RESUMO
Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression.
Assuntos
Monócitos , Neoplasias , Humanos , Monócitos/metabolismo , Medula Óssea , Colesterol/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , HipóxiaRESUMO
There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few µm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.
RESUMO
With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Fluorocarbonos , Transporte Biológico , Peptídeos Penetradores de Células/química , Aminoácidos/metabolismoRESUMO
N-Substituted peptides, such as peptoids and ß-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for ß-peptoids; to date, no preorganized ß-peptoid monomers have been reported. Here, we report the first design strategy for ß-peptoids, in which all four backbone dihedral angles (ω, Ï, θ, ψ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a ß-peptoid with a stable twisted strand shape.
Assuntos
Peptoides , Ciclopentanos , Peptídeos/química , Peptoides/químicaRESUMO
Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.
Assuntos
Alanina , Peptídeos , Alanina/análogos & derivados , Ligação de Hidrogênio , Peptídeos/química , Água/químicaRESUMO
We developed a photoreactive molecular glue, BPGlue-N3, which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BPGlue-N3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BPGlue-N3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu+) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BPGlue-N3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BPGlue-N3 and also a nitrene species, possibly generated by the BP-to-N3 energy transfer in BPGlue-N3. We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Aptâmeros de Nucleotídeos/química , Azidas/química , Benzofenonas/química , Linhagem Celular Tumoral , Movimento Celular , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Microscopia Confocal , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/química , Raios UltravioletaRESUMO
Backbone stereochemistry of cyclic peptides has been reported to have a great influence on microsomal stability and membrane permeability, two important factors that determine oral bioavailability. Here, we comprehensively investigated the correlation between the backbone stereochemistry of cyclic hexapeptide stereoisomers and their stability in liver microsomes, as well as passive membrane permeability.
Assuntos
Permeabilidade da Membrana CelularRESUMO
Here, we investigated the effect of CH3 to CF3 substitution on the membrane permeability of peptides. We synthesized a series of peptides with CF3 groups and corresponding nonfluorinated peptides and measured the membrane permeability of the peptides. As a result, we demonstrated that CH3 to CF3 substitution is useful for increasing the membrane permeability of di-/tri-peptides.
Assuntos
PeptídeosRESUMO
Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR spectroscopy. By application of this powerful tool, small molecules with stable isotopes have been used for highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to the rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with success in vitro or in vivo.
Assuntos
Imagem Molecular , Sondas Moleculares/síntese química , Espectroscopia de Ressonância Magnética , Sondas Moleculares/químicaRESUMO
Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/agonistas , Receptores de Citocinas/agonistas , Células A549 , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Movimento Celular/efeitos dos fármacos , Dimerização , Fator de Crescimento de Hepatócito/agonistas , Fator de Crescimento de Hepatócito/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Microscopia de Fluorescência , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/agonistas , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Unique folded structures of natural and synthetic oligomers are the most fundamental basis for their unique functions. N-Substituted ß-peptides, or ß-peptoids, are synthetic oligomers with great potential to fold into diverse three-dimensional structures because of the existence of four rotatable bonds in a monomer with highly modular synthetic accessibility. However, the existence of the four rotatable bonds poses a challenge for conformational control of ß-peptoids. Here, we report a strategy for per-residue programming of two dihedral angles of ß-peptoids, which is useful for restricting the conformational space of the oligomers. The oligomer was found to form a unique loop conformation that is stabilized by the backbone rotational restrictions. Circular dichroism and NMR spectroscopic analyses and X-ray crystallographic analysis of the oligomer are presented. The strategy would significantly facilitate the discovery of many more unique folded structures of ß-peptoids.
Assuntos
Peptoides/química , Dicroísmo Circular , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de ProteínaRESUMO
Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest.
Assuntos
DNA/análise , Desoxiuridina/análogos & derivados , Teoria Quântica , Desoxiuridina/química , Condutividade Elétrica , Estrutura MolecularRESUMO
We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.
Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Dendrímeros/farmacologia , Guanidinas/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/efeitos da radiação , Linhagem Celular Tumoral , Dendrímeros/síntese química , Dendrímeros/efeitos da radiação , Guanidinas/síntese química , Guanidinas/efeitos da radiação , Fator de Crescimento de Hepatócito/química , Humanos , Ligação Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-met/química , Raios UltravioletaRESUMO
The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.
Assuntos
Peptoides/química , Água/química , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação Proteica , Teoria QuânticaRESUMO
Here, we report a facile permeability assay to quantitatively evaluate the membrane permeability of multiple peptides in parallel. With a fluorogenic click reaction between azidocoumarin and a terminal alkyne tag introduced on a peptide, the peptide that crossed an artificial membrane or a cell monolayer was quantitatively detected. The method allows a rapid measurement of the permeability of multiple compounds on a plate reader even in the presence of a complex mixture of biological molecules.
Assuntos
Permeabilidade da Membrana Celular , Corantes Fluorescentes/análise , Células Madin Darby de Rim Canino/citologia , Membranas Artificiais , Imagem Óptica , Peptídeos/química , Alcinos/química , Animais , Células CACO-2 , Cumarínicos/química , Cães , Corantes Fluorescentes/química , Humanos , Estrutura MolecularRESUMO
Dynamic nuclear polarization with photo-excited triplet electrons (Triplet-DNP) is demonstrated using 6,13-diphenylpentacene (DPPentacene). DPPentacene is soluble in various organic solvents, while pentacene, which is used in most of the triplet-DNP experiments, has limited solubility. An enhancement factor of 81 is obtained for 1H spins in the glass of ethanol-d6 : water = 80 : 20 (w/w) doped with 0.1 mM DPPentacene at 90 K in 0.67 T.
RESUMO
α-Helix-mediated protein-protein interactions (PPIs) are important targets in biological research and drug development. Peptides containing d-amino acid residues are attractive molecules for inhibiting α-helix-mediated PPIs because of their wide surface area and high protease resistance. In this study, a peptide library was constructed using a one-bead one-compound format designed to isolate left-handed α-helical peptides, which are promising molecules as inhibitors of α-helix-mediated PPIs. Screening of the library against an α-helix-mediated PPI between MDM2 and p53 yielded an inhibitor of the PPI. Design and screening of the library, and biochemical and spectroscopic studies of the discovered peptide are presented.
Assuntos
Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Humanos , Ligantes , Conformação Proteica em alfa-Hélice , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Estereoisomerismo , Proteína Supressora de Tumor p53/químicaRESUMO
The reprogramming of receptor-ligand interactions affords an opportunity to direct cells to respond to user-defined external cues. Although this has often been achieved via the genetic engineering of receptors, an alternative, nongenetic approach is highly demanded. In this article, we propose the design of oligonucleotide-based synthetic switches that feature the ability to reprogram the ligand specificity of the growth factor receptor. We demonstrated that our synthetic switches induced growth factor signaling via the formation of the dynamic complex with specific external cues that would otherwise not induce the signaling. This chemical approach may be applied to designing a new class of chemical tools that can control the activities of native cells and represent smart and safer regenerative drugs.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Ligantes , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oligonucleotídeos/química , Receptores Proteína Tirosina Quinases/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
Oligonucleotide-based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss-0, which was composed solely of 100-mer single-stranded DNA, exhibited nanomolar potency. Aptamer ss-0 reproduced HGF-induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.
Assuntos
Fator de Crescimento de Hepatócito/química , Mimetismo Molecular , Oligonucleotídeos/química , Sequência de Bases , Linhagem Celular , Dimerização , Humanos , Oligonucleotídeos/genética , Fosforilação , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
Aminopeptidaseâ N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1-(13) C]Ala-NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN-induced chemical shift change. The [1-(13) C]Ala-NH2 probe allowed sensitive detection of APN activity using (13) Câ NMR spectroscopy.