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1.
J Exp Bot ; 69(3): 633-641, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29309615

RESUMO

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.


Assuntos
Cisteína Endopeptidases/metabolismo , Nicotiana/metabolismo , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Cisteína Endopeptidases/genética , Perfilação da Expressão Gênica , Peptídeos Cíclicos/genética , Proteínas de Plantas/genética , Nicotiana/genética
2.
Infect Immun ; 82(11): 4707-17, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25156737

RESUMO

Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários , Variação Antigênica/imunologia , Variação Genética , Malária/parasitologia , Modelos Moleculares , Mutagênese , Mutação , Conformação Proteica , Coelhos , Proteínas Recombinantes , Especificidade da Espécie
3.
J Fungi (Basel) ; 10(1)2024 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-38248963

RESUMO

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.

4.
PLoS One ; 17(11): e0269649, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36410013

RESUMO

INTRODUCTION: Drug development for neurodegenerative diseases such as Friedreich's ataxia (FRDA) is limited by a lack of validated, sensitive biomarkers of pharmacodynamic response in affected tissue and disease progression. Studies employing neuroimaging measures to track FRDA have thus far been limited by their small sample sizes and limited follow up. TRACK-FA, a longitudinal, multi-site, and multi-modal neuroimaging natural history study, aims to address these shortcomings by enabling better understanding of underlying pathology and identifying sensitive, clinical trial ready, neuroimaging biomarkers for FRDA. METHODS: 200 individuals with FRDA and 104 control participants will be recruited across seven international study sites. Inclusion criteria for participants with genetically confirmed FRDA involves, age of disease onset ≤ 25 years, Friedreich's Ataxia Rating Scale (FARS) functional staging score of ≤ 5, and a total modified FARS (mFARS) score of ≤ 65 upon enrolment. The control cohort is matched to the FRDA cohort for age, sex, handedness, and years of education. Participants will be evaluated at three study visits over two years. Each visit comprises of a harmonized multimodal Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS) scan of the brain and spinal cord; clinical, cognitive, mood and speech assessments and collection of a blood sample. Primary outcome measures, informed by previous neuroimaging studies, include measures of: spinal cord and brain morphometry, spinal cord and brain microstructure (measured using diffusion MRI), brain iron accumulation (using Quantitative Susceptibility Mapping) and spinal cord biochemistry (using MRS). Secondary and exploratory outcome measures include clinical, cognitive assessments and blood biomarkers. DISCUSSION: Prioritising immediate areas of need, TRACK-FA aims to deliver a set of sensitive, clinical trial-ready neuroimaging biomarkers to accelerate drug discovery efforts and better understand disease trajectory. Once validated, these potential pharmacodynamic biomarkers can be used to measure the efficacy of new therapeutics in forestalling disease progression. CLINICAL TRIAL REGISTRATION: ClinicalTrails.gov Identifier: NCT04349514.


Assuntos
Ataxia de Friedreich , Adulto , Humanos , Biomarcadores , Encéfalo/patologia , Progressão da Doença , Ataxia de Friedreich/patologia , Espectroscopia de Ressonância Magnética
5.
ACS Chem Biol ; 15(4): 962-969, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32203656

RESUMO

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.


Assuntos
Ciclotídeos/química , Cisteína Endopeptidases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Ciclização , Ciclotídeos/síntese química , Ciclotídeos/genética , Cisteína Endopeptidases/genética , Escherichia coli/genética , Oldenlandia/enzimologia , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Engenharia de Proteínas
6.
Methods Mol Biol ; 2012: 211-235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31161511

RESUMO

Cyclization of the peptide backbone by connecting the N- and C-terminus can endow target peptides with favorable properties, such as increased stability or potential oral bioavailability. However, there are few tools available for carrying out this modification. Asparaginyl endopeptidases (AEPs) are a class of enzymes that typically work as proteases, but a subset is highly efficient at cyclization of the peptide backbone. In this chapter we describe how to utilize a cyclizing AEP (OaAEP1b) to produce backbone-cyclized peptides both in planta and in vitro. Using the in planta method, OaAEP1b and the target precursor peptide are coexpressed in the leaves of the model plant Nicotiana benthamiana, and cyclization of the target peptide occurs in planta. Using the in vitro method, purified recombinant OaAEP1b produced in bacteria is used to cyclize the target precursor peptide in vitro.


Assuntos
Cisteína Endopeptidases/química , Oldenlandia/enzimologia , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Catálise , Ciclização , Ciclotídeos/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Ligases , Oldenlandia/genética , Engenharia de Proteínas , Proteínas Recombinantes , Relação Estrutura-Atividade
7.
Sci Rep ; 9(1): 10820, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346249

RESUMO

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1b. These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).


Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Antígenos de Protozoários/metabolismo , Ciclização , Modelos Moleculares , Engenharia de Proteínas , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo
8.
Nat Commun ; 6: 10199, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26680698

RESUMO

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Ciclização , Cisteína Endopeptidases/genética , Perfilação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oldenlandia , Peptídeos/metabolismo , Proteínas Recombinantes
9.
Biopolymers ; 95(5): 354-64, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21213258

RESUMO

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.


Assuntos
Antimaláricos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Antimaláricos/química , Sítios de Ligação/genética , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Técnicas In Vitro , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Virulência/efeitos dos fármacos
10.
J Biol Chem ; 284(14): 9361-71, 2009 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-19164290

RESUMO

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.


Assuntos
Antimaláricos/farmacologia , Peptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antimaláricos/química , Metilação , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Fatores de Tempo
11.
Infect Immun ; 73(10): 6981-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16177378

RESUMO

Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a "hot spot" on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.


Assuntos
Antimaláricos/farmacologia , Proteínas de Transporte/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Antígenos de Protozoários , Antimaláricos/química , Bioensaio , Proteínas de Transporte/química , Dados de Sequência Molecular , Biblioteca de Peptídeos , Conformação Proteica
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