RESUMO
Fortification of human milk (HM) is often necessary to meet the nutritional requirements of preterm infants. The present experiment aimed to establish whether the supplementation of HM with either an experimental donkey milk-derived fortifier containing whole donkey milk proteins, or with a commercial bovine milk-derived fortifier containing hydrolyzed bovine whey proteins, affects peptide release differently during digestion. The experiment was conducted using an in vitro dynamic system designed to simulate the preterm infant's digestion followed by digesta analysis by means of LC-MS-MS. The different fortifiers did not appear to influence the cumulative intensity of HM peptides. Fortification had a differential impact on the release of either donkey or bovine bioactive peptides. Donkey milk peptides showed antioxidant/ACE inhibitory activities, while bovine peptides showed opioid, dipeptil- and propyl endo- peptidase inhibitory and antimicrobial activity. A slight delay in peptide release from human lactoferrin and α-lactalbumin was observed when HM was supplemented with donkey milk-derived fortifier.
Assuntos
Digestão , Equidae , Proteínas do Leite , Leite Humano , Peptídeos , Humanos , Animais , Leite Humano/química , Leite Humano/metabolismo , Proteínas do Leite/química , Proteínas do Leite/metabolismo , Proteínas do Leite/análise , Bovinos , Peptídeos/química , Peptídeos/metabolismo , Alimentos Fortificados/análise , Espectrometria de Massas em Tandem , Modelos Biológicos , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismoRESUMO
This study isolated a novel peptide MMGGED with strong calcium-binding capacity from defatted walnut meal and synthesized a novel peptidecalcium chelate COS-MMGGED-Ca with high stability via glycation. Structural characterization and computer simulation identified binding sites, while in vitro digestion stability and calcium transport experiments explored the chelate's properties. Results showed that after glycation, COS-MMGGED bound Ca2+ with 88.75 ± 1.75 %, mainly via aspartic and glutamic acids. COS-MMGGED-Ca released Ca2+ steadily (60.27 %), with thermal denaturation temperature increased by 18 °C and 37 °C compared to MMGGED-Ca, indicating good processing performance. Furthermore, COS-MMGGED significantly enhanced Ca2+ transport across Caco-2 monolayers, 1.13-fold and 1.62-fold higher than CaCl2 and MMGGED, respectively, at 240 h. These findings prove glycation enhances structural properties, stability, calcium loading, and transport of peptidecalcium chelates, providing a scientific basis for developing novel efficient calcium supplements and high-value utilization of walnut meal.
Assuntos
Cálcio , Juglans , Peptídeos , Juglans/química , Humanos , Cálcio/química , Cálcio/metabolismo , Células CACO-2 , Peptídeos/química , Peptídeos/metabolismo , Glicosilação , Quelantes de Cálcio/químicaRESUMO
MYC promotes tumor growth through multiple mechanisms. Here, we show that, in human glioblastomas, the variant MYC transcript encodes a 114-amino acid peptide, MYC pre-mRNA encoded protein (MPEP), from the upstream open reading frame (uORF) MPEP. Secreted MPEP promotes patient-derived xenograft tumor growth in vivo, independent of MYC through direct binding, and activation of tropomyosin receptor kinase B (TRKB), which induces downstream AKT-mTOR signaling. Targeting MPEP through genetic ablation reduced growth of patient-derived 4121 and 3691 glioblastoma stem cells. Administration of an MPEP-neutralizing antibody in combination with a small-molecule TRKB inhibitor reduced glioblastoma growth in patient-derived xenograft tumor-bearing mice. The overexpression of MPEP in surgical glioblastoma specimens predicted a poor prognosis, supporting its clinical relevance. In summary, our results demonstrate that tumor-specific translation of a MYC-associated uORF promotes glioblastoma growth, suggesting a new therapeutic strategy for glioblastoma.
Assuntos
Glioblastoma , Fases de Leitura Aberta , Proteínas Proto-Oncogênicas c-myc , Receptor trkB , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Animais , Humanos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fases de Leitura Aberta/genética , Camundongos , Linhagem Celular Tumoral , Receptor trkB/metabolismo , Proliferação de Células/efeitos dos fármacos , Ligação Proteica , Transdução de Sinais , Peptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, biosensor discovery is slow and requires tedious steps in rational design, screening, and characterization. Here we report on a platform that streamlines biosensor discovery and unlocks directed nanosensor evolution through genetically encodable fluorogenic amino acids (FgAAs). Building on the classical knowledge-based semisynthetic approach, we engineer ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. An optimized genetic code expansion chemistry with FgAAs further enables rapid (~3 h) ribosomal nanosensor discovery via the cell-free translation of hundreds of candidates in parallel and directed nanosensor evolution with improved variant-specific sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform could accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.
Assuntos
Aminoácidos , Técnicas Biossensoriais , Corantes Fluorescentes , SARS-CoV-2 , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Humanos , SARS-CoV-2/genética , COVID-19/virologia , Nanotecnologia/métodos , Peptídeos/metabolismo , Peptídeos/química , Peptídeos/genéticaRESUMO
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Assuntos
Cisteína , Dissulfetos , Biblioteca de Peptídeos , Dissulfetos/química , Cisteína/química , Motivos de Aminoácidos , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Humanos , Oxirredução , Dobramento de ProteínaRESUMO
Cells contain intricate protein nanostructures, but replicating them outside of cells presents challenges. One such example is the vertical fibronectin pillars observed in embryos. Here, we demonstrate the creation of cell-free vertical fibronectin pillar mimics using nonequilibrium self-assembly. Our approach utilizes enzyme-responsive phosphopeptides that assemble into nanotubes. Enzyme action triggers shape changes in peptide assemblies, driving the vertical growth of protein nanopillars into bundles. These bundles, with peptide nanotubes serving as a template to remodel fibronectin, can then recruit collagen, which forms aggregates or bundles depending on their types. Nanopillar formation relies on enzyme-catalyzed nonequilibrium self-assembly and is governed by the concentrations of enzyme, protein, peptide, the structure of the peptide, and peptide assembly morphologies. Cryo-EM reveals unexpected nanotube thinning and packing after dephosphorylation, indicating a complex sculpting process during assembly. Our study demonstrates a cell-free method for constructing intricate, multiprotein nanostructures with directionality and composition.
Assuntos
Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Nanoestruturas/química , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Nanotubos/químicaRESUMO
Probiotics are used in cheese fermentation to endow the product with unique functional properties, such as enhanced flavor and aroma development through proteolysis and lipolysis. In this study, two probiotic Lactobacillus strains, Lactobacillus plantarum A3 and Lactobacillus reuteri WQY-1, were selected to develop new probiotic cheeses in the form of single- and mixed-strain starters. The results demonstrated that the L. plantarum A3 single-strain group and the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group exhibited superior product performance, particularly the release of functional hydrolysates during cheese ripening. Furthermore, Label-free quantitative proteomic analysis revealed 26 unique antioxidant peptides in the L. plantarum A3 single-strain group and 53 in the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group. Among these, CMENSAEPEQSLACQCL (ß-lactoglobulin), CMENSAEPEQSLVCQCL (ß-lactoglobulin), and IQYVLSR (κ-casein) have been found to possess potential antioxidant properties both in vitro and in vivo. This confirmed that milk-derived protein peptides in cheese products exhibit potential antioxidant functions through the hydrolysis of probiotic strains.
Assuntos
Antioxidantes , Queijo , Fermentação , Lactobacillus plantarum , Peptídeos , Probióticos , Queijo/microbiologia , Queijo/análise , Antioxidantes/metabolismo , Antioxidantes/química , Peptídeos/metabolismo , Peptídeos/química , Lactobacillus plantarum/metabolismo , Lactobacillus plantarum/química , Animais , Probióticos/metabolismo , Probióticos/química , Limosilactobacillus reuteri/metabolismo , Limosilactobacillus reuteri/química , Bovinos , Lactobacillus/metabolismo , CamundongosRESUMO
The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.
Assuntos
L-Lactato Desidrogenase , Ácido Láctico , Multimerização Proteica , Humanos , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/química , Ácido Láctico/metabolismo , Ácido Láctico/química , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Concentração de Íons de Hidrogênio , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Linhagem Celular TumoralRESUMO
The yeast pre1-1(ß4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with ß2- and ß5-propeptides, which together fill most of the antechambers between the α- and ß-rings. The ß5-propeptide is unprocessed and separates Ump1 from ß6 and ß7. The ß2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and ß3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, ß2 is activated first enabling processing of ß1-, ß6-, and ß7-propeptides. Subsequent maturation of ß5 and ß1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.
Assuntos
Microscopia Crioeletrônica , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Modelos Moleculares , Conformação Proteica , Peptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Chaperonas MolecularesRESUMO
Medin amyloid, prevalent in the vessel walls of 97â¯% of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.
Assuntos
Amiloide , Simulação de Dinâmica Molecular , Humanos , Amiloide/química , Amiloide/metabolismo , Aorta/metabolismo , Agregados Proteicos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica em Folha beta , Antígenos de Superfície/metabolismo , Antígenos de Superfície/química , Sequência de Aminoácidos , Proteínas do LeiteRESUMO
An important functionality of lifelike "synthetic cells" is to mimic cell division. Currently, specialized proteins that induce membrane fission in living cells are the primary candidates for dividing synthetic cells. However, interactions between lipid membranes and proteins that are not found in living cells may also be suitable. Here, we discuss the potential of short membrane-anchored peptides to induce cell division. Specifically, we used the coarse-grained MARTINI model to investigate the interaction between short membrane-anchored peptides and a lipid bilayer patch. The simulation revealed that the anchored peptide induces significant spontaneous curvature and suggests that the lipid-peptide complex can be considered as a conically shaped "bulky headgroup" lipid. By systematically increasing the electrostatic charge of the peptide, we find that membrane-anchored peptides may generate sufficiently large constriction forces even at dilute coverages. Finally, we show that when the peptide has an opposite charge to the membrane, the peptide may induce division by binding the inner membrane leaflet of a synthetic cell, that is, cell division from within.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Peptídeos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/metabolismo , Eletricidade EstáticaRESUMO
This review presents current knowledge related to the voltage-dependent anion channel-1 (VDAC1) as a multi-functional mitochondrial protein that acts in regulating both cell life and death. The location of VDAC1 at the outer mitochondrial membrane (OMM) allows control of metabolic cross-talk between the mitochondria and the rest of the cell, and also enables its interaction with proteins that are involved in metabolic, cell death, and survival pathways. VDAC1's interactions with over 150 proteins can mediate and regulate the integration of mitochondrial functions with cellular activities. To target these protein-protein interactions, VDAC1-derived peptides have been developed. This review focuses specifically on cell-penetrating VDAC1-based peptides that were developed and used as a "decoy" to compete with VDAC1 for its VDAC1-interacting proteins. These peptides interfere with VDAC1 interactions, for example, with metabolism-associated proteins such as hexokinase (HK), or with anti-apoptotic proteins such as Bcl-2 and Bcl-xL. These and other VDAC1-interacting proteins are highly expressed in many cancers. The VDAC1-based peptides in cells in culture selectively affect cancerous, but not non-cancerous cells, inducing cell death in a variety of cancers, regardless of the cancer origin or genetics. They inhibit cell energy production, eliminate cancer stem cells, and act very rapidly and at low micro-molar concentrations. The activity of these peptides has been validated in several mouse cancer models of glioblastoma, lung, and breast cancers. Their anti-cancer activity involves a multi-pronged attack targeting the hallmarks of cancer. They were also found to be effective in treating non-alcoholic fatty liver disease and diabetes mellitus. Thus, VDAC1-based peptides, by targeting VDAC1-interacting proteins, offer an affordable and innovative new conceptual therapeutic paradigm that can potentially overcome heterogeneity, chemoresistance, and invasive metastatic formation.
Assuntos
Diabetes Mellitus , Neoplasias , Canal de Ânion 1 Dependente de Voltagem , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/uso terapêutico , Peptídeos/metabolismo , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/química , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Ligação ProteicaRESUMO
The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.
Assuntos
Peptídeos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Receptor EphA2 , Motivo Estéril alfa , Receptor EphA2/metabolismo , Receptor EphA2/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Ligantes , Humanos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Modelos Moleculares , Ligação Proteica , Sequência de AminoácidosRESUMO
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.
Assuntos
Inibidores Enzimáticos , N-Acetilglucosaminiltransferases , Peptídeos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/química , Humanos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fator C1 de Célula Hospedeira/metabolismo , Fator C1 de Célula Hospedeira/química , Fator C1 de Célula Hospedeira/antagonistas & inibidores , Transferência Ressonante de Energia de Fluorescência , Modelos MolecularesRESUMO
Metastasis of breast cancer is the main cause of death for patients with breast cancer. The interaction between metadherin (MTDH) and staphylococcal nuclease domain 1 (SND1) plays a pivotal role in promoting breast cancer development. However, the binding details between MTDH and SND1 remain unclear. In this study, we employed all-atom molecular dynamics simulations (MDs) and conducted binding energy calculations to investigate the binding details and the impact of key residue mutations on binding. The mutations in key residues have not significantly affected the overall stability of the structure and the fluctuation of residues near the binding site; they have exerted a substantial impact on the binding of SND1 and MTDH peptide. The electrostatic interactions and van der Waals interactions play an important role in the binding of SND1 and the MTDH peptide. The mutations in the key residues have a significant impact on electrostatic and van der Waals interactions, resulting in weakened binding. The energy contributions of key residues mainly come from the electrostatic energy and van der Waals interactions of the side chain. In addition, the key residues form an intricate and stable network of hydrogen bonds and salt-bridge interactions with the MTDH peptide. The mutations in key residues have directly disrupt the interactions formed between SND1 and MTDH peptide, consequently leading to changes in the binding mode of the MTDH peptide. These analyses unveil the detailed atomic-level interaction mechanism between SND1 and the MTDH peptide, providing a molecular foundation for the development of antibreast cancer drugs.
Assuntos
Endonucleases , Proteínas de Membrana , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Humanos , Endonucleases/química , Endonucleases/metabolismo , Endonucleases/genética , Eletricidade Estática , Sítios de Ligação , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Ligação de Hidrogênio , Termodinâmica , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/genéticaRESUMO
Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Plastídeos , Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/metabolismo , Plastídeos/metabolismo , Plastídeos/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Proteico , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Cloroplastos/metabolismo , Proteínas de Cloroplastos/genética , Peptídeos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Sinais Direcionadores de ProteínasRESUMO
Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.
Assuntos
Fosfotreonina , Engenharia de Proteínas , Fosfotreonina/metabolismo , Fosfotreonina/química , Humanos , Engenharia de Proteínas/métodos , Ligação Proteica , Biblioteca de Peptídeos , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/genética , Domínios Proteicos , Fosforilação , Sequência de AminoácidosRESUMO
Here, we present a biosynthesized material M1 for immune checkpoint blocking therapy. M1 could realize a morphological transformation from globular to fibrous in situ in the presence of cathepsin B (CtsB) after entering tumor cells. The GO203 peptides of M1 are exposed, which could bind to mucin 1 (MUC1) to suppress the homodimerization process of MUC1, thereby downregulating PD-L1 expression.
Assuntos
Antígeno B7-H1 , Catepsina B , Regulação para Baixo , Peptídeos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/química , Humanos , Peptídeos/química , Peptídeos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Catepsina B/metabolismo , Mucina-1/metabolismo , Mucina-1/química , Linhagem Celular TumoralRESUMO
Recently, we have investigated the enzyme-assisted self-assembly of precursor peptides diffusing in an enzyme-containing host gel, leading to various self-assembly profiles within the gel. At high enzyme concentrations, the reaction-diffusion self-assembly processes result in the formation of a continuous non-monotonous peptide self-assembly profile. At low enzyme concentrations, they result in the formation of individual self-assembled peptide microglobules and at intermediate enzyme concentrations both kinds of self-assembled structures coexist. Herein, we develop a Liesegang-type model that considers four major points: (i) the diffusion of the precursor peptides within the host gel, (ii) the diffusion of the enzymes in the gel, (iii) the enzymatic transformation of the precursor peptides into the self-assembling ones and (iv) the nucleation of these building blocks as the starting point of the self-assembly process. This process is treated stochastically. Our model predicts most of the experimentally observed features and in particular (i) the transition from a continuous to a microglobular pattern of self-assembled peptides through five types of patterns by decreasing the enzyme concentration in the host hydrogel. (ii) It also predicts that when the precursor peptide concentration decreases, the enzyme concentration at which the continuous/microglobules transition appears increases. (iii) Finally, it predicts that for peptides whose critical self-assembly concentration in solution decreases, the peptide concentration at which the continuous-to-microglobular transition decreases too. All these predictions are observed experimentally.
Assuntos
Hidrogéis , Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Hidrogéis/química , Difusão , Enzimas/química , Enzimas/metabolismoRESUMO
Cortisol is established as a reliable biomarker for stress prompting intensified research in developing wearable sensors to detect it via eccrine sweat. Since cortisol is present in sweat in trace quantities, typically 8-140 ng/mL, developing such biosensors necessitates the design of bioreceptors with appropriate sensitivity and selectivity. In this work, we present a systematic biomimetic methodology and a semi-automated high-throughput screening tool which enables rapid selection of bioreceptors as compared to ab initio design of peptides via computational peptidology. Candidate proteins from databases are selected via molecular docking and ranked according to their binding affinities by conducting automated AutoDock Vina scoring simulations. These candidate proteins are then validated via full atomistic steered molecular dynamics computations including umbrella sampling to estimate the potential of mean force using GROMACS version 2022.6. These explicit molecular dynamic calculations are carried out in an eccrine sweat environment taking into consideration the protein dynamics and solvent effects. Subsequently, we present a candidate baseline peptide bioreceptor selected as a contiguous sequence of amino acids from the selected protein binding pocket favourably interacting with the target ligand (i.e., cortisol) from the active binding site of the proteins and maintaining its tertiary structure. A unique cysteine residue introduced at the N-terminus allows orientation-specific surface immobilization of the peptide onto the gold electrodes and to ensure exposure of the binding site. Comparative binding affinity simulations of this peptide with the target ligand along with commonly interfering species e.g., progesterone, testosterone and glucose are also presented to demonstrate the validity of this proposed peptide as a candidate baseline bioreceptor for future cortisol biosensor development.