RESUMEN
The protein-induced fluorescence change technique was employed to investigate the interactions between proteins and their DNA substrates modified with the Cy3 fluorophore. It has been reported that the human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal proline-tryptophan-tryptophan-proline (PWWP) domain (the N-terminal 100 amino acids of HDGF) capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancer. This project investigated the specific binding behavior of HDGF, the PWWP domain, and the C140 domain (the C-terminal 140 amino acids of HDGF) sequentially using protein-induced fluorescence change. We found that the binding of HDGF and its related proteins on Cy3-labeled 15 bp SMYD1 dsDNA will cause a significant decrease in the recorded Cy3 fluorophore intensity, indicating the occurrence of protein-induced fluorescence quenching. The dissociation equilibrium constant was determined by fitting the bound fraction curve to a binding model. An approximate 10-time weaker SMYD1 binding affinity of the PWWP domain was found in comparison to HDGF. Moreover, the PWWP domain is required for DNA binding, and the C140 domain can enhance the DNA binding affinity. Furthermore, we found that the C140 domain can regulate the sequence-specific binding capability of HDGF on SMYD1.
Asunto(s)
Proteínas de Unión al ADN , ADN , Péptidos y Proteínas de Señalización Intercelular , Unión Proteica , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Dominios Proteicos , Sitios de Unión , Carbocianinas/química , Proteínas Musculares , Factores de TranscripciónRESUMEN
Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment protocols. Studies have highlighted the potential of antimicrobial peptides sourced from Vespula lewisii venom, particularly those belonging to the mastoparan family, as effective against HSV-1. This study aimed to demonstrate the antiviral properties of mastoparans, including mastoparan-L [I5, R8], mastoparan-MO, and [I5, R8] mastoparan, against HSV-1. Initially, Vero cell viability was assessed in the presence of these peptides, followed by the determination of antiviral activity, mechanism of action, and dose-response curves through plaque assays. Structural analyses via circular dichroism and nuclear magnetic resonance were conducted, along with evaluating membrane fluidity changes induced by [I5, R8] mastoparan using fluorescence-labeled lipid vesicles. Cytotoxic assays revealed high cell viability (>80%) at concentrations of 200 µg/mL for mastoparan-L and mastoparan-MO and 50 µg/mL for [I5, R8] mastoparan. Mastoparan-MO and [I5, R8] mastoparan exhibited over 80% HSV-1 inhibition, with up to 99% viral replication inhibition, particularly in the early infection stages. Structural analysis indicated an α-helical structure for [I5, R8] mastoparan, suggesting effective viral particle disruption before cell attachment. Mastoparans present promising prospects for HSV-1 infection control, although further investigation into their mechanisms is warranted.
Asunto(s)
Antivirales , Herpesvirus Humano 1 , Péptidos y Proteínas de Señalización Intercelular , Péptidos , Venenos de Avispas , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/fisiología , Antivirales/farmacología , Antivirales/química , Animales , Células Vero , Chlorocebus aethiops , Péptidos/farmacología , Péptidos/química , Venenos de Avispas/farmacología , Venenos de Avispas/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Supervivencia Celular/efectos de los fármacos , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
Pulp and periapical diseases can lead to the cessation of tooth development, resulting in compromised tooth structure and functions. Despite numerous efforts to induce pulp regeneration, effective strategies are still lacking. Growth factors (GFs) hold considerable promise in pulp regeneration due to their diverse cellular regulatory properties. However, the limited half-lives and susceptibility to degradation of exogenous GFs necessitate the administration of supra-physiological doses, leading to undesirable side effects. In this research, a heparin-functionalized bioactive glass (CaO-P2O5-SiO2-Heparin, abbreviated as PSC-Heparin) with strong bioactivity and a stable neutral pH is developed as a promising candidate to addressing challenges in pulp regeneration. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis reveal the successful synthesis of PSC-Heparin. Scanning electron microscopy and X-ray diffraction show the hydroxyapatite formation can be observed on the surface of PSC-Heparin after soaking in simulated body fluid for 12 h. PSC-Heparin is capable of harvesting various endogenous GFs and sustainably releasing them over an extended duration by the enzyme-linked immunosorbent assay. Cytological experiments show that developed PSC-Heparin can facilitate the adhesion, migration, proliferation, and odontogenic differentiation of stem cells from apical papillae. Notably, the histological analysis of subcutaneous implantation in nude mice demonstrates PSC-Heparin is capable of promoting the odontoblast-like layers and pulp-dentin complex formation without the addition of exogenous GFs, which is vital for clinical applications. This work highlights an effective strategy of harvesting endogenous GFs and avoiding the involvement of exogenous GFs to achieve pulp-dentin complex regeneration, which may open a new horizon for regenerative endodontic therapy.
Asunto(s)
Pulpa Dental , Heparina , Regeneración , Heparina/química , Heparina/farmacología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/metabolismo , Animales , Regeneración/efectos de los fármacos , Ratones , Vidrio/química , Humanos , Ratones Desnudos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Células Madre/efectos de los fármacos , Células Madre/citología , Células Madre/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacosRESUMEN
The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.
Asunto(s)
Integrasa de VIH , VIH-1 , Integración Viral , Integrasa de VIH/metabolismo , Integrasa de VIH/química , Integrasa de VIH/genética , VIH-1/metabolismo , VIH-1/fisiología , Humanos , ADN Viral/metabolismo , ADN Viral/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/químicaRESUMEN
In the conventional secretory pathway, cargo receptors play important roles in exporting newly synthesized secretory proteins from the endoplasmic reticulum (ER). We previously showed that a cargo receptor, surfeit locus protein 4 (SURF4), promotes ER export of a soluble signaling molecule, sonic hedgehog, via recognizing the polybasic residues within its Cardin-Weintraub motif. In addition to sonic hedgehog, we found 30 more secretory proteins containing the polybasic motif (K/R)(K/R)(K/R)XX(K/R)(K/R), but whether SURF4 plays a general role in mediating ER export of these secretory proteins is unclear. Here, we analyzed the trafficking of four of these secretory proteins: desert hedgehog, Indian hedgehog, bone morphogenetic protein 8A (BMP8A), and secreted frizzled-related protein 1 (SFRP1). We found that the polybasic motifs contained in these cargo proteins are important for their ER export. Further analyses indicated that the polybasic motifs of BMP8A and SFRP1 interact with the triacidic motif on the predicted first luminal domain of SURF4. These interactions with SURF4 are essential and sufficient for the ER-to-Golgi trafficking of BMP8A and SFRP1. Moreover, we demonstrated that SURF4 localizes at a subpopulation of ER exit sites to regulate the ER export of its clients. Taken together, these results suggest that SURF4 is recruited to specific ER exit sites and plays a general role in capturing polybasic motif-containing secretory cargo proteins through electrostatic interactions.
Asunto(s)
Retículo Endoplásmico , Proteínas Hedgehog , Humanos , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Secuencias de AminoácidosRESUMEN
Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.
Asunto(s)
Antiarrítmicos , Péptidos y Proteínas de Señalización Intercelular , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Neurotoxinas , Péptidos , Venenos de Araña , Animales , Antiarrítmicos/química , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Lípidos , Ratones , Neurotoxinas/química , Neurotoxinas/farmacología , Péptidos/química , Péptidos/farmacología , Venenos de Araña/química , Venenos de Araña/farmacología , Venenos de Araña/uso terapéuticoRESUMEN
The high mortality associated with invasive fungal infections, narrow spectrum of available antifungals, and increasing evolution of antifungal resistance necessitate the development of alternative therapies. Host defense peptides are regarded as the first line of defense against microbial invasion in both vertebrates and invertebrates. In this work, we investigated the effectiveness of four naturally occurring pore-forming antimicrobial peptides (melittin, magainin 2, cecropin A, and mastoparan B) against a panel of clinically relevant pathogens, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida glabrata. We present data on the antifungal activities of the four pore-forming peptides, assessed with descriptive statistics, and their cytocompatibility with cultured human cells. Among the four peptides, mastoparan B (MB) displayed potent antifungal activity, whereas cecropin A was the least potent. We show that MB susceptibility of phylogenetically distant non-candida albicans can vary and be described by different intrinsic physicochemical parameters of pore-forming α-helical peptides. These findings have potential therapeutic implications for the design and development of safe antifungal peptide-based drugs.
Asunto(s)
Antifúngicos/química , Antifúngicos/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Proteica , Análisis Espectral , Relación Estructura-ActividadRESUMEN
Sulfated glycosaminoglycan (GAG) analogues derived from plant, algae or microbial sourced polysaccharides are highly interesting in order to gain bioactivities similar to sulfated GAGs but without risks and concerns derived from their typical animal sources. Since the exopolysaccharide (EPS) produced by the bacterium Vibrio diabolicus HE800 strain from deep-sea hydrothermal vents is known to have a GAG-like structure with a linear backbone composed of unsulfated aminosugar and uronic acid monomers, its structural modification through four different semi-synthetic sulfation strategies has been performed. A detailed structural characterization of the six obtained polysaccharides revealed that three different sulfation patterns (per-O-sulfation, a single N-sulfation and a selective primary hydroxyls sulfation) were achieved, with molecular weights ranging from 5 to 40 kDa. A Surface Plasmonic Resonance (SPR) investigation of the affinity between such polysaccharides and a set of growth factors revealed that binding strength is primarily depending on polysaccharide sulfation degree.
Asunto(s)
Glicosaminoglicanos/química , Polisacáridos Bacterianos/química , Vibrio , Amino Azúcares/química , Animales , Péptidos y Proteínas de Señalización Intercelular/química , Espectroscopía de Resonancia Magnética/métodos , Peso Molecular , Sulfatos/química , Resonancia por Plasmón de Superficie/métodos , Ácidos Urónicos/químicaRESUMEN
Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.
Asunto(s)
Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Cromatografía de Afinidad/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Recombinantes/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/agonistas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Proteínas Recombinantes/genéticaRESUMEN
Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.
Asunto(s)
Anticoagulantes/química , Heparitina Sulfato/química , Mucosa Intestinal/química , Animales , Disacáridos/análisis , Factor Xa/química , Péptidos y Proteínas de Señalización Intercelular/química , PorcinosRESUMEN
This work aims to use carboxymethyl cellulose (CMC) as main structural and functional component of 3D printed scaffolds for healing of diabetic wounds. Differently from previous inks involving small contents in CMC, herein sterile (steam-heated) concentrated CMC solely dispersions (10-20%w/v) were screened regarding printability and fidelity properties. CMC (15%w/v)-citric acid inks showed excellent self-healing rheological properties and stability during storage. CMC scaffolds loaded with platelet rich plasma (PRP) sustained the release of relevant growth factors. CMC scaffolds both with and without PRP promoted angiogenesis in ovo, stem cell migration in vitro, and wound healing in a diabetic model in vivo. Transparent CMC scaffolds allowed direct monitoring of bilateral full-thickness wounds created in rat dorsum. CMC scaffolds facilitated re-epithelialization, granulation, and angiogenesis in full-thickness skin defects, and the performance was improved when combined with PRP. Overall, CMC is pointed out as outstanding component of active dressings for diabetic wounds.
Asunto(s)
Carboximetilcelulosa de Sodio/química , Sistemas de Liberación de Medicamentos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Impresión Tridimensional , Andamios del Tejido/química , Cicatrización de Heridas/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 1 , Péptidos y Proteínas de Señalización Intercelular/química , Masculino , Tamaño de la Partícula , Plasma Rico en Plaquetas/química , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta1/química , Factores de Crecimiento Endotelial Vascular/químicaRESUMEN
Antimicrobial peptides are an important class of therapeutic agent used against a wide range of pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses. Mastoparan (MpVT) is an α-helix and amphipathic tetradecapeptide obtained from Vespa tropica venom. This peptide exhibits antibacterial activity. In this work, we investigate the effect of amino acid substitutions and deletion of the first three C-terminal residues on the structure-activity relationship. In this in silico study, the predicted structure of MpVT and its analog have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bonds. The secondary structure and the biological activity of six designed analogs are studied. The biological activity assays show that the substitution of phenylalanine (MpVT1) results in a higher antibacterial activity than that of MpVT without increasing toxicity. The analogs with the first three deleted C-terminal residues showed decreased antibacterial and hemolytic activity. The CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 40% 2,2,2-trifluoroethanol (TFE). In conclusion, the first three C-terminal deletions reduced the length of the α-helix, explaining the decreased biological activity. MpVTs show that the hemolytic activity of mastoparan is correlated to mean hydrophobicity and mean hydrophobic moment. The position and spatial arrangement of specific hydrophobic residues on the non-polar face of α-helical AMPs may be crucial for the interaction of AMPs with cell membranes.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Venenos de Avispas/química , Venenos de Avispas/farmacología , Sustitución de Aminoácidos , Animales , Antibacterianos/síntesis química , Péptidos Antimicrobianos/síntesis química , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Estructurales , Estructura Secundaria de Proteína , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Avispas/químicaRESUMEN
The clinical success of platelet-rich plasma (PRP) is constrained by its limited mechanical strength, rapid disintegration by lytic enzymes, and the consequent short-term release of bioactive growth factors (GFs). Recently, attempts to formulate PRP and other hemoderivatives, such as platelet lysate (PL) have been underway. The current study aimed to formulate allogeneic freeze-dried human platelet lysate (HPL) onto lyophilized chitosan - dipotassium hydrogen orthophosphate (CS/DHO) thermo-sensitive scaffolds. A systemic approach was employed to optimize freeze-drying (FD) procedures targeting predefined critical quality attributes (CQAs). Thermal behavior, vibrational spectroscopy, morphological and moisture content analyses were used to detect possible protein destabilization during formulation and suboptimal cake properties. The effect of CS/DHO concentrations on thermo-responsiveness and release kinetics were investigated. Finally, six-months stability and cytotoxicity studies were carried out. An optimized lyophilizate was attainable with residual moisture of less than 5% and thermoresponsive to 33 °C in less than 3 min. HPL proteins were sustainedly released over five days in a pH-sensitive manner. The stability study indicated plausible physical and biochemical stability. Cell viability testing supported the cytocompatibility of the system. Finally, the lyophilizate variant of CS/DHO thermogel overcomes limited storage stability previously posed as a challenge in freshly prepared thermogels. The developed system overcomes the drawbacks of currently used PRP treatment and provides a novel GF-rich scaffold for wound repair.
Asunto(s)
Plaquetas/química , Quitosano/química , Geles/química , Péptidos y Proteínas de Señalización Intercelular/química , Fosfatos/química , Plasma Rico en Plaquetas/química , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Liofilización , Humanos , Concentración de Iones de Hidrógeno , Tecnología FarmacéuticaRESUMEN
Hormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes, hrmI/belK and hrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase-like dinuclear iron enzymes, catalyze oxidation of the ϵ-amino group of l-lysine to afford l-6-nitronorleucine. Subsequently, HrmJ and BelL, which are iron- and α-ketoglutarate-dependent oxygenases, effectively convert l-6-nitronorleucine into 3-(trans-2-nitrocyclopropyl)-alanine through C4-C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.
Asunto(s)
Ciclopropanos/metabolismo , Depsipéptidos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Metaloproteínas/metabolismo , Catálisis , Ciclopropanos/química , Depsipéptidos/química , Péptidos y Proteínas de Señalización Intercelular/química , Metaloproteínas/química , Estructura MolecularRESUMEN
AMPA receptor mediate most fast excitatory synaptic transmission and play a key role in synaptic plasticity in the central nervous system (CNS) by trafficking and targeting of its subunits to individual postsynaptic membrane. Collapsing response mediator protein 2 (CRMP2), an intracellular phospho-protein, has been reported to promote the maturation of the dendritic spine and transfer AMPA receptors to the membrane. However, our knowledge about the molecular mechanisms of CRMP2 regulating AMPA receptors trafficking is limited. Here, we reported that CRMP2 promoted the surface expression of AMPA receptor GluA1 subunit in cultured hippocampal neurons and in HEK293T cells expressing GluA1 subunits. Furthermore, we found that CRMP2 interacted with GluA1, and their interaction was inhibited by CRMP2 phosphorylation at ser522. Moreover, our results showed that phosphorylation of CRMP2 at ser522 by cyclin-dependent kinase 5 (Cdk5) decreased the fluorescence intensity of surface GluA1 and the amplitude and frequency of miniature excitatory synaptic currents (mEPSCs) in cultured hippocampal neurons, indicating a reduction levels and synaptic function of AMPA receptors. Taken together, our data demonstrated that phosphorylation of CRMP2 by Cdk5 is important for AMPA receptor surface delivery in hippocampal neurons.
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Quinasa 5 Dependiente de la Ciclina , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso , Receptores AMPA , Quinasa 5 Dependiente de la Ciclina/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Proteínas del Tejido Nervioso/química , Fosforilación , Receptores AMPA/metabolismo , Transmisión SinápticaRESUMEN
BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs. MATERIALS AND METHODS: OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array. RESULTS: The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM. CONCLUSION: DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.
Asunto(s)
Adenocarcinoma Escirroso/genética , Dipeptidil Peptidasa 4/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Receptores CXCR4/genética , Neoplasias Gástricas/genética , Adenocarcinoma Escirroso/patología , Fibroblastos Asociados al Cáncer/química , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Medios de Cultivo Condicionados/química , Dipeptidil Peptidasa 4/química , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Proteínas de Neoplasias/genética , Neoplasias Gástricas/patologíaRESUMEN
Myocardial infarction (MI) is one of the leading causes of heart-related deaths worldwide. Following MI, the hypoxic microenvironment triggers apoptosis, disrupts the extracellular matrix and forms a non-functional scar that leads towards adverse left ventricular (LV) remodelling. If left untreated this eventually leads to heart failure. Besides extensive advancement in medical therapy, complete functional recovery is never accomplished, as the heart possesses limited regenerative ability. In recent decades, the focus has shifted towards tissue engineering and regenerative strategies that provide an attractive option to improve cardiac regeneration, limit adverse LV remodelling and restore function in an infarcted heart. Acellular scaffolds possess attractive features that have made them a promising therapeutic candidate. Their application in infarcted areas has been shown to improve LV remodelling and enhance functional recovery in post-MI hearts. This review will summarise the updates on acellular scaffolds developed and tested in pre-clinical and clinical scenarios in the past five years with a focus on their ability to overcome damage caused by MI. It will also describe how acellular scaffolds alone or in combination with biomolecules have been employed for MI treatment. A better understanding of acellular scaffolds potentialities may guide the development of customised and optimised therapeutic strategies for MI treatment.
Asunto(s)
Cardiotónicos/farmacología , Andamios del Tejido , Remodelación Ventricular , Animales , Materiales Biocompatibles , Cardiotónicos/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/química , Vesículas Extracelulares/química , Terapia Genética/métodos , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Infarto del Miocardio/patología , Polímeros/química , Proteínas/químicaRESUMEN
BACKGROUND: Platelet-rich fibrin (PRF) has gained popularity in craniofacial surgery, as it provides an excellent reservoir of autologous growth factors (GFs) that are essential for bone regeneration. However, the low elastic modulus, short-term clinical application, poor storage potential and limitations in emergency therapy use restrict its more widespread clinical application. This study fabricates lyophilised PRF (Ly-PRF), evaluates its physical and biological properties, and explores its application for craniofacial tissue engineering purposes. MATERIAL AND METHODS: A lyophilisation method was applied, and the outcome was evaluated and compared with traditionally prepared PRF. We investigated how lyophilisation affected PRF's physical characteristics and biological properties by determining: (1) the physical and morphological architecture of Ly-PRF using SEM, and (2) the kinetic release of PDGF-AB using ELISA. RESULTS: Ly-PRF exhibited a dense and homogeneous interconnected 3D fibrin network. Moreover, clusters of morphologically consistent cells of platelets and leukocytes were apparent within Ly-PRF, along with evidence of PDGF-AB release in accordance with previously reports. CONCLUSIONS: The protocol established in this study for Ly-PRF preparation demonstrated versatility, and provides a biomaterial with growth factor release for potential use as a craniofacial bioscaffold.
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Péptidos y Proteínas de Señalización Intercelular/química , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Fibrina Rica en Plaquetas/química , Ingeniería de Tejidos , Adulto , Plaquetas/química , Plaquetas/metabolismo , Regeneración Ósea/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Liofilización , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Leucocitos/química , Masculino , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Fibrina Rica en Plaquetas/metabolismo , Donantes de Tejidos , Adulto JovenRESUMEN
Corosolic acid (CA), a bioactive compound obtained from Actinidia chinensis, has potential anti-cancer activities. Glioblastoma (GBM) is a malignant brain tumor and whether CA exerts anti-cancer activity on GBM remains unclear. This study was aimed to explore the anticancer activity and its underlying mechanism of CA in GBM cells. Our findings showed that CA ≤ 20 µM did not affect cell viability and cell proliferative rate of normal astrocyte and four GBM cells. Notably, 10 or 20 µM CA significantly inhibited cell migration and invasion of three GBM cells, decreased the protein level of F-actin and disrupted F-actin polymerization in these GBM cells. Further investigation revealed that CA decreased AXL level by promoting ubiquitin-mediated proteasome degradation and upregulating the carboxyl terminus of Hsc70-interacting protein (CHIP), an inducer of AXL polyubiquitination. CHIP knock-down restored the CA-reduced AXL and invasiveness of GBM cells. Additionally, we observed that CA-reduced Growth arrest-specific protein 6 (GAS6) and inhibited JAK2/MEK/ERK activation, and GAS6 pre-treatment restored attenuated JAK2/MEK/ERK activation and invasiveness of GBM cells. Furthermore, molecular docking analysis revealed that CA might bind to GAS6 and AXL. These findings collectively indicate that CA attenuates the invasiveness of GBM cells, attributing to CHIP upregulation and binding to GAS6 and AXL and subsequently promoting AXL degradation and downregulating GAS6-mediated JAK2/MEK/ERK cascade. Conclusively, this suggests that CA has potential anti-metastatic activity on GBM cells by targeting the CHIP/GAS6/AXL axis.
Asunto(s)
Glioblastoma/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Quinasas Janus/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Triterpenos/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Simulación del Acoplamiento Molecular , Invasividad Neoplásica , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Ratas , Proteínas Tirosina Quinasas Receptoras/química , Transducción de Señal/efectos de los fármacos , Triterpenos/química , Ensayo de Tumor de Célula Madre , Ubiquitina/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
Ostrinia nubilalis, a lepidopteran moth, also known as the European corn borer, has a major impact on the production of economically important crops in the United States and Europe. The female moth invites the male moth for mating through the release of pheromones, a volatile chemical signal. Pheromone binding proteins (PBPs) present in the male moth antennae are believed to pick up the pheromones, transport them across the aqueous sensillum lymph, and deliver them to the olfactory receptor neurons. Here we report for the first time the cloning, expression, refolding, purification, and structural characterization of Ostrinia nubilalis PBP3 (OnubPBP3). The recombinant protein showed nanomolar affinity to each isomer of the Ostrinia pheromones, E- and Z-11-tetradecenyl acetate. In a pH titration study by nuclear magnetic resonance, the protein exhibited an acid-induced unfolding at pH below 5.5. The molecular dynamics simulation study demonstrated ligand-induced conformational changes in the protein with both E- and Z-isomers of the Ostrinia pheromone. The simulation studies showed that while protein flexibility decreases upon binding to E-pheromone, it increases when bound to Z-pheromone. This finding suggests that the OnubPBP3 complex with E-pheromone is more stable than with Z-pheromone.