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1.
Nat Commun ; 12(1): 5204, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471136

RESUMO

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.


Assuntos
Retículo Endoplasmático/metabolismo , Canais de Translocação SEC/metabolismo , Via Secretória/fisiologia , Animais , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteoma/metabolismo , Proteômica
2.
Nat Commun ; 12(1): 5101, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429411

RESUMO

3' untranslated regions (3' UTRs) post-transcriptionally regulate mRNA stability, localization, and translation rate. While 3'-UTR isoforms have been globally quantified in limited cell types using bulk measurements, their differential usage among cell types during mammalian development remains poorly characterized. In this study, we examine a dataset comprising ~2 million nuclei spanning E9.5-E13.5 of mouse embryonic development to quantify transcriptome-wide changes in alternative polyadenylation (APA). We observe a global lengthening of 3' UTRs across embryonic stages in all cell types, although we detect shorter 3' UTRs in hematopoietic lineages and longer 3' UTRs in neuronal cell types within each stage. An analysis of RNA-binding protein (RBP) dynamics identifies ELAV-like family members, which are concomitantly induced in neuronal lineages and developmental stages experiencing 3'-UTR lengthening, as putative regulators of APA. By measuring 3'-UTR isoforms in an expansive single cell dataset, our work provides a transcriptome-wide and organism-wide map of the dynamic landscape of alternative polyadenylation during mammalian organogenesis.


Assuntos
Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Poliadenilação , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Células NIH 3T3 , Neurônios/metabolismo , Organogênese , Isoformas de Proteínas , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Transcriptoma
3.
Int J Mol Sci ; 22(15)2021 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-34360683

RESUMO

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.


Assuntos
Adesão Celular , Domínios Proteicos , Transdução de Sinais , Sindecana-2/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Sindecana-2/fisiologia , Quinases da Família src/metabolismo
4.
Nat Commun ; 12(1): 4789, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373451

RESUMO

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.


Assuntos
Neoplasias Pulmonares/genética , Oncogenes/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Terapia de Alvo Molecular , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras
5.
Nat Commun ; 12(1): 4693, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344862

RESUMO

Many cellular processes, including cell division, development, and cell migration require spatially and temporally coordinated forces transduced by cell-surface receptors. Nucleic acid-based molecular tension probes allow one to visualize the piconewton (pN) forces applied by these receptors. Building on this technology, we recently developed molecular force microscopy (MFM) which uses fluorescence polarization to map receptor force orientation with diffraction-limited resolution (~250 nm). Here, we show that structured illumination microscopy (SIM), a super-resolution technique, can be used to perform super-resolution MFM. Using SIM-MFM, we generate the highest resolution maps of both the magnitude and orientation of the pN traction forces applied by cells. We apply SIM-MFM to map platelet and fibroblast integrin forces, as well as T cell receptor forces. Using SIM-MFM, we show that platelet traction force alignment occurs on a longer timescale than adhesion. Importantly, SIM-MFM can be implemented on any standard SIM microscope without hardware modifications.


Assuntos
Microscopia de Fluorescência/métodos , Receptores de Superfície Celular/metabolismo , Animais , Fenômenos Biomecânicos , Plaquetas/metabolismo , Linfócitos T CD8-Positivos , Corantes Fluorescentes/metabolismo , Humanos , Integrinas/metabolismo , Camundongos , Sondas Moleculares/metabolismo , Células NIH 3T3 , Paxilina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Imagem com Lapso de Tempo
6.
Molecules ; 26(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34361644

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease with multiple causes, characterized by excessive myofibrocyte aggregation and extracellular matrix deposition. Related studies have shown that transforming growth factor-ß1 (TGF-ß1) is a key cytokine causing fibrosis, promoting abnormal epithelial-mesenchymal communication and fibroblast-to-myofibroblast transition. Fedratinib (Fed) is a marketed drug for the treatment of primary and secondary myelofibrosis, targeting selective JAK2 tyrosine kinase inhibitors. However, its role in pulmonary fibrosis remains unclear. In this study, we investigated the potential effects and mechanisms of Fed on pulmonary fibrosis in vitro and in vivo. In vitro studies have shown that Fed attenuates TGF-ß1- and IL-6-induced myofibroblast activation and inflammatory response by regulating the JAK2/STAT3 signaling pathway. In vivo studies have shown that Fed can reduce bleomycin-induced inflammation and collagen deposition and improve lung function. In conclusion, Fed inhibited inflammation and fibrosis processes induced by TGF-ß1 and IL-6 by targeting the JAK2 receptor.


Assuntos
Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Janus Quinase 2/metabolismo , Pirrolidinas/farmacologia , Sulfonamidas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Bleomicina , Movimento Celular/efeitos dos fármacos , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/induzido quimicamente , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3
7.
Nat Commun ; 12(1): 4964, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400628

RESUMO

Immunological adjuvants are essential for successful cancer vaccination. However, traditional adjuvants have some limitations, such as lack of controllability and induction of systemic toxicity, which restrict their broad application. Here, we present a light-activable immunological adjuvant (LIA), which is composed of a hypoxia-responsive amphiphilic dendrimer nanoparticle loaded with chlorin e6. Under irradiation with near-infrared light, the LIA not only induces tumour cell lysis and tumour antigen release, but also promotes the structural transformation of 2-nitroimidazole containing dendrimer to 2-aminoimidazole containing dendrimer which can activate dendritic cells via the Toll-like receptor 7-mediated signaling pathway. The LIA efficiently inhibits both primary and abscopal tumour growth and induces strong antigen-specific immune memory effect to prevent tumour metastasis and recurrence in vivo. Furthermore, LIA localizes the immunological adjuvant effect at the tumour site. We demonstrate this light-activable immunological adjuvant offers a safe and potent platform for in situ cancer vaccination.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Anticâncer/imunologia , Dendrímeros/farmacologia , Vacinação , Animais , Antígenos de Neoplasias , Antitussígenos , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Humanos , Hipóxia , Imunoterapia , Luz , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Nanopartículas/química , Metástase Neoplásica/prevenção & controle , Recidiva Local de Neoplasia , Neoplasias/genética , Neoplasias/prevenção & controle , Porfirinas , Transcriptoma
8.
In Vivo ; 35(5): 2719-2728, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34410961

RESUMO

BACKGROUND/AIM: Acellular dermal matrices (ADMs) have become popular in implant-based breast reconstruction. The aim of this study was to compare three commonly used ADM products in vivo in an animal model. MATERIALS AND METHODS: The nucleic acid content (residual double-stranded DNA) and the levels of the remaining growth factors after decellularization were measured for each ADM. Cytocompatibility with ADMs was documented using NIH 3T3 mouse fibroblast cells. In vivo, the implanted ADMs were histologically evaluated at 1, 2, 3, and 6 months (n=5) using male 8-week-old Sprague-Dawley rats. RESULTS: Fibroblasts grew in the SureDerm HD and DermACELL with no cytotoxicity. In a rat model, SureDerm HD and DermACELL incorporated more readily into the surrounding host tissue, as measured by rapid cell influx and collagen deposition, and showed more delayed tissue remodeling with decreased matrix metalloproteinases levels compared to AlloDerm. CONCLUSION: SureDerm HD and DermACELL can be used as biological materials for breast reconstruction.


Assuntos
Derme Acelular , Mamoplastia , Animais , Humanos , Masculino , Camundongos , Células NIH 3T3 , Ratos , Ratos Sprague-Dawley
9.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445307

RESUMO

Hypoxic conditions induce the activation of hypoxia-inducible factor-1α (HIF-1α) to restore the supply of oxygen to tissues and cells. Activated HIF-1α translocates into the nucleus and binds to hypoxia response elements to promote the transcription of target genes. Cathepsin L (CTSL) is a lysosomal protease that degrades cellular proteins via the endolysosomal pathway. In this study, we attempted to determine if CTSL is a hypoxia responsive target gene of HIF-1α, and decipher its role in melanocytes in association with the autophagic pathway. The results of our luciferase reporter assay showed that the expression of CTSL is transcriptionally activated through the binding of HIF1-α at its promoter. Under autophagy-inducing starvation conditions, HIF-1α and CTSL expression is highly upregulated in melan-a cells. The mature form of CTSL is closely involved in melanosome degradation through lysosomal activity upon autophagosome-lysosome fusion. The inhibition of conversion of pro-CTSL to mature CTSL leads to the accumulation of gp100 and tyrosinase in addition to microtubule-associated protein 1 light chain 3 (LC3) II, due to decreased lysosomal activity in the autophagic pathway. In conclusion, we have identified that CTSL, a novel target of HIF-1α, participates in melanosome degradation in melanocytes through lysosomal activity during autophagosome-lysosome fusion.


Assuntos
Catepsina L/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Melanossomas/metabolismo , Animais , Catepsina L/genética , Hipóxia Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Melanócitos/metabolismo , Camundongos , Células NIH 3T3
10.
Methods Mol Biol ; 2312: 277-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228296

RESUMO

There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.


Assuntos
Engenharia Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas , Proteínas de Ligação a Tacrolimo/genética , Animais , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Agregados Proteicos , Estabilidade Proteica , Proteostase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Transfecção
11.
Chemistry ; 27(48): 12395-12409, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34213045

RESUMO

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.


Assuntos
Sulfatos de Condroitina , Oligossacarídeos , Animais , Glicosaminoglicanos , Camundongos , Midkina , Células NIH 3T3
12.
Nat Methods ; 18(7): 799-805, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34226721

RESUMO

A growing appreciation of the importance of cellular metabolism and revelations concerning the extent of cell-cell heterogeneity demand metabolic characterization of individual cells. We present SpaceM, an open-source method for in situ single-cell metabolomics that detects >100 metabolites from >1,000 individual cells per hour, together with a fluorescence-based readout and retention of morpho-spatial features. We validated SpaceM by predicting the cell types of cocultured human epithelial cells and mouse fibroblasts. We used SpaceM to show that stimulating human hepatocytes with fatty acids leads to the emergence of two coexisting subpopulations outlined by distinct cellular metabolic states. Inducing inflammation with the cytokine interleukin-17A perturbs the balance of these states in a process dependent on NF-κB signaling. The metabolic state markers were reproduced in a murine model of nonalcoholic steatohepatitis. We anticipate SpaceM to be broadly applicable for investigations of diverse cellular models and to democratize single-cell metabolomics.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Metabolômica/métodos , Análise de Célula Única/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Técnicas de Cocultura , Células Epiteliais , Ácidos Graxos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Células NIH 3T3 , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais , Estresse Fisiológico
13.
Int J Pharm ; 606: 120892, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34274455

RESUMO

The main objective of this study was to assess the therapeutic activity of gum odina and gelatin based biomimetic scaffold which was previously established as an excellent wound dressing material. In the accelerated stability study, the changes in physicochemical properties were found to be negligible. The cytotoxicity studies were carried out in-vitro and the results showed that upto 90% of the cells remained viable in presence of the scaffold, confirming its biocompatibility. Moreover, results depicted the superior ability of the scaffold to promote cutaneous healing by increasing the rate of wound contraction (about 98%), granulation formation, collagen deposition and formation of an intact epidermis within 18 days. A satisfactory amount of hydroxyproline (240.2 ± 6.67 µg/100 mg tissue) in scaffold treated groups at 21 days ensured the significant deposition of collagen to re-epithelialization. Further it can be hypothesized that the controlled levels of antioxidant enzymes (SOD, CAT) to diminish the oxidative stress in the wounded sites were due to the innate antioxidant properties of both blank and drug loaded scaffold. These results strongly indicated that the prepared scaffolds have strong potential for biomedical applications and it may serve as promising candidate for the next generation of wound treatment systems.


Assuntos
Antibacterianos/administração & dosagem , Biomimética , Gelatina , Tecidos Suporte , Cicatrização/efeitos dos fármacos , Animais , Bandagens , Camundongos , Células NIH 3T3 , Ratos Wistar
14.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208348

RESUMO

Antioxidants play a critical role in the treatment of degenerative diseases and delaying the aging of dermal tissue. Caffeic acid (CA) is a representative example of the antioxidants found in plants. However, CA is unsuitable for long-term storage because of its poor stability under ambient conditions. Caffeoyl-Pro-His-NH2 (CA-Pro-His-NH2, CA-PH) exhibits the highest antioxidant activity, free radical scavenging and lipid peroxidation inhibition activity among the histidine-containing CA-conjugated dipeptides reported to date. The addition of short peptides to CA, such as Pro-His, is assumed to synergistically enhance its antioxidative activity. In this study, several caffeoyl-prolyl-histidyl-Xaa-NH2 derivatives were synthesized and their antioxidative activities evaluated. CA-Pro-His-Asn-NH2 showed enhanced antioxidative activity and higher structural stability than CA-PH, even after long-term storage. CA-Pro-His-Asn-NH2 was stable for 3 months, its stability being evaluated by observing the changes in its NMR spectra. Moreover, the solid-phase synthetic strategy used to prepare these CA-Pro-His-Xaa-NH2 derivatives was optimized for large-scale production. We envision that CA-Pro-His-Xaa-NH2 derivatives can be used as potent dermal therapeutic agents and useful cosmetic ingredients.


Assuntos
Ácidos Cafeicos/síntese química , Ácidos Cafeicos/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Compostos de Bifenilo/química , Ácidos Cafeicos/química , Morte Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Peróxidos/metabolismo , Picratos/química , Espectroscopia de Prótons por Ressonância Magnética , Técnicas de Síntese em Fase Sólida , Espectrometria de Massas por Ionização por Electrospray
15.
Nat Commun ; 12(1): 4339, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267198

RESUMO

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.


Assuntos
Fosfatidilinositóis/metabolismo , Domínios de Homologia à Plecstrina , Proteínas/química , Proteínas/metabolismo , Algoritmos , Animais , Sítios de Ligação , Biologia Computacional/métodos , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Fosfatidilinositóis/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteínas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sensibilidade e Especificidade , Proteína rhoA de Ligação ao GTP/metabolismo
16.
Appl Microbiol Biotechnol ; 105(13): 5419-5431, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34244814

RESUMO

In recent years, an increasing number of studies have shown that fibroblast growth factor 12 (FGF12) plays important roles in regulating neural development and function. Importantly, changes of FGF12 expression are thought to be related to the pathophysiology of many neurological diseases. However, little research has been performed to explore the protective effect of FGF12 on nerve damage. This study aims to explore its neuroprotective effects using our recombinant humanized FGF12 (rhFGF12). The hFGF12 gene was cloned and ligated into an expression vector to construct a recombinant plasmid pET-3a-hFGF12. Single colonies were screened to obtain high expression engineering strains, and fermentation and purification protocols for rhFGF12 were designed and optimized. The biological activities and related mechanisms of rhFGF12 were investigated by MTT assay using NIH3T3 and PC12 cell lines. The in vitro neurotoxicity model of H2O2-induced oxidative injury in PC12 cells was established to explore the protective effects of rhFGF12. The results indicate that the beneficial effects of rhFGF12 were most likely achieved by promoting cell proliferation and reducing apoptosis. Moreover, a transgenic zebrafish (islet) with strong GFP fluorescence in the motor neurons of the hindbrain was used to establish a central injury model caused by mycophenolate mofetil (MMF). The results suggested that rhFGF12 could ameliorate central injury induced by MMF in zebrafish. In conclusion, we have established an efficient method to express and purify active rhFGF12 using an Escherichia coli expression system. Besides, rhFGF12 plays a protective effect of on nerve damage, and it provides a promising therapeutic approach for nerve injury. KEY POINTS: • Effective expression and purification of bioactive rhFGF12 protein in E. coli. • ERK/MAPK pathway is involved in rhFGF12-stimulated proliferation on PC12 cells. • The rhFGF12 has the neuroprotective effects by inhibiting apoptosis.


Assuntos
Fármacos Neuroprotetores , Animais , Escherichia coli/genética , Fatores de Crescimento de Fibroblastos/genética , Humanos , Peróxido de Hidrogênio , Camundongos , Células NIH 3T3 , Fármacos Neuroprotetores/farmacologia , Ratos , Peixe-Zebra
17.
Commun Biol ; 4(1): 846, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267305

RESUMO

Dental plaques are biofilms that cause dental caries by demineralization with acidogenic bacteria. These bacteria reside inside a protective sheath which makes any curative treatment challenging. We propose an antibiotic-free strategy to disrupt the biofilm by engineered clustered carbon dot nanoparticles that function in the acidic environment of the biofilms. In vitro and ex vivo studies on the mature biofilms of Streptococcus mutans revealed >90% biofilm inhibition associated with the contact-mediated interaction of nanoparticles with the bacterial membrane, excessive reactive oxygen species generation, and DNA fragmentation. An in vivo examination showed that these nanoparticles could effectively suppress the growth of S. mutans. Importantly, 16S rRNA analysis of the dental microbiota showed that the diversity and richness of bacterial species did not substantially change with nanoparticle treatment. Overall, this study presents a safe and effective approach to decrease the dental biofilm formation without disrupting the ecological balance of the oral cavity.


Assuntos
Biofilmes/efeitos dos fármacos , Microbiota/fisiologia , Nanopartículas/toxicidade , Polímeros/toxicidade , Streptococcus mutans/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Feminino , Humanos , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Microbiota/genética , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células NIH 3T3 , Nanopartículas/química , Nanopartículas/ultraestrutura , Polímeros/química , RNA Ribossômico 16S/genética , Ratos Sprague-Dawley , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/ultraestrutura
18.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200910

RESUMO

To increase the half-life of growth hormones, we proposed its long-lasting regulation through the ubiquitin-proteasome system (UPS). We identified lysine residues (K67, K141, and K166) that are involved in the ubiquitination of human growth hormone (hGH) using ubiquitination site prediction programs to validate the ubiquitination sites, and then substituted these lysine residues with arginine residues. We identified the most effective substituent (K141R) to prevent ubiquitination and named it AUT-hGH. hGH was expressed and purified in the form of hGH-His, and ubiquitination was first verified at sites containing K141 in the blood stream. Through the study, we propose that AUT-hGH with an increased half-life could be used as a long-lasting hGH in the blood stream.


Assuntos
Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Citoplasma/metabolismo , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Células HEK293 , Meia-Vida , Humanos , Masculino , Camundongos , Células NIH 3T3 , Ratos , Ratos Sprague-Dawley
19.
Nat Commun ; 12(1): 4416, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285220

RESUMO

In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 104 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Análise Espacial , Transcriptoma/genética , Animais , Encéfalo/crescimento & desenvolvimento , Embrião de Mamíferos , Estudos de Viabilidade , Técnicas Genéticas , Células HeLa , Humanos , Masculino , Camundongos , Células NIH 3T3 , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/efeitos da radiação , Transcrição Reversa/efeitos da radiação , Transcriptoma/efeitos da radiação , Raios Ultravioleta
20.
Nat Commun ; 12(1): 4404, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285225

RESUMO

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-ß (TGFß) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFß induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFß on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFß-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFß signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.


Assuntos
Autofagia/genética , Epigênese Genética , Histona Acetiltransferases/metabolismo , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Biópsia , Estudos de Casos e Controles , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Fibroblastos , Fibrose , Técnicas de Inativação de Genes , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Células NIH 3T3 , Cultura Primária de Células , Receptores de Fatores de Crescimento Transformadores beta , Transdução de Sinais/genética , Pele/citologia , Pele/patologia , Proteína Smad3/metabolismo , Adulto Jovem
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