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Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.
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BACKGROUND: The identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4. METHODS: In vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4's interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations. RESULTS: PARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4's tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss. CONCLUSIONS: PARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex-unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.
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Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Neoplasias Pulmonares , Proteínas Nucleares , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação , Ligação Proteica , Splicing de RNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
Retinoic acid (RA) signaling is a master regulator of vertebrate development with crucial roles in directing body axis orientation and tissue differentiation, including in the reproductive system. However, a mechanistic understanding of how RA signaling promotes cell lineage identity in different tissues is often missing. Here, leveraging prostate organoid technology, we demonstrated that RA signaling orchestrates the commitment of adult mouse prostate progenitors to glandular identity, epithelial barrier integrity, and ultimately, proper specification of the prostatic lumen. Mechanistically, RA-dependent RARγ activation promotes the expression of the pioneer factor Foxa1, which synergizes with the androgen pathway for proper luminal expansion, cytoarchitecture and function. FOXA1 nucleotide variants are common in human prostate and breast cancers and considered driver mutations, though their pathogenic mechanism is incompletely understood. Combining functional genetics experiments with structural modeling of FOXA1 folding and chromatin binding analyses, we discovered that FOXA1 F254E255 is a loss-of-function mutation leading to compromised transcriptional function and lack of luminal fate commitment of prostate progenitors. Overall, we define RA as a crucial instructive signal for glandular identity in adult prostate progenitors. We propose deregulation of vitamin A metabolism as a risk factor for benign and malignant prostate disease, and identified cancer associated FOXA1 indels affecting residue F254 as loss-of-function mutations promoting dedifferentiation of adult prostate progenitors. Summary: Retinoic acid signaling orchestrates luminal differentiation of adult prostate progenitors.
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Proteoglycans contain glycosaminoglycans (GAGs) which are negatively charged linear polymers made of repeating disaccharide units of uronic acid and hexosamine units. They play vital roles in numerous physiological and pathological processes, particularly in governing cellular communication and attachment. Depending on their sulfonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hamper their characterization at atomic resolution, but this may be circumvented via coarse-grained (CG) approaches. In this work, we report a CG model for a library of common GAG types in their isolated or proteoglycan-linked states compatible with version 2.2 (v2.2) of the widely popular CG Martini force field. The model reproduces conformational and thermodynamic properties for a wide variety of GAGs, as well as matching structural and binding data for selected proteoglycan test systems. The parameters developed here may thus be employed to study a range of GAG-containing biomolecular systems, thereby benefiting from the efficiency and broad applicability of the Martini framework.
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Glicosaminoglicanos , Simulação de Dinâmica Molecular , Termodinâmica , Glicosaminoglicanos/química , Proteoglicanas/químicaRESUMO
The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.
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Relaxina , Relaxina/farmacologia , Relaxina/química , Relaxina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismo , Domínios Proteicos , beta-Arrestinas/metabolismoRESUMO
Stapled peptides are a promising class of molecules with potential as highly specific probes of protein-protein interactions and as therapeutics. Hydrocarbon stapling affects the peptide properties through the interplay of two factors: enhancing the overall hydrophobicity and constraining the conformational flexibility. By constructing a series of virtual peptides, we study the role of each factor in modulating the structural properties of a hydrocarbon-stapled peptide PM2, which has been shown to enter cells, engage its target Mouse Double Minute 2 (MDM2), and activate p53. Hamiltonian replica exchange molecular dynamics (HREMD) simulations suggest that hydrocarbon stapling favors helical populations of PM2 through a combination of the geometric constraints and the enhanced hydrophobicity of the peptide. To further understand the conformational landscape of the stapled peptides along the binding pathway, we performed HREMD simulations by restraining the peptide at different distances from MDM2. When the peptide approaches MDM2, the binding pocket undergoes dehydration which appears to be greater in the presence of the stapled peptide compared with the linear peptide. In the binding pocket, the helicity of the stapled peptide is increased due to the favorable interactions between the peptide residues as well as the staple and the microenvironment of the binding pocket, contributing to enhanced affinity. The dissection of the multifaceted mechanism of hydrocarbon stapling into individual factors not only deepens fundamental understanding of peptide stapling, but also provides guidelines for the design of new stapled peptides.
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Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology. Workflow application gives peptides with >292x improved cell proliferation potencies and no off-target cell proliferation effects ( > 3800x on-target index). Application of these 'design rules' to a distinct Mdm2(X) peptide series improves ( > 150x) cellular potencies and removes off-target toxicities. The outlined workflow should facilitate therapeutic impacts, especially for those targets such as Mdm2(X) that have hydrophobic interfaces and are targetable with a helical motif.
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Peptídeos , Proteínas Proto-Oncogênicas c-mdm2 , Peptídeos/farmacologia , Peptídeos/químicaRESUMO
Summary: Protein structures carry signal of common ancestry and can therefore aid in reconstructing their evolutionary histories. To expedite the structure-informed inference process, a web server, Structome, has been developed that allows users to rapidly identify protein structures similar to a query protein and to assemble datasets useful for structure-based phylogenetics. Structome was created by clustering â¼94% of the structures in RCSB PDB using 90% sequence identity and representing each cluster by a centroid structure. Structure similarity between centroid proteins was calculated, and annotations from PDB, SCOP, and CATH were integrated. To illustrate utility, an H3 histone was used as a query, and results show that the protein structures returned by Structome span both sequence and structural diversity of the histone fold. Additionally, the pre-computed nexus-formatted distance matrix, provided by Structome, enables analysis of evolutionary relationships between proteins not identifiable using searches based on sequence similarity alone. Our results demonstrate that, beginning with a single structure, Structome can be used to rapidly generate a dataset of structural neighbours and allows deep evolutionary history of proteins to be studied. Availability and Implementation: Structome is available at: https://structome.bii.a-star.edu.sg.
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DAXX (Death Domain Associated Protein 6) is frequently upregulated in various common cancers, and its suppression has been linked to reduced tumor progression. Consequently, DAXX has gained significant interest as a therapeutic target in such cancers. DAXX is known to function in several critical biological pathways including chromatin remodelling, transcription regulation, and DNA repair. Leveraging structural information, we have designed and developed a novel set of stapled/stitched peptides that specifically target a surface on the N-terminal helical bundle domain of DAXX. This surface serves as the anchor point for binding to multiple interaction partners, such as Rassf1C, p53, Mdm2, and ATRX, as well as for the auto-regulation of the DAXX N-terminal SUMO interaction motif (SIM). Our experiments demonstrate that these peptides effectively bind to and inhibit DAXX with a higher affinity than the known interaction partners. Furthermore, these peptides release the auto-inhibited SIM, enabling it to interact with SUMO-1. Importantly, we have developed stitched peptides that can enter cells, maintaining their intracellular concentrations at nanomolar levels even after 24 hours, without causing any membrane perturbation. Collectively, our findings suggest that these stitched peptides not only serve as valuable tools for probing the molecular interactions of DAXX but also hold potential as precursors to the development of therapeutic interventions.
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The scaffolding protein CARD11 is a critical mediator of antigen receptor signaling in lymphocytes. Hypomorphic (partial loss-of-function) mutations in CARD11 are associated with the development of severe atopic dermatitis, in which T cell receptor signaling is reduced and helper T cell differentiation is skewed to an allergy-associated type 2 phenotype. Here, we found that the docking protein DOK3 plays a key role in the pathogenesis of atopic dermatitis by suppressing CARD11 activity. DOK3 interacted with CARD11 and decreased its phosphorylation in T cells by recruiting the catalytic subunit of protein phosphatase 4, thereby dampening downstream signaling. Knocking out Dok3 enhanced the production of the cytokine IFN-γ by T cells, which conferred protection against experimental atopic dermatitis-like skin inflammation in mice. The expression of DOK3 was increased in T cells isolated from patients with atopic dermatitis and inversely correlated with IFNG expression. A subset of hypomorphic CARD11 variants found in patients with atopic dermatitis bound more strongly than wild-type CARD11 to DOK3. Our findings suggest that the strength of the interaction of DOK3 with CARD11 may predispose individuals to developing atopic dermatitis.
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Dermatite Atópica , Linfócitos T , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Guanilato Ciclase/genética , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/genética , Linfócitos T/metabolismoRESUMO
The c-Jun-NH2-terminal kinases (JNKs) regulate cell death, generally through the direct phosphorylation of both pro- and anti-apoptotic substrates. In this report, we demonstrate an alternate mechanism of JNK-mediated cell death involving the anti-apoptotic protein human apurinic/apyrimidinic endonuclease 1 (APE1). Treatment of cells with a variety of genotoxic stresses enhanced APE1-JNK (all isoforms of JNK1 or JNK2) interaction, specifically in cells undergoing apoptosis. Steady-state APE1 levels were decreased in these cells, in which APE1 is ubiquitinated and degraded in a JNK-dependent manner. Absence of JNKs reduced APE1 ubiquitination and increased its abundance. Mechanistically, the E3 ligase ITCH associates with both APE1 and JNK and is necessary for JNK-dependent APE1 ubiquitination and degradation. Structural models of the JNK-APE1 interaction support the observation of enhanced association of the complex in the presence of ubiquitin. The data together show a mechanism of JNK-mediated cell death by the degradation of APE1 through ITCH.
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Dano ao DNA , Endonucleases , MAP Quinase Quinase 4 , Humanos , Morte Celular , Fosforilação , Ubiquitinação , MAP Quinase Quinase 4/metabolismoRESUMO
Cyclic peptides are poised to target historically difficult to drug intracellular protein-protein interactions, however, their general cell impermeability poses a challenge for characterizing function. Recent advances in microfluidics have enabled permeabilization of the cytoplasmic membrane by physical cell deformation (i.e., mechanoporation), resulting in intracellular delivery of impermeable macromolecules in vector- and electrophoretic-free approaches. However, the number of payloads (e.g., peptides) and/or concentrations delivered via microfluidic mechanoporation is limited by having to pre-mix cells and payloads, a manually intensive process. In this work, we show that cells are momentarily permeable (t 1/2 = 1.1-2.8 min) after microfluidic vortex shedding (µVS) and that lower molecular weight macromolecules can be cytosolically delivered upon immediate exposure after cells are processed/permeabilized. To increase the ability to screen peptides, we built a system, dispensing-microfluidic vortex shedding (DµVS), that integrates a µVS chip with inline microplate-based dispensing. To do so, we synced an electronic pressure regulator, flow sensor, on/off dispense valve, and an x-y motion platform in a software-driven feedback loop. Using this system, we were able to deliver low microliter-scale volumes of transiently mechanoporated cells to hundreds of wells on microtiter plates in just several minutes (e.g., 96-well plate filled in <2.5 min). We validated the delivery of an impermeable peptide directed at MDM2, a negative regulator of the tumor suppressor p53, using a click chemistry- and NanoBRET-based cell permeability assay in 96-well format, with robust delivery across the full plate. Furthermore, we demonstrated that DµVS could be used to identify functional, low micromolar, cellular activity of otherwise cell-inactive MDM2-binding peptides using a p53 reporter cell assay in 96- and 384-well format. Overall, DµVS can be combined with downstream cell assays to investigate intracellular target engagement in a high-throughput manner, both for improving structure-activity relationship efforts and for early proof-of-biology of non-optimized peptide (or potentially other macromolecular) tools.
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Invasive fungal disease is an emerging and serious public health threat globally. The expanding population of susceptible individuals, together with the rapid emergence of multidrug-resistant fungi pathogens, call for the development of novel therapeutic strategies beyond the limited repertoire of licensed antifungal drugs. Card9 is a critical signaling molecule involved in antifungal defense; we have previously identified Dok3 to be a key negative regulator of Card9 activity in neutrophils. In this study, we identified two synthetic peptides derived from the coiled-coil domain of Card9, which can specifically block Dok3-Card9 binding. We showed that these peptides are cell-permeable, non-toxic, and can enhance antifungal cytokine production and the phagocytosis of human neutrophils upon fungal infection. Collectively, these data provide a proof of concept that disrupting the Dok3-Card9 interaction can boost the antifungal effector functions of neutrophils; they further suggest the potential utility of these peptide inhibitors as an immune-based therapeutic to fight fungal infection.
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Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.
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Receptores ErbB , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transdução de Sinais , Ativação Transcricional , FosforilaçãoRESUMO
New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species.
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Anti-Infecciosos , Peptídeos , Animais , Camundongos , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos , Biofilmes , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Mamíferos/metabolismoRESUMO
SUMMARY: p53 mutant proteins are widely expressed in human cancer. In this issue, Guiley and Shokat describe the development of compounds that rescue the function of the Y220C mutant p53 protein by forming covalent complexes with the target protein. See related article by Guiley and Shokat, p. 56 (3).
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Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
CDK4/6 inhibitors combined with endocrine therapy have demonstrated higher antitumor activity than endocrine therapy alone for the treatment of advanced estrogen receptor-positive breast cancer. Some of these tumors are de novo resistant to CDK4/6 inhibitors and others develop acquired resistance. Here, we show that p16 overexpression is associated with reduced antitumor activity of CDK4/6 inhibitors in patient-derived xenografts (n = 37) and estrogen receptor-positive breast cancer cell lines, as well as reduced response of early and advanced breast cancer patients to CDK4/6 inhibitors (n = 89). We also identified heterozygous RB1 loss as biomarker of acquired resistance and poor clinical outcome. Combination of the CDK4/6 inhibitor ribociclib with the PI3K inhibitor alpelisib showed antitumor activity in estrogen receptor-positive non-basal-like breast cancer patient-derived xenografts, independently of PIK3CA, ESR1 or RB1 mutation, also in drug de-escalation experiments or omitting endocrine therapy. Our results offer insights into predicting primary/acquired resistance to CDK4/6 inhibitors and post-progression therapeutic strategies.
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Antineoplásicos , Neoplasias da Mama , Inibidores de Proteínas Quinases , Antineoplásicos/uso terapêutico , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Estrogênio/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
An attractive approach to target intracellular macromolecular interfaces and to model putative drug interactions is to design small high-affinity proteins. Variable domains of the immunoglobulin heavy chain (VH domains) are ideal miniproteins, but their development has been restricted by poor intracellular stability and expression. Here we show that an autonomous and disufhide-free VH domain is suitable for intracellular studies and use it to construct a high-diversity phage display library. Using this library and affinity maturation techniques we identify VH domains with picomolar affinity against eIF4E, a protein commonly hyper-activated in cancer. We demonstrate that these molecules interact with eIF4E at the eIF4G binding site via a distinct structural pose. Intracellular overexpression of these miniproteins reduce cellular proliferation and expression of malignancy-related proteins in cancer cell lines. The linkage of high-diversity in vitro libraries with an intracellularly expressible miniprotein scaffold will facilitate the discovery of VH domains suitable for intracellular applications.
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Fator de Iniciação 4E em Eucariotos , Fator de Iniciação 4F em Eucariotos , Técnicas de Visualização da Superfície Celular , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Biblioteca Gênica , Cadeias Pesadas de Imunoglobulinas/genéticaRESUMO
Identifying new binding sites and poses that modify biological function are an important step towards drug discovery. We have identified a novel disulphide constrained peptide that interacts with the cap-binding site of eIF4E, an attractive therapeutic target that is commonly overexpressed in many cancers and plays a significant role in initiating a cancer specific protein synthesis program though binding the 5'cap (7'methyl-guanoisine) moiety found on mammalian mRNAs. The use of disulphide constrained peptides to explore intracellular biological targets is limited by their lack of cell permeability and the instability of the disulphide bond in the reducing environment of the cell, loss of which results in abrogation of binding. To overcome these challenges, the cap-binding site interaction motif was placed in a hypervariable loop on an VH domain, and then selections performed to select a molecule that could recapitulate the interaction of the peptide with the target of interest in a process termed Peptide Epitope Linker Evolution (PELE). A novel VH domain was identified that interacted with the eIF4E cap binding site with a nanomolar affinity and that could be intracellularly expressed in mammalian cells. Additionally, it was demonstrated to specifically modulate eIF4E function by decreasing cap-dependent translation and cyclin D1 expression, common effects of eIF4F complex disruption.