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1.
Biomolecules ; 14(7)2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-39062465

RESUMO

Safe and eco-friendly preservatives are crucial to preventing food spoilage and illnesses, as foodborne diseases caused by pathogens result in approximately 600 million cases of illness and 420,000 deaths annually. ε-Poly-L-lysine (ε-PL) is a novel food preservative widely used in many countries. However, its commercial application has been hindered by high costs and low production. In this study, ε-PL's biosynthetic capacity was enhanced in Streptomyces albulus WG608 through metabolic engineering guided by multi-omics techniques. Based on transcriptome and metabolome data, differentially expressed genes (fold change >2 or <0.5; p < 0.05) and differentially expressed metabolites (fold change >1.2 or <0.8) were separately subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The integrative analysis of transcriptome, metabolome, and overexpression revealed the essential roles of isocitrate lyase, succinate dehydrogenase, flavoprotein subunit, diaminopimelate dehydrogenase, polyphosphate kinase, and polyP:AMP phosphotransferase in ε-PL biosynthesis. Subsequently, a strain with enhanced ATP supply, L-lysine supply, and ε-PL synthetase expression was constructed to improve its production. Finally, the resulting strain, S. albulus WME10, achieved an ε-PL production rate of 77.16 g/L in a 5 L bioreactor, which is the highest reported ε-PL production to date. These results suggest that the integrative analysis of the transcriptome and metabolome can facilitate the identification of key pathways and genetic elements affecting ε-PL synthesis, guiding further metabolic engineering and thus significantly enhancing ε-PL production. The method presented in this study could be applicable to other valuable natural antibacterial agents.


Assuntos
Engenharia Metabólica , Polilisina , Streptomyces , Streptomyces/metabolismo , Streptomyces/genética , Engenharia Metabólica/métodos , Polilisina/biossíntese , Polilisina/metabolismo , Metaboloma , Transcriptoma , Metabolômica/métodos , Multiômica
2.
Theranostics ; 14(1): 203-219, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38164146

RESUMO

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.


Assuntos
Nanopartículas , Neoplasias da Bexiga Urinária , Camundongos , Animais , Humanos , Bexiga Urinária/patologia , Polilisina/metabolismo , Sistemas CRISPR-Cas/genética , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Terapia Genética
3.
J Periodontol ; 94(12): 1436-1449, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37133980

RESUMO

BACKGROUND: The aim of this study was to construct crosslinked polylysine-hyaluronic acid microspheres (pl-HAM) ladened with gingival mesenchymal stem cells (GMSCs) and explore its biologic behavior in soft tissue regeneration. METHODS: The effects of the crosslinked pl-HAM on the biocompatibility and the recruitment of L-929 cells and GMSCs were detected in vitro. Moreover, the regeneration of subcutaneous collagen tissue, angiogenesis and the endogenous stem cells recruitment were investigated in vivo. We also detected the cell developing capability of pl-HAMs. RESULTS: The crosslinked pl-HAMs appeared to be completely spherical-shaped particles and had good biocompatibility. L-929 cells and GMSCs grew around the pl-HAMs and increased gradually. Cell migration experiments showed that pl-HAMs combined with GMSCs could promote the migration of vascular endothelial cells significantly. Meanwhile, the green fluorescent protein-GMSCs in the pl-HAM group still remain in the soft tissue regeneration area 2 weeks after surgery. The results of in vivo studies showed that denser collagen deposition and more angiogenesis-related indicator CD31 expression in the pl-HAMs+ GMSCs + GeL group compared with the pl-HAMs + GeL group. Immunofluorescence showed that CD44, CD90, CD73 co-staining positive cells surrounded the microspheres in both pl-HAMs + GeL group and pl-HAM + GMSCs + GeL group. CONCLUSIONS: The crosslinked pl-HAM ladened with GMSCs system could provide a suitable microenvironment for collagen tissue regeneration, angiogenesis and endogenous stem cells recruitment, which may be an alternative to autogenous soft tissue grafts for minimally invasive treatments for periodontal soft tissue defects in the future.


Assuntos
Células-Tronco Mesenquimais , Polilisina , Polilisina/metabolismo , Polilisina/farmacologia , Ácido Hialurônico/farmacologia , Microesferas , Células Endoteliais , Angiogênese , Diferenciação Celular , Gengiva/metabolismo , Células-Tronco , Colágeno/metabolismo , Engenharia Tecidual
4.
Cells Tissues Organs ; 212(1): 8-20, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-34937023

RESUMO

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.


Assuntos
Heparina , Células-Tronco Mesenquimais , Humanos , Heparina/farmacologia , Heparina/metabolismo , Polilisina/farmacologia , Polilisina/química , Polilisina/metabolismo , Interferon gama/farmacologia , Interferon gama/metabolismo , Osteogênese , Diferenciação Celular
5.
Appl Environ Microbiol ; 88(20): e0095222, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36190251

RESUMO

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.


Assuntos
Polilisina , Transcriptoma , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos , Butanóis , Carbono , Citrato (si)-Sintase/metabolismo , Ácido Diaminopimélico/metabolismo , Etanol , Fermentação , Substâncias Perigosas , Metaboloma , NAD/metabolismo , Polilisina/metabolismo , Fator sigma/metabolismo , Ácidos Tricarboxílicos
6.
J Biochem ; 172(4): 205-216, 2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-35792074

RESUMO

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Histonas/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Peptidil Dipeptidase A/metabolismo , Polilisina/metabolismo , Ligação Proteica , SARS-CoV-2 , Proteínas e Peptídeos Salivares/metabolismo , Proteínas e Peptídeos Salivares/farmacologia , Glicoproteína da Espícula de Coronavírus
7.
J Biol Chem ; 298(6): 102039, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35595100

RESUMO

Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.


Assuntos
Biossíntese Peptídica , Polilisina , RNA Mensageiro , RNA de Transferência , Ribossomos , Peptídeos/metabolismo , Polilisina/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/metabolismo , Ribossomos/metabolismo
8.
Biochem Biophys Res Commun ; 596: 43-48, 2022 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-35108653

RESUMO

ε-poly-l-lysine (ε-PL) synthetase (Pls) is a membrane protein that possesses both adenylation and thiolation domains, characteristic of non-ribosomal peptide synthetases (NRPSs). Pls catalyzes the polymerization of l-Lys molecules in a highly specific manner within proteinogenic amino acids. However, this enzyme accepts certain l-Lys analogs which contain small substituent groups at the middle position of the side chain. From the crystal structures of the adenylation domain from NRPSs, the amino acid residues involved in substrate binding can be assumed; however, the precise interactions for better understanding the Pls recognition of l-Lys and its analogs have not yet been fully elucidated. Here, we determined the crystal structure of the adenylation domain of Pls in complex with the intermediate lysyl adenylate at 2.3 Å resolution. This is the first structure determination of the l-Lys activating adenylation domain. The crystal structure reveals that the shape of the substrate-binding pocket determines the specific recognition of l-Lys and its analogs and the electrostatic and hydrogen-bonding interactions further strengthen substrate binding. This study helps us understand the ε-PL synthesis mechanism and contributes to improving our knowledge of the molecular mechanism of NRPS adenylation domains towards their successful application in bioengineering.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Polilisina/metabolismo , Streptomyces/enzimologia , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Peptídeo Sintases/química , Peptídeo Sintases/genética , Ligação Proteica , Domínios Proteicos , Streptomyces/genética , Especificidade por Substrato
9.
Adv Sci (Weinh) ; 9(1): e2104128, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34738744

RESUMO

Diabetic ulcers, a difficult problem faced by clinicians, are strongly associated with an increase in cellular senescence. Few empirical studies have focused on exploring a targeted strategy to cure diabetic wounds by eliminating senescent fibroblasts (SFs) and reducing side effects. In this study, poly-l-lysine/sodium alginate (PLS) is modified with talabostat (PT100) and encapsulates a PARP1 plasmid (PARP1@PLS-PT100) for delivery to target the dipeptidyl peptidase 4 (DPP4) receptor and eliminate SFs. PARP1@PLS-PT100 releases encapsulated plasmids, displaying high selectivity for SFs over normal fibroblasts by targeting the DPP4 receptor, decreasing senescence-associated secretory phenotypes (SASPs), and stimulating the secretion of anti-inflammatory factors. Furthermore, the increased apoptosis of SFs and the disappearance of cellular senescence alleviates SASPs, accelerates re-epithelialization and collagen deposition, and significantly induces macrophage M2 polarization, which mediates tissue repair and the inflammatory response. This innovative strategy has revealed the previously undefined role of PARP1@PLS-PT100 in promoting diabetic wound healing, suggesting its therapeutic potential in refractory wound repair.


Assuntos
Alginatos/metabolismo , Senescência Celular/genética , Diabetes Mellitus Experimental/metabolismo , Dipeptidil Peptidase 4/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Polilisina/análogos & derivados , Cicatrização/genética , Animais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Dipeptidil Peptidase 4/genética , Modelos Animais de Doenças , Nanosferas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Polilisina/metabolismo , Ratos , Ratos Sprague-Dawley , Cicatrização/efeitos dos fármacos
10.
J Tissue Eng Regen Med ; 16(3): 279-289, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34788485

RESUMO

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. Neutralized CECM (N-CECM) suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than 4 weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions.


Assuntos
Condrócitos , Polilisina , Adesivos/análise , Adesivos/metabolismo , Animais , Células Endoteliais , Matriz Extracelular/metabolismo , Polilisina/análise , Polilisina/metabolismo , Polilisina/farmacologia , Aderências Teciduais/metabolismo , Aderências Teciduais/prevenção & controle
11.
J Immunother Cancer ; 9(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34193567

RESUMO

BACKGROUND: While adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based immunotherapy for central nervous system (CNS) tumors, such as glioblastoma (GBM), still needs to overcome multiple challenges, including effective homing and persistence of T-cells. Based on previous observations that interleukin (IL)-17-producing T-cells can traffic to the CNS in autoimmune conditions, we evaluated CD8+ T-cells that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells in a preclinical GBM model. METHODS: We differentiated Pmel-1 CD8+ T-cells into Tc17-1 cells and compared their phenotypic and functional characteristics with those of IFN-γ-producing CD8+ T (Tc1) and IL-17-producing CD8+ T (Tc17) cells. We also evaluated the therapeutic efficacy, persistence, and tumor-homing of Tc17-1 cells in comparison to Tc1 cells using a mouse GL261 glioma model. RESULTS: In vitro, Tc17-1 cells demonstrated profiles of both Tc1 and Tc17 cells, including production of both IFN-γ and IL-17, although Tc17-1 cells demonstrated lesser degrees of antigen-specific cytotoxic activity compared with Tc1 cells. In mice-bearing intracranial GL261-Quad tumor and treated with temozolomide, Tc1 cells, but not Tc17-1, showed a significant prolongation of survival. However, when the T-cell transfer was combined with poly-ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17-1 cells exhibited significantly prolonged survival associated with upregulation of very late activation antigen-4 on Tc17-1 cells in vivo. Glioma cells that recurred following the therapy lost the susceptibility to Pmel-1-derived cytotoxic T-cells, indicating that immuno-editing was a mechanism of the acquired resistance. CONCLUSIONS: Tc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Carboximetilcelulose Sódica/análogos & derivados , Glioma/terapia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Poli I-C/metabolismo , Polilisina/análogos & derivados , Vacinas de Subunidades Antigênicas/uso terapêutico , Carboximetilcelulose Sódica/metabolismo , Humanos , Polilisina/metabolismo
12.
ChemMedChem ; 16(15): 2345-2353, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34061468

RESUMO

The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6 h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics.


Assuntos
Antivirais/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Glicoconjugados/farmacologia , Lectinas Tipo C/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicoconjugados/síntese química , Glicoconjugados/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Manose/análogos & derivados , Manose/metabolismo , Manose/farmacologia , Testes de Sensibilidade Microbiana , Polilisina/análogos & derivados , Polilisina/metabolismo , Polilisina/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células THP-1 , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo
13.
Biophys Chem ; 266: 106440, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32771805

RESUMO

Thylakoid membranes of photosynthetic plant chloroplasts are involved in a wide range of energy producing pathways. Their stacking can be employed in order to provide increased surface area for biocatalytic purposes. Here we probe the aggregate formation of higher plants' thylakoids using low-molecular poly-l-lysine as an electrostatic polymer linker in low ionic strength media. Microelectrophoresis, actinic light scattering, millisecond-delayed fluorescence and free radical production of thylakoid membranes are measured and analyzed in the presence of the cationic polypeptide to track its influence on the surface electrical properties, the electron-transport processes and the proton gradient accumulation across membranes. Enhanced proton gradient in polylysine-treated thylakoids is obtained upon illumination due to alterations of the proton intake across the membrane resulting from the non-specific electrostatic interactions of the cationic polypeptide with thylakoids. We report lower rates of lipid peroxidation in polylysine-treated thylakoids measured both in the dark and under illumination in salt-free medium. The gained insight on the effect of polycations on photosynthetic membranes may be used in future developments of thylakoid-based approaches for energy transfer applications.


Assuntos
Polilisina/metabolismo , Tilacoides/metabolismo , Transporte de Elétrons , Folhas de Planta/química , Folhas de Planta/metabolismo , Polilisina/química , Prótons , Spinacia oleracea/química , Spinacia oleracea/metabolismo , Tilacoides/química
14.
ACS Appl Mater Interfaces ; 12(17): 19337-19344, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32249578

RESUMO

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from in vitro experiments probing fundamental molecular mechanisms of cell adhesion to more applied in vivo strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant αVß3 integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces via surface plasmon resonance (SPR). Next, SPR experiments were utilized to measure the extent of blocking of cRGD-functionalized surfaces by the commonly used agents BSA, PLL-g-PEG, and fetal calf serum (FCS)-supplemented media, using recombinant αVß3 integrin as a probe for cRGD binding activity in the presence of blocking agents. All three additives were highly efficient blockers of cRGD activity, as exemplified by cell culture media containing 1% FCS which reduced the cRGD activity by 33-fold. We then developed a strategy to combat these deleterious effects by employing the recombinant integrins as a protective cap. We show that the unblocked cRGD activity can be preserved in the presence of PLL-g-PEG by employing the αVß3 integrin as a removable protective cap, both in cell-free and in vitro experiments. In vitro studies with MDA-MB-231 cells cultured atop cRGD-functionalized surfaces found that cell adhesion and migration prevented by PLL-g-PEG were restored when this protective cap approach was used.


Assuntos
Integrina alfaVbeta3/metabolismo , Peptídeos Cíclicos/antagonistas & inibidores , Peptídeos Cíclicos/metabolismo , Polietilenoglicóis/metabolismo , Polilisina/análogos & derivados , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Polilisina/metabolismo , Ligação Proteica , Ressonância de Plasmônio de Superfície
15.
ACS Appl Mater Interfaces ; 12(15): 17302-17313, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32212678

RESUMO

Trypsin-responsive near-infrared fluorescent (NIRF) and magnetic resonance (MR) dual-imaging composite nanoparticle/polypeptide coacervate nanoprobes with tunable sizes, have been constructed herein via electrostatic interaction-induced self-assembly. Considering the requirements of in vivo metabolism on nanoparticle size, three coacervate nanoprobes with diameters of around 100, 200, and 300 nm were fabricated with a polydispersity of around 0.2. These coacervate nanoprobes consist of Fe3O4 magnetic nanoparticles surface-decorated with poly acrylic acid and Cy5.5-modified poly-l-lysine (PLL-g-Cy5.5) serving as MR imaging and trypsin-responsive substrate/NIRF agents, respectively. The notable fluorescence signal from PLL-g-Cy5.5 is self-quenched due to the short distances between the fluorescent Cy5.5 molecules after construction of the coacervate nanoprobes. Remarkably, coacervate nanoprobes with a diameter of around 100 nm are selectively disintegrated into fragmented segments upon the hydrolysis of PLL by trypsin, resulting in an 18-fold amplification of the NIRF intensity in comparison with the self-assembled coacervate nanoprobes in the quenched state. Moreover, the MR imaging enhancement is also related to the disintegration of the coacervate nanoprobes. Cellular experiments and in vivo studies demonstrate that the coacervate nanoprobes exhibit remarkable trypsin-sensitive NIRF and MR dual-imaging capabilities and thus have excellent potential to serve as dual-imaging nanoprobes for the efficient mapping of malignant tumors in which trypsin is often overexpressed. In consideration of their excellent capability to enrich charged molecules, the coacervate nanoprobes provide a conceptually novel and promising platform toward in vivo trypsin mapping and controlled delivery of targeted payloads.


Assuntos
Corantes Fluorescentes/química , Nanopartículas/química , Tripsina/metabolismo , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Feminino , Óxido Ferroso-Férrico/química , Corantes Fluorescentes/metabolismo , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Polilisina/química , Polilisina/metabolismo , Distribuição Tecidual , Transplante Heterólogo
16.
Colloids Surf B Biointerfaces ; 190: 110895, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32145605

RESUMO

Multistage delivery systems with size reduction capacity have been proposed as a powerful strategy for improving tissue drug penetration. Here we developed a simple and fast supramolecular approach to construct size-shrinkable polyamine-salt aggregates by ionic cross-linking of biodegradable poly-L-lysine dendrigraft with tripolyphosphate anion. The use of a peptide dendrimer as a nanobuilding block (∼7 nm in diameter) allows the formation of supraparticles (SPs) with well-defined dimensions (∼200 nm in diameter), narrow size distribution and great capacity to encapsulate different molecules, including chemotherapeutic agents as Curcumin and Doxorubicin. When exposed to slightly acidic environments, the crosslinked matrix is instantaneously disassembled to free dendrimer units. Subsequently, model cargo molecules entrapped in the dendrimer architecture can be released by the action of trypsin enzyme through peptide biodegradation. Therefore, these SPs with proved sequential pH and enzyme-responsiveness could be exploited as nanocarriers in multistage drug delivery systems.


Assuntos
Curcumina/química , Dendrímeros/química , Doxorrubicina/química , Peptídeos/química , Tripsina/química , Curcumina/metabolismo , Dendrímeros/síntese química , Dendrímeros/metabolismo , Doxorrubicina/metabolismo , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Estrutura Molecular , Tamanho da Partícula , Peptídeos/síntese química , Peptídeos/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Polilisina/química , Polilisina/metabolismo , Propriedades de Superfície , Tripsina/metabolismo
17.
Spectrochim Acta A Mol Biomol Spectrosc ; 227: 117563, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31689607

RESUMO

Phenothiazines are very effective antipsychotic drugs, which also have anticancer and antimicrobial activities. Despite being used in human treatment, the molecular mechanism of the biological actions of these molecules is not yet understood in detail. The role of the interactions between phenothiazines and proteins or lipid membranes has been much discussed. Herein, fourier-transform infrared (FTIR) spectroscopic studies were used to investigate the effect of three phenothiazines: fluphenazine (FPh); chlorpromazine (ChP); and propionylpromazine (PP) on the structures of a positively charged poly-l-lysine (PLL) peptide, a negatively charged dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) membrane, and on the mutual interactions between electrostatically associated PLL molecules and DPPC/DPPG membranes. Phenothiazine-induced alterations in the secondary structure of PLL, the conformational state (trans/gauche) of the hydrocarbon lipid chains, and the hydration of the DPPC/DPPG membrane interface were studied on the basis of amide I' vibrations, antisymmetric and symmetric stretching vibrations of the CH2 groups of the lipid hydrocarbon chains (νsCH2), and stretching vibrations of the lipid C=O groups (νC = O), respectively. It was shown that in the presence of negatively charged DPPC/DPPG membranes, the phenothiazines were able to modify the secondary structure of charged PLL molecules. Additionally, the effect of PLL on the structure of DPPC/DPPG membranes was also altered by the presence of the phenothiazine molecules.


Assuntos
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Antipsicóticos/farmacologia , Clorpromazina/farmacologia , Flufenazina/farmacologia , Fosfatidilgliceróis/metabolismo , Promazina/análogos & derivados , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Polilisina/metabolismo , Promazina/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier
18.
Small GTPases ; 11(3): 220-224, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-29239694

RESUMO

Ras proteins must localize to the plasma membrane (PM) for biological function. The membrane anchor of the K-Ras4B isoform comprises a farnesylated and methylated C-terminal cysteine together with an adjacent hexa-lysine polybasic domain (PBD). Traditionally, polybasic sequences have been thought to interact electrostatically with negatively charged membranes showing no specificity for anionic lipid head groups. By contrast we recently showed that the K-Ras membrane anchor actually exhibits a very high degree of specificity for phosphatidylserine (PtdSer). The selectivity for PtdSer is determined by a combinatorial code comprising the PBD sequence plus the prenyl anchor. Lipid binding specificity is therefore altered by PBD point mutations that in turn modulate signaling output. For example, mutating Lys177 or Lys178 to glutamine switches K-Ras4B lipid affinity from PtdSer to phosphoinositol 4,5-bisphosphate (PIP2). Changing the lipid anchor from farnesyl to geranylgeranyl or the PBD lysines to arginines also changes lipid binding specificity. All-atom molecular dynamics simulations reveal the structural basis for these K-Ras anchor lipid-binding preferences. Here we examine the PM interactions of a series of geranylgeranylated PBD mutants and provide further evidence that the precise PBD sequence and prenyl lipid determines lipid sorting specificity of the K-Ras anchor and hence biological function.


Assuntos
Membrana Celular/metabolismo , Lipídeos/química , Neopreno/metabolismo , Polilisina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Humanos , Neopreno/química , Polilisina/química , Polilisina/genética
19.
Nat Struct Mol Biol ; 26(12): 1132-1140, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768042

RESUMO

Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.


Assuntos
Peptídeos/metabolismo , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/química , Poli A/química , Poliadenilação , Polilisina/química , Polilisina/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/química
20.
Molecules ; 24(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627389

RESUMO

The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.


Assuntos
Quitosana/metabolismo , Técnicas de Transferência de Genes/classificação , Nanopartículas/metabolismo , Polietilenoimina/metabolismo , Polilisina/metabolismo , Animais , Transporte Biológico , Quitosana/química , Dextranos/química , Dextranos/metabolismo , Endossomos/metabolismo , Terapia Genética/métodos , Glucanos/química , Glucanos/metabolismo , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hidrólise , Lisossomos/metabolismo , Nanopartículas/química , Polietilenoimina/química , Polilisina/química
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