RESUMO
Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , DNARESUMO
α-synuclein (αS) is the key component of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-ß amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity. Furthermore, lipids are known to be important for native αS function, where they play a key role in the regulation of synaptic vesicle docking to presynaptic membranes and dopamine transmission. However, the role of lipids in the function of mutant αS is unclear. Here, we studied αS aggregation properties of WT αS and five of the most predominant single-point missense mutants associated with early onset PD in the presence of anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine lipid vesicles. Our results highlight significant differences between aggregation rates, the number of aggregates produced, and overall fibril morphologies of WT αS and the A30P, E46K, H50Q, G51D, and A53T missense mutants in the presence of lipid vesicles. These findings have important implications regarding the interplay between the lipids required for αS function and the individual point mutations known to accelerate PD and related diseases.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Expressão Gênica , Lipídeos , Doença de Parkinson/metabolismo , Mutação Puntual , Mutação de Sentido IncorretoRESUMO
The AP-1 transcription factor family crucially regulates progression of the cell cycle, as well as playing roles in proliferation, differentiation, and the stress response. The two best described AP-1 family members, cFos and cJun, are known to dimerize to form a functional AP-1 heterodimer that binds to a consensus response element sequence. Although cJun can also homodimerize and bind to DNA, the canonical view is that cFos cannot bind DNA without heterodimerizing with cJun. Here, we show that cFos can actually bind to DNA in the absence of cJun in vitro. Using dual color single molecule imaging of cFos alone, we directly visualize binding to and movement on DNA. Of all these DNA-bound proteins, detailed analysis suggested 30 to 46% were homodimers. Furthermore, we constructed fluorescent protein fusions of cFos and cJun for Förster resonance energy transfer experiments. These constructs indicated complete dimerization of cJun, but although cFos could dimerize, its extent was reduced. Finally, to provide orthogonal confirmation of cFos binding to DNA, we performed bulk-phase circular dichroism experiments that showed clear structural changes in DNA; these were found to be specific to the AP-1 consensus sequence. Taken together, our results clearly show cFos can interact with DNA both as monomers and dimers independently of its archetypal partner, cJun.
Assuntos
Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Fator de Transcrição AP-1 , Dicroísmo Circular , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Lactate dehydrogenase 5 (LDH5) is overexpressed in many cancers and is a potential target for anticancer therapy due to its role in aerobic glycolysis. Small-molecule drugs have been developed as competitive inhibitors to bind substrate/cofactor sites of LDH5, but none reached the clinic to date. Recently, we designed the first LDH5 non-competitive inhibitor, cGmC9, a peptide that inhibits protein-protein interactions required for LDH5 enzymatic activity. Peptides are gaining a large interest as anticancer agents to modulate intracellular protein-protein interactions not targetable by small molecules; however, delivery of these peptides to the cytosol, where LDH5 and other anticancer targets are located, remains a challenge for this class of therapeutics. In this study, we focused on the cellular internalisation of cGmC9 to achieve LDH5 inhibition in the cytosol. We designed cGmC9 analogues and compared them for LDH5 inhibition, cellular uptake, toxicity, and antiproliferation against a panel of cancer cell lines. The lead analogue, [R/r]cGmC9, specifically impairs proliferation of cancer cell lines with high glycolytic profiles. Proteomics analysis showed expected metabolic changes in response to decreased glycolysis. This is the first report of a peptide-based LDH5 inhibitor able to modulate cancer metabolism and kill cancer cells that are glycolytic. The current study demonstrates the potential of using peptides as inhibitors of intracellular protein-protein interactions relevant for cancer pathways and shows that active peptides can be rationally designed to improve their cell permeation.
Assuntos
L-Lactato Desidrogenase , Neoplasias , Humanos , Lactato Desidrogenase 5 , Peptídeos/farmacologia , Neoplasias/tratamento farmacológico , Proliferação de CélulasRESUMO
c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70% of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy. Here we reassess the challenges of directly targeting c-Myc, evaluate lessons learned from current inhibitors, and explore how future strategies such as miniaturisation of Omomyc and targeting E-box binding could facilitate translation of c-Myc inhibitors into the clinic.
Assuntos
Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Humanos , Terapia de Alvo Molecular , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.
Assuntos
Biologia Computacional/métodos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células , Simulação por Computador , Dimerização , Genes fos/fisiologia , Genes jun/fisiologia , Humanos , Oncogenes , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismoRESUMO
Basic leucine zipper (bZIP) proteins reside at the end of cell-signaling cascades and function to modulate transcription of specific gene targets. bZIPs are recognized as important regulators of cellular processes such as cell growth, apoptosis, and cell differentiation. One such validated transcriptional regulator, activator protein-1, is typically comprised of heterodimers of Jun and Fos family members and is key in the progression and development of a number of different diseases. The best described component, cJun, is upregulated in a variety of diseases such as cancer, osteoporosis, and psoriasis. Toward our goal of inhibiting bZIP proteins implicated in disease pathways, we here describe the first use of a novel in silico peptide library screening platform that facilitates the derivation of sequences exhibiting a high affinity for cJun while disfavoring homodimer formation or formation of heterodimers with other closely related Fos sequences. In particular, using Fos as a template, we have computationally screened a peptide library of more than 60 million members and ranked hypothetical on/off target complexes according to predicted stability. This resulted in the identification of a sequence that bound cJun but displayed little homomeric stability or preference for cFos. The computationally selected sequence maintains an interaction stability similar to that of a previous experimentally derived cJun antagonist while providing much improved specificity. Our study provides new insight into the use of tandem in silico screening/ in vitro validation and the ability to create a peptide that is capable of satisfying conflicting design requirements.
Assuntos
Simulação por Computador , Zíper de Leucina , Biblioteca de Peptídeos , Multimerização Proteica , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/química , Humanos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
A major biochemical goal is the ability to mimic nature in engineering highly specific protein-protein interactions (PPIs). We previously devised a computational interactome screen to identify eight peptides that form four heterospecific dimers despite 32 potential off-targets. To expand the speed and utility of our approach and the PPI toolkit, we have developed new software to derive much larger heterospecific sets (≥24 peptides) while directing against antiparallel off-targets. It works by predicting Tm values for every dimer on the basis of core, electrostatic, and helical propensity components. These guide interaction specificity, allowing heterospecific coiled coil (CC) sets to be incrementally assembled. Prediction accuracy is experimentally validated using circular dichroism and size exclusion chromatography. Thermal denaturation data from a 22-CC training set were used to improve software prediction accuracy and verified using a 136-CC test set consisting of eight predicted heterospecific dimers and 128 off-targets. The resulting software, qCIPA, individually now weighs core a-a' (II/NN/NI) and electrostatic g-e'+1 (EE/EK/KK) components. The expanded data set has resulted in emerging sequence context rules for otherwise energetically equivalent CCs; for example, introducing intrahelical electrostatic charge blocks generated increased stability for designed CCs while concomitantly decreasing the stability of off-target CCs. Coupled with increased prediction accuracy and speed, the approach can be applied to a wide range of downstream chemical and synthetic biology applications, in addition more generally to impose specificity on structurally unrelated PPIs.
Assuntos
Modelos Estatísticos , Peptídeos/química , Mapeamento de Interação de Proteínas/estatística & dados numéricos , Software , Biblioteca de Peptídeos , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Eletricidade Estática , TermodinâmicaRESUMO
α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson's disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.
Assuntos
FMN Redutase/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , alfa-Sinucleína/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Biocatálise , Linhagem Celular Tumoral , FMN Redutase/química , FMN Redutase/genética , Humanos , Lipossomos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peso Molecular , Mutação , Nanoestruturas/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71-82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.
Assuntos
Biblioteca de Peptídeos , Peptídeos/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Corpos de Lewy/metabolismo , Microscopia de Força Atômica , Doença de Parkinson/metabolismo , Peptídeos/química , Reação em Cadeia da Polimerase , alfa-Sinucleína/metabolismoRESUMO
Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8-14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Proteína de Morte Celular Associada a bcl/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Células Jurkat , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Proteína bcl-X/antagonistas & inibidoresRESUMO
Correction for 'Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiency' by Nicholas E. Shepherd et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob02185a.
RESUMO
The aggregation of ß-amyloid (Aß) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind Aß1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving Aß-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Animais , Dicroísmo Circular , Modelos Animais de Doenças , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Atividade Motora/efeitos dos fármacos , Células PC12 , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/isolamento & purificação , Placa Amiloide/tratamento farmacológico , Estrutura Secundária de Proteína , RatosRESUMO
Tauopathies are characterised by the pathological accumulation of misfolded tau. The emerging view is that toxic tau species drive synaptic dysfunction and potentially tau propagation before measurable neurodegeneration is evident, but the underlying molecular events are not well defined. Human non-mutated 0N4R tau (tauWT) and P301L mutant 0N4R tau (tauP301L) were expressed in mouse primary cortical neurons using adeno-associated viruses to monitor early molecular changes and synaptic function before the onset of neuronal loss. In this model tauP301L was differentially phosphorylated relative to tauwt with a notable increase in phosphorylation at ser262. Affinity purification - mass spectrometry combined with tandem mass tagging was used to quantitatively compare the tauWT and tauP301L interactomes. This revealed an enrichment of tauP301L with ribosomal proteins but a decreased interaction with the proteasome core complex and reduced tauP301L degradation. Differences in the interaction of tauP301L with members of a key synaptic calcium-calmodulin signalling pathway were also identified, most notably, increased association with CaMKII but reduced association with calcineurin and the candidate AD biomarker neurogranin. Decreased association of neurogranin to tauP301L corresponded with the appearance of enhanced levels of extracellular neurogranin suggestive of potential release or leakage from synapses. Finally, analysis of neuronal network activity using micro-electrode arrays showed that overexpression of tauP301L promoted basal hyperexcitability coincident with these changes in the tau interactome and implicating tau in specific early alterations in synaptic function.
Assuntos
Neurônios , Proteínas tau , Animais , Proteínas tau/metabolismo , Proteínas tau/genética , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Tauopatias/metabolismo , Tauopatias/patologia , Tauopatias/genética , Sinapses/metabolismo , Neurogranina/metabolismo , Neurogranina/genética , Calcineurina/metabolismoRESUMO
Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents â¼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.
Assuntos
Fator 3 Ativador da Transcrição , Biblioteca de Peptídeos , Fator 3 Ativador da Transcrição/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos/metabolismoRESUMO
Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.
Assuntos
Citocromos c6/química , Dissulfetos/química , Heme/química , Dobramento de Proteína , Termodinâmica , Sequência de Aminoácidos , Arabidopsis/química , Cianobactérias/química , Cisteína/metabolismo , Citocromos c6/metabolismo , Dissulfetos/metabolismo , Heme/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Oxirredução , Estrutura Terciária de Proteína , Serina/metabolismoRESUMO
Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. However, chemical synthesis of head-to-tail cyclic peptides and proteins is challenging, is often low yielding, and employs toxic and unsustainable reagents. Plant derived asparaginyl endopeptidases such as OaAEP1 have been employed to catalyze the head-to-tail cyclization of peptides in vitro, offering a safer and more sustainable alternative to chemical methods. However, while asparaginyl endopeptidases have been used in vitro and in native and transgenic plant species, they have never been used to generate recombinant cyclic proteins in live recombinant organisms outside of plants. Using dihydrofolate reductase as a proof of concept, we show that a truncated OaAEP1 variant C247A is functional in the Escherichia coli physiological environment and can therefore be coexpressed with a substrate protein to enable concomitant in situ cyclization. The bacterial system is ideal for cyclic protein production owing to the fast growth rate, durability, ease of use, and low cost. This streamlines cyclic protein production via a biocatalytic process with fast kinetics and minimal ligation scarring, while negating the need to purify the enzyme, substrate, and reaction mixtures individually. The resulting cyclic protein was characterized in vitro, demonstrating enhanced thermal stability compared to the corresponding linear protein without impacting enzyme activity. We anticipate this convenient method for generating cyclic peptides will have broad utility in a range of biochemical and chemical applications.
RESUMO
Peptides and their mimetics are increasingly recognised as drug-like molecules, particularly for intracellular protein-protein interactions too large for inhibition by small molecules, and inaccessible to larger biologics. In the past two decades, evidence associating the misfolding and aggregation of alpha-synuclein strongly implicates this protein in disease onset and progression of Parkinson's disease and related synucleinopathies. The subsequent formation of toxic, intracellular, Lewy body deposits, in which alpha-synuclein is a major component, is a key diagnostic hallmark of the disease. To reach their therapeutic site of action, peptides must both cross the blood-brain barrier and enter dopaminergic neurons to prevent the formation of these intracellular inclusions. In this review, we describe and summarise the current efforts made in the development of peptides and their mimetics to directly engage with alpha-synuclein with the intention of modulating aggregation, and importantly, toxicity. This is a rapidly expanding field with great socioeconomic impact; these molecules harbour significant promise as therapeutics, or as early biomarkers during prodromal disease stages, or both. As these are age-dependent conditions, an increasing global life expectancy means disease prevalence is rising. No current treatments exist to either prevent or slow disease progression. It is therefore crucial that drugs are developed for these conditions before health care and social care capacities become overrun.
Assuntos
Doença de Parkinson , Sinucleinopatias , Humanos , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Corpos de Inclusão/metabolismo , PeptídeosRESUMO
Parkinson's is the second most common neurodegenerative disease, with the number of individuals susceptible due to increase as a result of increasing life expectancy and a growing worldwide population. However, despite the number of individuals affected, all current treatments for PD are symptomatic-they alleviate symptoms, but do not slow disease progression. A major reason for the lack of disease-modifying treatments is that there are currently no methods to diagnose individuals during the earliest stages of the disease, nor are there any methods to monitor disease progression at a biochemical level. Herein, we have designed and evaluated a peptide-based probe to monitor αS aggregation, with a particular focus on the earliest stages of the aggregation process and the formation of oligomers. We have identified the peptide-probe K1 as being suitable for further development to be applied to number of applications including: inhibition of αS aggregation; as a probe to monitor αS aggregation, particularly at the earliest stages before Thioflavin-T is active; and a method to detect early-oligomers. With further development and in vivo validation, we anticipate this probe could be used for the early diagnosis of PD, a method to evaluate the effectiveness of potential therapeutics, and as a tool to help in the understanding of the onset and development of PD.
Assuntos
Doenças Neurodegenerativas , Humanos , alfa-Sinucleína , PeptídeosRESUMO
The DNA binding activity of the transcriptional regulator activator protein-1 shows considerable promise as a target in cancer therapy. A number of different strategies have been employed to inhibit the function of this protein with promise having been demonstrated both in vitro and in vivo. Peptide-based therapeutics have received renewed interest in the last few years, and a number of 37-amino acid peptides capable of binding to the coiled coil dimerization domain of Jun and Fos have been derived. Here, we demonstrate how truncation and semi-rational library design, followed by protein-fragment complementation, can be used to produce a leucine zipper binding peptide by iterative means. To this end, we have implemented this strategy on the FosW peptide to produce 4hFosW. This peptide is truncated by four residues with comparably favorable binding properties and demonstrates the possibility to design progressively shorter peptides to serve as leucine zipper antagonists while retaining many of the key features of the parent peptide. Whether or not the necessity for low molecular weight antagonists is required from the perspective of druggability and efficacy is subject to debate. However, antagonists of reduced length are worthy of perusal from the point of view of synthetic cost as well as identifying the smallest functional unit that is required for binding.