Pinpointing disulfide connectivities in cysteine-rich proteins

Bernardino, Kalil; Pinto, Mauro Sérgio Vianello; Bolzani, Vanderlan S.; Moura, André Farias de; Batista, João M.

doi:10.1039/c7cc02333b

Cited by 3 publications

(8 citation statements)

References 29 publications

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“…Figure reproduced with permission from Čemažar et al 105 Most experimental work on cyclotide folding kinetics was done in early studies, [38][39][40]60,84,[103][104][105][106] and it is only recently that theoretical methods have been applied to cyclotide folding. 365 That study used thermodynamic integration simulations to examine different cystine knot connectivities of cycloviolacin O4 and identified three disulfide pairings with the lowest energy (in order of formation): (1) III-IV, II-V, I-VI; (2) III-V, II-VI, I-IV; and (3) I-IV, II-III, V-VI. 365 However, none of these disulfide pairings correspond to the native form (in no implied order of formation): I-IV, II-V, III-VI.…”

Section: Disulfide Connectivity and Oxidative Foldingmentioning

confidence: 99%

“…365 That study used thermodynamic integration simulations to examine different cystine knot connectivities of cycloviolacin O4 and identified three disulfide pairings with the lowest energy (in order of formation): (1) III-IV, II-V, I-VI; (2) III-V, II-VI, I-IV; and (3) I-IV, II-III, V-VI. 365 However, none of these disulfide pairings correspond to the native form (in no implied order of formation): I-IV, II-V, III-VI. 9 Separate analyses focused on the native disulfide connectivity indicated two possible orders of formation: I-IV, II-V, III-VI and II-V, I-IV, III-VI, suggesting that cycloviolacin O4 could fold via one or both of these paths.…”

Section: Disulfide Connectivity and Oxidative Foldingmentioning

confidence: 99%

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Cyclotides: From Structure to Function

2019

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Early studies of cyclotides [7][8][9][13][14][15] were based on discovery and characterization efforts at the peptide level. The general approach for these studies involved either bioassay-guided or mass-guided fractionation of plant extracts, followed by peptide isolation and purification, amino acid analysis, Edman sequencing and chemical, NMR 9 or mass spectrometry-based 10 approaches to deduce disulfide connectivity. The first step in many of the early discovery programs was a pre-screening procedure to determine if a given plant tissue might contain cyclotides (Figure 3). Various protocols 13,15,57,69,116,123 have been reported for quickly screening if a plant might be cyclotide-positive, with the simplest based on extraction (using dichloromethane/methanol, aqueous acetonitrile, or aqueous buffer) and purification on a C18 reverse phase column followed by a main-screen. The latter typically involves (A) examination of a reversed phase HPLC-MS profile for late-eluting peaks that are (B) in the mass range 2800-4000 Da and (C) contain six Cys residues as judged by a mass shift of 348 Da after reduction and S-alkylation, as shown in Figure 3. 116 The rationale for the target HPLC elution range was that all of the cyclotides discovered in early studies were somewhat hydrophobic and eluted relatively late on HPLC. Similarly, many of the initial cyclotides were in the mass range 2500-4000 Da and it was assumed that new cyclotides would fall within a similar range. Finally, by definition, a cyclotide must contain six Cys residues so the mass shift test in step C of the screen provided a simple way of testing for that criterion. An example of the 348 Da mass shift after reduction and alkylation is illustrated for the identification of the cyclotide Cter A from Clitoria ternatea (butterfly pea) seeds in Figure 3. Chassalia chartacea chassatides 235 Chassalia curviflora chacur c Chassalia discolor CD-1 c Chassalia parvifolia circulins 7,22,406 Hedyotis biflora hedyotides 207,213,280,350 H. centranthoides hcf-1 c H. terminalis htf-1 c Oldenlandia affinis kalata peptides 1-5,9 Palicourea condensata palicoureins 25 P. rigida parigidin-br1 236,329,345 P. tetragona paltet 1 c Psychotria brachiata psybra 1 c Psychotria brachyceras Psyleio and psybry peptides c Psychotria leiocarpa Psyleio peptides 338 Psychotria leptothyrsa psyle peptides 157,158 Psychotria longipes cyclopsychotride A 8 Psychotria poeppigiana psypoe 1 Psychotria solitudinum psysol 2 305 Psychotria suterella PS-1 c Cucurbitaceae Momordica clarkeana TI peptides 315 These trypsin inhibitor cyclotides are also referred to as cyclic knottins; typically, these peptides are found only in seeds. M. cochinchinensis MCoTI peptides 24 M. denticulate TI peptides 315 M. diocia Modi peptides 424 M. gilgiana TI peptides 315 M. macrophylla TI peptides 315 M. subangulata TI peptides 315 Fabaceae Clitoria ternatea Cter peptides; 204 cliotides 206 These cyclotides are biosynthetically processed from 'hijacked' genes (i.e. from albumin genes in which an ancestral albumin subun...

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Section: Disulfide Connectivity and Oxidative Foldingmentioning

confidence: 99%

Section: Disulfide Connectivity and Oxidative Foldingmentioning

confidence: 99%

Cyclotides: From Structure to Function

2019

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“…Although Brazil has the largest biodiversity in the world and is thus an ideal “playground” for phytochemists to discover peptides from plants, there are still few studies describing the isolation of cyclotides from Brazilian plants. ,− The relative lack of study of cyclotides in Brazilian plants probably reflects an earlier focus among Brazilian researchers on nonpeptidic natural products based on available training and equipment, but with opportunities for international internships in cyclotide-specialist laboratories, as occurred in this project, cyclotide research is expanding in Brazil. CyBase, a database that showcases cyclic protein sequences and structures, reports approximately 500 natural cyclotides discovered from five plant families, i.e., Rubiaceae, Fabaceae, Poaceae, Solanaceae, and Violaceae .…”

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Cyclotides from Brazilian Palicourea sessilis and Their Effects on Human Lymphocytes

et al. 2021

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Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus Palicourea Aubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilis and their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A–E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A–D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.

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“…35,36 Despite this difference, in the current literature there are only two published studies describing the distribution of the cyclotides in plant tissues by MALDI imaging 37 and immunolocalization. 38 In this study, an analytical characterization was carried out on three cyclotides from P. calceolaria, namely, Poca A (1), Poca B (2), and the previously known CyO4 5,39 (3). We investigated their effects on breast cancer cell migration and cytotoxicity.…”

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confidence: 99%

“…In this study, an analytical characterization was carried out on three cyclotides from P. calceolaria , namely, Poca A ( 1 ), Poca B ( 2 ), and the previously known CyO4 , ( 3 ). We investigated their effects on breast cancer cell migration and cytotoxicity.…”

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Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria

et al. 2018

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Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 μM, and the concentration-related cytotoxic responses were observed with IC50 values of 1.8, 2.7, and 9.8 μM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 μM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

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Pinpointing disulfide connectivities in cysteine-rich proteins

Cited by 3 publications

References 29 publications

Cyclotides: From Structure to Function

Cyclotides: From Structure to Function

Cyclotides from Brazilian Palicourea sessilis and Their Effects on Human Lymphocytes

Inhibition of Breast Cancer Cell Migration by Cyclotides Isolated from Pombalia calceolaria

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