The structural conformation of the tachykinin domain drives the anti-tumoural activity of an octopus peptide in melanoma BRAFV600E.

BACKGROUND AND PURPOSE: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma. EXPERIMENTAL APPROACH: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). KEY RESULTS: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. CONCLUSION AND IMPLICATIONS: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo.

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

ERK and mTORC1 Inhibitors Enhance the Anti-Cancer Capacity of the Octpep-1 Venom-Derived Peptide in Melanoma BRAF(V600E) Mutations.

Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna-derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.

Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising therapeutic against breast cancer.

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

Helminth Immunomodulation in Autoimmune Disease.

Helminths have evolved to become experts at subverting immune surveillance. Through potent and persistent immune tempering, helminths can remain undetected in human tissues for decades. Redirecting the immunomodulating "talents" of helminths to treat inflammatory human diseases is receiving intensive interest. Here, we review therapies using live parasitic worms, worm secretions, and worm-derived synthetic molecules to treat autoimmune disease. We review helminth therapy in both mouse models and clinical trials and discuss what is known on mechanisms of action. We also highlight current progress in characterizing promising new immunomodulatory molecules found in excretory/secretory products of helminths and their potential use as immunotherapies for acute and chronic inflammatory diseases.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Distinct miRNA signatures associate with subtypes of cholangiocarcinoma from infection with the tumourigenic liver fluke Opisthorchis viverrini.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a significant public health problem in East Asia, where it is strongly associated with chronic infection by the food-borne parasite Opisthorchis viverrini (OV). We report the first comprehensive miRNA expression profiling by microarray of the most common histologic grades and subtypes of ICC: well differentiated, moderately differentiated, and papillary ICC. METHODS: MicroRNA expression profiles from FFPE were compared among the following: ICC tumour tissue (n = 16), non-tumour tissue distally macrodissected from the same ICC tumour block (n = 15), and normal tissue (n = 13) from individuals undergoing gastric bypass surgery. A panel of deregulated miRNAs was validated by qPCR. RESULTS: Each histologic grade and subtype of ICC displayed a distinct miRNA profile, with no cohort of miRNAs emerging as commonly deregulated. Moderately differentiated ICC showed the greatest miRNA deregulation in quantity and magnitude, followed by the papillary subtype, and then well differentiated ICC. Moreover, when ICC tumour tissues were compared to adjacent non-tumour tissue, similar miRNA dysregulation profiles were observed. CONCLUSIONS: We show that common histologic grades and subtypes of ICC have distinct miRNA profiles. As histological grade and subtypes are associated with ICC aggressiveness, these profiles could be used to enhance the early detection and improve the personalised treatment for ICC. These findings also suggest the involvement of specific miRNAs during ICC tumour progression and differentiation. We plan to use these insights to (a) detect these profiles in circulation and (b) conduct functional analyses to decipher the roles of miRNAs in ICC tumour differentiation.

Circumventing qPCR inhibition to amplify miRNAs in plasma.

BACKGROUND: Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance. FINDINGS: Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures. CONCLUSION: Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.

Proteomic and genomic analyses suggest the association of apolipoprotein C1 with abdominal aortic aneurysm.

PURPOSE: Abdominal aortic aneurysm (AAA) is an important cause of mortality in the elderly. Mouse models are widely used to investigate AAA pathogenesis but their suitability for biomarker discovery is unexplored. EXPERIMENTAL DESIGN: We conducted a three-phase study. Phase 1: Aortas from angiotensin-II-infused apolipoprotein E deficient (ApoE(-/-) ) mice with and without AAA were assessed via iTRAQ and analyzed in silico to identify potential circulating markers. Microarray data from ApoE(-/-) mice and human patients were analyzed in parallel. Phase 2: Putative markers were compared between datasets to shortlist common candidates. Phase 3: The relationship of two shortlisted markers and AAA presence was assessed. RESULTS: iTRAQ identified eight proteins with biomarker potential. Microarray data identified 72 and 96 potential biomarkers from ApoE(-/-) mice and human patients, respectively. All three datasets suggested apolipoprotein C1 (ApoC1) as a marker for AAA; microarray data identified matrix metalloproteinase 9 (MMP9) as a second potential marker. Plasma ApoC1 and MMP9 concentrations positively correlated with AAA diameter in ApoE(-/-) mice. CONCLUSIONS AND CLINICAL RELEVANCE: ApoC1 may be a novel biomarker for AAA.

Methods and matrices: approaches to identifying miRNAs for nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC. METHODS: We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set. RESULTS: Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating "unknown" miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC. CONCLUSIONS: We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.

Semienzymatic cyclization of disulfide-rich peptides using Sortase A.

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

The miRNAome of Opisthorchis viverrini induced intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer, arising in the biliary ducts that extend into the liver. The highest incidence of ICC occurs in Southeast Asia, particularly in the Mekong River Basin countries of Thailand, Laos, Cambodia, and Vietnam, where it is strongly associated with chronic infection by the food-borne liver fluke Opisthorchis viverrini (OV), one of only three eukaryote pathogens considered Group one carcinogens. Intrahepatic cholangiocarcinoma is usually diagnosed at an advanced stage, with a poor prognosis and survival often less than 24 months. Hence, biomarkers that enable the early detection of ICC would be desirable and have a potentially important impact on the public health in the resource-poor regions where this cancer is most prevalent. As microRNAs (miRNAs) remain well preserved after formalin fixation, there is much interest in developing them as biomarkers that can be investigated using tumor biopsy samples preserved in formalin fixed paraffin embedded (FFPE) tumor blocks. Recently, we reported the first comprehensive profiling of tissue-based miRNA expression using FFPE from the three most common subtypes of OV-induced ICC tumors: moderately differentiated ICC, papillary ICC, and well-differentiated ICC. We observed that each subtype of OV-induced ICC exhibited a distinct miRNA profile, which suggested the involvement of specific sets of miRNAs in the progression of this cancer. In addition, non-tumor tissue adjacent to ICC tumor tissue on the same FFPE block shared a similar miRNA dysregulation profile with the tumor tissue than with normal (non-tumor) liver tissue (individuals without ICC or OV infection). Herein, we provide a detailed description of the microarray analysis procedures used to derive these findings.

Profiling miRNAs in nasopharyngeal carcinoma FFPE tissue by microarray and Next Generation Sequencing.

Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous-cell carcinoma that occurs in the epithelial lining of the nasopharynx. Nasopharyngeal carcinoma has a geographically well-defined distribution worldwide, with the highest prevalence in China, Southeast Asia, and Northern Africa. Symptoms of nascent NPC may be unapparent or trivial, with diagnosis based on the histopathology of biopsied tissue following endoscopy of the nasopharynx. The tumor node metastasis (TNM) staging system is the benchmark for the prognosis of NPC and guides treatment strategy. However, there is a consensus that the TNM system is not sufficiently specific for the prognosis of NPC, as it does not reflect the biological heterogeneity of this tumor, making another biomarker for the detection of NPC a priority. We have previously reported on different approaches for microRNA (miRNA) biomarker discovery for Formalin Fixed Paraffin Embedded (FFPE) NPC tissue samples by both a targeted (microarray) and an untargeted (small RNA-Seq) discovery platform. Both miRNA discovery platforms produced similar results, narrowing the miRNA signature to 1-5% of the known mature human miRNAs, with untargeted (small RNA-Seq approach) having the advantage of indicating "unknown" miRNAs associated with NPC. Both miRNA profiles strongly associated with NPC, providing two potential discovery platforms for biomarker signatures for NPC. Herein, we provide a detailed description of the methods that we used to interrogate FFPE samples to discover biomarkers for NPC.

Infection with the carcinogenic liver fluke Opisthorchis viverrini modifies intestinal and biliary microbiome.

Opisthorchis viverrini is a fish-borne trematode endemic in East Asia. Following ingestion, the flukes locate to the biliary tre where chronic infection frequently leads to cholangiocarcinoma (CCA). The mechanisms by which O. viverrini infection culminates in CCA remain unknown. An unexplored aspect is its influence on the host microbiome. In the hamster, infection with this pathogen reliably leads to CCA. Genomic DNAs of microbiota from colorectal contents and bile of hamsters and from whole O. viverrini were examined in this model of fluke-induced CCA. Microbial communities were characterized by high-throughput sequencing of variable regions 7-9 of prokaryotic 16S ribosomal DNA. Of ∼1 million sequences, 536,009 with useable reads were assignable to 29,776 operational taxonomy units (OTUs) and, in turn, to 20 phyla and 273 genera of Bacteria or Archaea. Microbial community analyses revealed that fluke infection perturbed the gastrointestinal tract microbiome, increasing Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae, while decreasing Porphyromonadaceae, Erysipelotrichaceae, and Eubacteriaceae (P≤0.05). More than 60 OTUs were detected in the biliary system, which confirmed bacteriobilia and a noteworthy community of microbes associated with the parasites. The fluke-associated microorganisms included potential pathogens from the Enterobacteriaceae and Listeriaceae and others, including Cyanobacteria and Deinococci, usually found in external environments. Given that opisthorchiasis is distinguished from other helminth infections by a robust inflammatory phenotype with conspicuously elevated IL-6, and that inflammation of the biliary system leads to periductal fibrosis, which is a precursor of CCA, the flukes and their microbiota may together drive this distinctive immune response.

Expression, refolding and purification of Ov-GRN-1, a granulin-like growth factor from the carcinogenic liver fluke, that causes proliferation of mammalian host cells.

Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver fluke of humans, Opisthorchis viverrini, and showed that recombinant Ov-GRN-1 expressed and refolded from Escherichia coli caused proliferation of mammalian cell lines at nanomolar concentrations. We now report on an optimized method to express and purify monomeric Ov-GRN-1 in E. coli using a straightforward and scalable purification and refolding process. Purified monomeric protein caused proliferation at nanomolar concentrations of cancerous and non-cancerous cell lines derived from human bile duct tissue. The expression and purification method we describe herein will serve as a backbone upon which to develop expression and purification processes for recombinant GRNs from other organisms, accelerating research on this intriguing family of proteins.

Vaccinomics for the major blood feeding helminths of humans.

Approximately one billion people are infected with hookworms and/or blood flukes (schistosomes) in developing countries. These two parasites are responsible for more disability adjusted life years lost than most other neglected tropical diseases (NTDs), and together, are second only to malaria. Although anthelmintic drugs are effective and widely available, they do not protect against reinfection, resistant parasites are likely to emerge, and mass drug administration programs are unsustainable. Therefore, there is a pressing need for the development of vaccines against these parasites. In recent years, there have been major advances in our understanding of hookworms and schistosomes at the molecular level through the use of "omics" technologies. The secretomes of these parasites have been characterized using transcriptomics, genomics, proteomics, and newly developed gene manipulation and silencing techniques, and the proteins of interest are now the target of novel antigen discovery approaches, notably immunomics. This research has resulted in the discovery, development, and early stage clinical trials of subunit vaccines against hookworms and schistosomes.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley (Hordeum vulgare) have tissue-specific expression patterns.

CyBase: a database of cyclic protein sequence and structure.

CyBase is a curated database and information source for backbone-cyclized proteins. The database incorporates naturally occurring cyclic proteins as well as synthetic derivatives, grafted analogues and acyclic permutants. The database provides a centralized repository of information on all aspects of cyclic protein biology and addresses issues pertaining to the management and searching of topologically circular sequences. The database is freely available at http://research.imb.uq.edu.au/cybase.

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1.

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A.

Cyclotides are a family of plant proteins that have the unusual combination of head-to-tail backbone cyclization and a cystine knot motif. They are exceptionally stable and show resistance to most chemical, physical, and enzymatic treatments. The structure of tricyclon A, a previously unreported cyclotide, is described here. In this structure, a loop that is disordered in other cyclotides forms a beta sheet that protrudes from the globular core. This study indicates that the cyclotide fold is amenable to the introduction of a range of structural elements without affecting the cystine knot core of the protein, which is essential for the stability of the cyclotides. Tricyclon A does not possess a hydrophobic patch, typical of other cyclotides, and has minimal hemolytic activity, making it suitable for pharmaceutical applications. The 22 kDa precursor protein of tricyclon A was identified and provides clues to the processing of these fascinating miniproteins.