RESUMO
In this study, we propose a reversible covalent conjugation method for peptides, proteins, and even live cells based on specific recognition between natural amino acid sequences. Two heptad sequences can specifically recognize each other and induce the formation of a disulfide bond between cysteine residues. We show the covalent bond formation and dissociation between peptides and proteins in cell-free conditions and on the surface of live cells. Because heptad sequences consist of natural amino acids, they are expressed in cells without additional preparation and can be used to selectively conjugate peptides, proteins, and cells.
Assuntos
Cisteína , Peptídeos , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos , Domínios ProteicosRESUMO
Quorum sensing (QS) inhibitor-based therapy is an attractive strategy to inhibit bacterial biofilm formation without excessive induction of antibiotic resistance. Thus, we designed Ca2+-binding poly(lactide- co-glycolide) (PLGA) microparticles that can maintain a sufficient concentration of QS inhibitors around hydroxyapatite (HA) surfaces in order to prevent biofilm formation on HA-based dental or bone tissues or implants and, therefore, subsequent pathogenesis. Poly(butyl methacrylate- co-methacryloyloxyethyl phosphate) (PBMP) contains both Ca2+-binding phosphomonoester groups and PLGA-interacting butyl groups. The PBMP-coated PLGA (PLGA/PBMP) microparticles exhibited superior adhesion to HA surfaces without altering the sustained release properties of uncoated PLGA microparticles. PLGA/PBMP microparticle-encapsulating furanone C-30, a representative QS inhibitor, effectively inhibited the growth of Streptococcus mutans and its ability to form biofilms on HA surface for prolonged periods of up to 100 h, which was much longer than either furanone C-30 in its free form or when encapsulated in noncoated PLGA microparticles.
Assuntos
Biofilmes/efeitos dos fármacos , Cálcio/química , Durapatita/química , Furanos/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Polímeros/química , Percepção de Quorum/efeitos dos fármacos , Animais , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Furanos/química , Metacrilatos/química , Camundongos , Polímeros/síntese química , Streptococcus mutans/fisiologia , Propriedades de SuperfícieRESUMO
Cetuximab (CTX), a monoclonal antibody against epidermal growth factor receptor, is being widely used for colorectal cancer (CRC) with wild-type (WT) KRAS. However, its responsiveness is still very limited and WT KRAS is not enough to indicate such responsiveness. Here, by analyzing the gene expression data of CRC patients treated with CTX monotherapy, we have identified DUSP4, ETV5, GNB5, NT5E, and PHLDA1 as potential targets to overcome CTX resistance. We found that knockdown of any of these five genes can increase CTX sensitivity in KRAS WT cells. Interestingly, we further found that GNB5 knockdown can increase CTX sensitivity even for KRAS mutant cells. We unraveled that GNB5 overexpression contributes to CTX resistance by modulating the Akt signaling pathway from experiments and mathematical simulation. Overall, these results indicate that GNB5 might be a promising target for combination therapy with CTX irrespective of KRAS mutation.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Biomarcadores Tumorais/genética , Cetuximab/farmacologia , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Modelos Teóricos , Mutação , 5'-Nucleotidase/genética , Apoptose , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/genética , Fosfatases de Especificidade Dupla/genética , Proteínas Ligadas por GPI/genética , Perfilação da Expressão Gênica , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Transdução de Sinais , Análise de Sistemas , Fatores de Transcrição/genéticaRESUMO
An amphipathic leucine (L) and lysine (K)-rich α-helical peptide is multimerized based on helix-loop-helix structures to maximize the penetrating activities. The multimeric LK-based cell penetrating peptides (LK-CPPs) can penetrate cells as protein-fused forms at 100-1000-fold lower concentrations than Tat peptide. The enhanced penetrating activity is increased through multimerization by degrees up to the tetramer level. The multimeric LK-CPPs show rapid cell penetration through macropinocytosis at low nanomolar concentrations, unlike the monomeric LK, which have slower penetrating kinetics at much higher concentrations. The heparan sulfate proteoglycan (HSPG) receptors are highly involved in the rapid internalization of multimeric LK-CPPs. As a proof of concept of biomedical applications, an adipogenic transcription factor, peroxisome proliferator-activated receptor gamma 2 (PPAR-γâ2), is delivered into preadipocytes, and highly enhanced expression of adipogenic genes at nanomolar concentrations is induced. The multimeric CPPs can be a useful platform for the intracellular delivery of bio-macromolecular reagents that have difficulty with penetration in order to control biological reactions in cells at feasible concentrations for biomedical purposes.
RESUMO
We stapled an amphipathic peptide mainly consisting of leucine (L) and lysine (K) by an azobenzene (Ab) linker for photocontrol of the secondary structure. The cis- trans isomerization of the Ab moieties could stabilize and destabilize the α-helical conformation of the LK peptide along with dramatic change of associated peptide structures in a reversible manner by UV-vis irradiation. The cell-penetrating activities of the LK peptide can be readily regulated by the photocontrol, as the stabilized cis-Ab-LK peptide showed remarkable increase of cell penetration compared to the destabilized trans-Ab-LK peptide. The photoswitchable cell-penetrating peptides would provide important structural information for cell permeability as well as an effective targeting strategy for peptide-based pharmaceuticals with spatiotemporal specificity.