cystine-knot miniprotein libraries with phage display technology we discover exquisitely EDB-specific ligands that share a distinctive motif. Probes with a binding constant in the picomolar range are generated by chemical oligomerization of selected ligands and fluorophore conjugation. We show by fluorescence imaging that the probes stain EDB in tissue sections derived from human U-87 MG glioblastoma xenografts in mice. Moreover, we demonstrate selective accumulation and retention of intravenously administered probes in the tumor tissue of mice with U-87 MG glioblastoma xenografts by in vivo and ex vivo fluorescence imaging. These data warrants further pursuit of the selected cystine-knot miniproteins for in vivo imaging applications.

Weaponisation 'on the fly': Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies.

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the KV11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of KV11.1 channel activation to more depolarised potentials, but has no effect on KV1.3, KV2.1, KV10.1, KCa1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these KV channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

KNOTTIN: the database of inhibitor cystine knot scaffold after 10 years, toward a systematic structure modeling.

Knottins, or inhibitor cystine knots (ICKs), are ultra-stable miniproteins with multiple applications in drug design and medical imaging. These widespread and functionally diverse proteins are characterized by the presence of three interwoven disulfide bridges in their structure, which form a unique pseudoknot. Since 2004, the KNOTTIN database (www.dsimb.inserm.fr/KNOTTIN/) has been gathering standardized information about knottin sequences, structures, functions and evolution. The website also provides access to bibliographic data and to computational tools that have been specifically developed for ICKs. Here, we present a major upgrade of our database, both in terms of data content and user interface. In addition to the new features, this article describes how KNOTTIN has seen its size multiplied over the past ten years (since its last publication), notably with the recent inclusion of predicted ICKs structures. Finally, we report how our web resource has proved usefulness for the researchers working on ICKs, and how the new version of the KNOTTIN website will continue to serve this active community.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

The Whitefly Bemisia tabaci Knottin-1 Gene Is Implicated in Regulating the Quantity of Tomato Yellow Leaf Curl Virus Ingested and Transmitted by the Insect.

The whitefly Bemisia tabaci is a major pest to agricultural crops. It transmits begomoviruses, such as Tomato yellow leaf curl virus (TYLCV), in a circular, persistent fashion. Transcriptome analyses revealed that B. tabaci knottin genes were responsive to various stresses. Upon ingestion of tomato begomoviruses, two of the four knottin genes were upregulated, knot-1 (with the highest expression) and knot-3. In this study, we examined the involvement of B. tabaci knottin genes in relation to TYLCV circulative transmission. Knottins were silenced by feeding whiteflies with knottin dsRNA via detached tomato leaves. Large amounts of knot-1 transcripts were present in the abdomen of whiteflies, an obligatory transit site of begomoviruses in their circulative transmission pathway; knot-1 silencing significantly depleted the abdomen from knot-1 transcripts. Knot-1 silencing led to an increase in the amounts of TYLCV ingested by the insects and transmitted to tomato test plants by several orders of magnitude. This effect was not observed following knot-3 silencing. Hence, knot-1 plays a role in restricting the quantity of virions an insect may acquire and transmit. We suggest that knot-1 protects B. tabaci against deleterious effects caused by TYLCV by limiting the amount of virus associated with the whitefly vector.

The immune strategy and stress response of the Mediterranean species of the Bemisia tabaci complex to an orally delivered bacterial pathogen.

BACKGROUND: The whitefly, Bemisia tabaci, a notorious agricultural pest, has complex relationships with diverse microbes. The interactions of the whitefly with entomopathogens as well as its endosymbionts have received great attention, because of their potential importance in developing novel whitefly control technologies. To this end, a comprehensive understanding on the whitefly defense system is needed to further decipher those interactions. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a comprehensive investigation of the whitefly's defense responses to infection, via oral ingestion, of the pathogen, Pseudomonas aeruginosa, using RNA-seq technology. Compared to uninfected whiteflies, 6 and 24 hours post-infected whiteflies showed 1,348 and 1,888 differentially expressed genes, respectively. Functional analysis of the differentially expressed genes revealed that the mitogen associated protein kinase (MAPK) pathway was activated after P. aeruginosa infection. Three knottin-like antimicrobial peptide genes and several components of the humoral and cellular immune responses were also activated, indicating that key immune elements recognized in other insect species are also important for the response of B. tabaci to pathogens. Our data also suggest that intestinal stem cell mediated epithelium renewal might be an important component of the whitefly's defense against oral bacterial infection. In addition, we show stress responses to be an essential component of the defense system. CONCLUSIONS/SIGNIFICANCE: We identified for the first time the key immune-response elements utilized by B. tabaci against bacterial infection. This study provides a framework for future research into the complex interactions between whiteflies and microbes.

Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvß3, αvß5, and α5ß1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5ß1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.

Evolution of a horizontally acquired legume gene, albumin 1, in the parasitic plant Phelipanche aegyptiaca and related species.

BACKGROUND: Parasitic plants, represented by several thousand species of angiosperms, use modified structures known as haustoria to tap into photosynthetic host plants and extract nutrients and water. As a result of their direct plant-plant connections with their host plant, parasitic plants have special opportunities for horizontal gene transfer, the nonsexual transmission of genetic material across species boundaries. There is increasing evidence that parasitic plants have served as recipients and donors of horizontal gene transfer (HGT), but the long-term impacts of eukaryotic HGT in parasitic plants are largely unknown. RESULTS: Here we show that a gene encoding albumin 1 KNOTTIN-like protein, closely related to the albumin 1 genes only known from papilionoid legumes, where they serve dual roles as food storage and insect toxin, was found in Phelipanche aegyptiaca and related parasitic species of family Orobanchaceae, and was likely acquired by a Phelipanche ancestor via HGT from a legume host based on phylogenetic analyses. The KNOTTINs are well known for their unique "disulfide through disulfide knot" structure and have been extensively studied in various contexts, including drug design. Genomic sequences from nine related parasite species were obtained, and 3D protein structure simulation tests and evolutionary constraint analyses were performed. The parasite gene we identified here retains the intron structure, six highly conserved cysteine residues necessary to form a KNOTTIN protein, and displays levels of purifying selection like those seen in legumes. The albumin 1 xenogene has evolved through >150 speciation events over ca. 16 million years, forming a small family of differentially expressed genes that may confer novel functions in the parasites. Moreover, further data show that a distantly related parasitic plant, Cuscuta, obtained two copies of albumin 1 KNOTTIN-like genes from legumes through a separate HGT event, suggesting that legume KNOTTIN structures have been repeatedly co-opted by parasitic plants. CONCLUSIONS: The HGT-derived albumins in Phelipanche represent a novel example of how plants can acquire genes from other plants via HGT that then go on to duplicate, evolve, and retain the specialized features required to perform a unique host-derived function.

Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

Engineering knottins as novel binding agents.

Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.

Natural and engineered cystine knot miniproteins for diagnostic and therapeutic applications.

Cystine knot miniproteins define a class of peptides in the size range of approximately 28-35 amino acid residues that often combine high chemical and biological stability with high potency and selectivity. They share a common structural motif that is defined by three intramolecular disulfide bonds that gives rise to a very stable scaffold. Members of this family cover a broad spectrum of natural bioactivities ranging from antimicrobial and antiviral activities to selective blockage or activation of ion channels, cell surface receptors and extracelluar proteases. In recent years, the spectrum of natural bioactivities of this class of miniproteins was further expanded by application of protein design and directed evolution technologies. Miniproteins have been developed that inhibit platelet aggregation, block asthma-related proteases, act as growth factor mimics or address human tumor targets. Recent reports on miniproteins binding to cancer specific targets indicate that these biomolecules due to their particularly high in vivo stability, high target affinity, good tissue distribution, and fast body clearance are very promising agents that can be endowed with important beneficial features for imaging and therapeutic applications. With the first cystine-knot miniprotein already marketed as an analgesic, more candidates can be expected to find their way into the clinic for diagnostic and therapeutic applications over next years.

Structure and modeling of knottins, a promising molecular scaffold for drug discovery.

The knottins are extremely stable miniproteins present in many species and are able to perform various tasks. Owing to its small size and its amazing stability, the knottin structural domain is considered as an excellent scaffold for drug development. Several recent databases and web servers dedicated to or aware of knottins have appeared and are shortly described. Altogether they provide a valuable ensemble of data and of specific tools that greatly facilitate knottin-based studies. The essential structural features of the knottin scaffold, which heavily rest on the three knotted disulfide bridges for its stability, are reviewed. These include small but well-conserved secondary structures and hydrogen bonding networks, but also several further interactions that have been shown to be essential for stability and/or activity. Examples are supplementary disulfide bridges, side chain hydrogen bonds, or circularization. General structure prediction and modeling tools are not well fitted to knottins, and several specific tools have been developed. Specifically, methods to assign a disulfide connectivity pattern to small disulfide-rich sequences or to build accurate 3D models of knottins are available and are discussed in the review. Although more works are still needed to better understand sequence-structure-function relationships, recent studies strongly suggest that existing applications of knottins as drugs (i.e. painkillers), molecules for diagnosis, or insecticidal crop treatment should rapidly generalize and extend to other fields as well, e.g. as antimicrobials.