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1.
Biochim Biophys Acta Gen Subj ; 1868(2): 130535, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38103757

RESUMO

BACKGROUND: Calcimycin (A23187) is a polyether antibiotic and divalent cation ionophore, extracted from Streptomyces chartrecensis. With wide variety of antimicrobial activities, it also exhibits cytotoxicity of tumor cells. Calcimycin exhibit therapeutic potential against tumor cell growth; however, the molecular mechanism remains to be fully elucidated. Present study explores the mechanism of calcimycin-induced apoptosis cancer cell lines. METHODS: Apoptotic induction in a dose-dependent manner were recorded with MTT assays, Phase contrast imaging, wound healing assay, fluorescence imaging by DAPI and AO/EB staining and FACS using cell line model. Mitochondrial potential was analyzed by TMRM assay as Ca2+ signaling is well known to be influenced and synchronized by mitochondria also. RESULTS: Calcimycin induces apoptosis in dose dependent manner, also accompanied by increased intracellular calcium-level and expression of purinergic receptor-P2RX4, a ligand-gated ion channel. CONCLUSION: Calcimycin tends to increase the intracellular calcium level, mRNA expression of ATP receptor P2RX4, and phosphorylation of p38. Blocking of either intracellular calcium by BAPTA-AM, P2RX4 expression by antagonist 5-BDBD, and phospho-p38 by SB203580, abrogated the apoptotic activity of calcimycin. GENERAL SIGNIFICANCE: Taken together, these results show that calcimycin induces apoptosis in P2RX4 and ATP mediated intracellular Ca2+ and p38 MAPK mediated pathway in both the cancer cell lines. This study explored a new mode of action for calcimycin in cancer that could be potentially employed in future studies for cancer therapeutic research. This study disentangles that the calcimycin-induced apoptotic cell death is P2RX4 and ATP involved, intracellular Ca2+ and p38 MAPK mediated pathway.


Assuntos
Apoptose , Calcimicina , Cálcio , Receptores Purinérgicos P2X4 , Células MCF-7 , Linhagem Celular Tumoral , Humanos , Calcimicina/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Espaço Intracelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
PLoS One ; 18(11): e0291331, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38011105

RESUMO

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.


Assuntos
Bordetella parapertussis , Coqueluche , Humanos , Bordetella parapertussis/metabolismo , Toxina Adenilato Ciclase/metabolismo , Bordetella pertussis/metabolismo , Espaço Intracelular/metabolismo , Coqueluche/microbiologia , Células Epiteliais/metabolismo
3.
Int J Mol Sci ; 24(17)2023 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-37685957

RESUMO

Liquid-liquid phase separation (LLPS, also known as biomolecular condensation) and the related biogenesis of various membraneless organelles (MLOs) and biomolecular condensates (BMCs) are now considered fundamental molecular mechanisms governing the spatiotemporal organization of the intracellular space [...].


Assuntos
Condensados Biomoleculares , Espaço Intracelular
4.
Anal Chem ; 95(33): 12427-12434, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37560995

RESUMO

Reactive sulfur species (RSS) are emerging as a potential key gasotransmitter in diverse physiological processes linking two signaling molecules H2S and SO2. However, the exact roles of H2S and SO2 remain unclear. A major hurdle is the shortage of accurate and robust approaches for sensing of H2S and SO2 in biological systems. Herein, we report a reaction-mediated dual-recognition strategy-based nanosensor, silver nanoparticles (AgNPs)-loaded MIL-101 (Fe) (ALM) hybrids, for the simultaneous detection of H2S and SO2 in a living cell. Upon exposure to H2S, AgNPs can be oxidized to form Ag2S, causing a decrease of surface enhanced Raman spectroscopy (SERS) signals of p,p'-dimercaptoazobenzene. Moreover, SO2 reacts with the amino moiety of MIL-101 to form charge-transfer complexes, resulting in an increment of fluorescent (FL) intensity. The ALM with dual-modal signals can simultaneously analyze H2S and SO2 at a concentration as low as 2.8 × 10-6 and 0.003 µM, respectively. Most importantly, the ALM sensing platform enables targeting mitochondria and detection multiple RSS simultaneously in living cells under external stimulation, as well as displays indiscernible crosstalk between SERS and FL signals, which is very beneficial for the comprehension of physiological issues related with RSS.


Assuntos
Espaço Intracelular , Espaço Intracelular/química , Enxofre/química , Humanos , Linhagem Celular Tumoral , Prata/química , Nanopartículas Metálicas , Dióxido de Enxofre/química
5.
J Mater Chem B ; 11(33): 7873-7912, 2023 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-37551112

RESUMO

Intracellular cargo delivery, the introduction of small molecules, proteins, and nucleic acids into a specific targeted site in a biological system, is an important strategy for deciphering cell function, directing cell fate, and reprogramming cell behavior. With the advancement of nanotechnology, many researchers use nanoparticles (NPs) to break through biological barriers to achieving efficient targeted delivery in biological systems, bringing a new way to realize efficient targeted drug delivery in biological systems. With a similar size to many biomolecules, NPs possess excellent physical and chemical properties and a certain targeting ability after functional modification on the surface of NPs. Currently, intracellular cargo delivery based on NPs has emerged as an important strategy for genome editing regimens and cell therapy. Although researchers can successfully deliver NPs into biological systems, many of them are delivered very inefficiently and are not specifically targeted. Hence, the development of efficient, target-capable, and safe nanoscale drug delivery systems to deliver therapeutic substances to cells or organs is a major challenge today. In this review, on the basis of describing the research overview and classification of NPs, we focused on the current research status of intracellular cargo delivery based on NPs in biological systems, and discuss the current problems and challenges in the delivery process of NPs in biological systems.


Assuntos
Espaço Intracelular , Nanoestruturas , Animais , Espaço Intracelular/química , Sistemas de Liberação de Medicamentos , Nanoestruturas/química , Nanopartículas/química , Endocitose , Lipossomos/química , Inativação Gênica
6.
J Biol Chem ; 299(9): 105147, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37567478

RESUMO

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.


Assuntos
Proteínas de Bactérias , Dissulfetos , Exotoxinas , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas , Espaço Intracelular , Compostos de Sulfidrila , Ativação Transcricional , Vibrio cholerae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Exotoxinas/genética , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Ativação Transcricional/efeitos dos fármacos , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Dissulfetos/metabolismo , Dissulfetos/farmacologia , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/farmacologia , Espaço Intracelular/metabolismo , Espectrometria de Massas , Metabolômica , Dissulfeto de Glutationa/farmacologia , Microbioma Gastrointestinal/imunologia
7.
Biomaterials ; 301: 122262, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37542857

RESUMO

Localized corrosion has become a concerning issue in orthopedic implants as it is associated with peri-implant adverse tissue reactions and implant failure. Here, the pitting corrosion of 316 L stainless steels (316 L SSs) was initiated by electrochemical polarization to simulate the in vivo localized corrosion of orthopedic implants. The effect of localized corrosion on osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMSCs) was systematically studied. The results suggest that pitting corrosion of 316 L SS reduced the viability, adhesion, proliferation, and osteogenic differentiation abilities of BMSCs, especially for the cells around the corrosion pits. The relatively high concentrations of metallic ions such as Cr3+ and Ni2+ released by pitting corrosion could cause cytotoxicity to the BMSCs. The inhomogeneous electrochemical environment resulted from localized corrosion could promote reactive oxygen species (ROS) generation around the corrosion pits and cause oxidative stress of BMSCs. In addition, localized corrosion could also electrochemically interact with the cells and lead to cell membrane depolarization. The depolarized cell membranes and relatively high levels of ROS mediated the degradation of the osteogenic capacity of BMSCs. This work provides new insights into corrosion-mediated cell function degeneration as well as the material-cell interactions.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Aço Inoxidável , Corrosão , Aço Inoxidável/química , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Ratos , Ratos Sprague-Dawley , Células Cultivadas , Apoptose , Espaço Intracelular , Cálcio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Sci Adv ; 9(35): eadi4517, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37647406

RESUMO

Signal perception is a key function in regulating biological activities and adapting to changing environments. Per-Arnt-Sim (PAS) domains are ubiquitous sensors found in diverse receptors in bacteria, archaea, and eukaryotes, but their origins, distribution across the tree of life, and extent of their functional diversity are not fully characterized. Here, we show that using sequence conservation and structural information, it is possible to propose specific and potential functions for a large portion of nearly 3 million PAS domains. Our analysis suggests that PAS domains originated in bacteria and were horizontally transferred to archaea and eukaryotes. We reveal that gas sensing via a heme cofactor evolved independently in several lineages, whereas redox and light sensing via flavin adenine dinucleotide and flavin mononucleotide cofactors have the same origin. The close relatedness of human PAS domains to those in bacteria provides an opportunity for drug design by exploring potential natural ligands and cofactors for bacterial homologs.


Assuntos
Bactérias , Eucariotos , Espaço Intracelular , Domínios Proteicos , Proteínas , Eucariotos/química , Humanos , Animais , Sistemas de Liberação de Medicamentos , Bactérias/química , Filogenia , Flavina-Adenina Dinucleotídeo/metabolismo , Espaço Intracelular/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo
9.
Methods Mol Biol ; 2700: 77-92, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37603175

RESUMO

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.


Assuntos
Células Dendríticas , Sistemas CRISPR-Cas , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Células Dendríticas/metabolismo , Animais , Camundongos , Citocinas/metabolismo , Espaço Intracelular/metabolismo
10.
Genes (Basel) ; 14(7)2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37510296

RESUMO

Charcot-Marie-Tooth disease (CMT) and associated neuropathies are the most predominant genetically transmitted neuromuscular conditions; however, effective pharmacological treatments have not established. The extensive genetic heterogeneity of CMT, which impacts the peripheral nerves and causes lifelong disability, presents a significant barrier to the development of comprehensive treatments. An estimated 100 loci within the human genome are linked to various forms of CMT and its related inherited neuropathies. This review delves into prospective therapeutic strategies used for the most frequently encountered CMT variants, namely CMT1A, CMT1B, CMTX1, and CMT2A. Compounds such as PXT3003, which are being clinically and preclinically investigated, and a broad array of therapeutic agents and their corresponding mechanisms are discussed. Furthermore, the progress in established gene therapy techniques, including gene replacement via viral vectors, exon skipping using antisense oligonucleotides, splicing modification, and gene knockdown, are appraised. Each of these gene therapies has the potential for substantial advancements in future research.


Assuntos
Doença de Charcot-Marie-Tooth , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/terapia , Humanos , Mutação , Espaço Intracelular/metabolismo , Cálcio/metabolismo , Inativação Gênica , Terapia Genética , Animais
11.
Chem Commun (Camb) ; 59(48): 7463-7466, 2023 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-37254719

RESUMO

Herein, we report an indocyanine green (ICG)-decorated and glucose oxidase (GOx)-loaded nanoscale composite COF material via a stepwise post-synthetic modification. The obtained GOx@COF-ICG can achieve synergistic inhibition of intracellular heat defense systems through starvation therapy to enhance photothermal therapy of tumors.


Assuntos
Resposta ao Choque Térmico , Espaço Intracelular , Nanoestruturas , Neoplasias , Espaço Intracelular/química , Estruturas Metalorgânicas/química , Sobrevivência Celular , Humanos , Linhagem Celular Tumoral , Animais , Terapia Fototérmica , Neoplasias/terapia
12.
Microb Biotechnol ; 16(7): 1524-1535, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37212362

RESUMO

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.


Assuntos
Brucelose , Exossomos , Macrófagos , Brucella melitensis , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Exossomos/imunologia , Exossomos/microbiologia , Animais , Camundongos , Polaridade Celular , Antígenos de Bactérias/imunologia , Brucelose/imunologia , Brucelose/metabolismo , Transdução de Sinais , Espaço Intracelular/microbiologia , Viabilidade Microbiana
13.
Curr Opin Immunol ; 82: 102306, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36989589

RESUMO

Persistent bacterial infections constitute an enormous challenge for public health. Amongst infections with other bacteria, infections with typhoidal and nontyphoidal Salmonella enterica serovars can result in long-term infections of the human and animal host. Persistent infections that are asymptomatic are difficult to identify and thus can serve as a silent reservoir for transmission. Symptomatic persistent infections are often difficult to treat as they harbor a combination of antibiotic-tolerant and antibiotic-resistant bacteria and boost the spread of genetic antibiotic resistance. In the last couple of years, the field has made some major progress in understanding the role of persisters, their reservoirs as well as their interplay with host factors in persistent Salmonella infections.


Assuntos
Infecção Persistente , Infecções por Salmonella , Humanos , Espaço Intracelular , Salmonella/genética
14.
ACS Appl Mater Interfaces ; 15(12): 15893-15906, 2023 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-36940438

RESUMO

The hypopermeability and hypoxia in the tumor milieu are important factors that limit multiple treatments. Herein, the reactive oxygen species (ROS)-triggered self-assembled nanoparticles (RP-NPs) was constructed. The natural small molecule Rhein (Rh) was encapsulated into RP-NPs as a sonosensitizer highly accumulated at the tumor site. Then highly tissue-permeable ultrasound (US) irradiation induced apoptosis of tumor cells through the excitation of Rh and acoustic cavitation, which prompted the rapid production of large amounts of ROS in the hypoxic tumor microenvironment. In addition, the thioketal bond structures in the innovatively designed prodrug LA-GEM were triggered and broken by ROS to achieve rapid targeted release of the gemcitabine (GEM). Sonodynamic therapy (SDT) increased the tissue permeability of solid tumors and actively disrupted redox homeostasis via mitochondrial pathways to kill hypoxic tumor cells, and the triggered response mechanism to GEM synergistically amplified the effect of chemotherapy. The chemo-sonodynamic combinational treatment approach is highly effective and noninvasive, with promising applications for hypoxic tumor elimination, such as in cervical cancer (CCa) patients who want to maintain their reproductive function.


Assuntos
Nanopartículas , Neoplasias , Hipóxia Tumoral , Espécies Reativas de Oxigênio/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Espaço Intracelular , Microambiente Tumoral , Sistemas de Liberação de Medicamentos , Gencitabina/química , Gencitabina/farmacologia , Terapia Combinada , Humanos , Animais , Camundongos , Células HeLa
15.
Analyst ; 148(8): 1682-1693, 2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-36912705

RESUMO

The spatiotemporal visualization of intracellular microRNA (miRNA) plays a critical role in the diagnosis and treatment of malignant disease. Although DNAzyme-based biosensing has been regarded as the most promising candidate, inefficient analytical resolution is frequently encountered. Here, we propose a bioorthogonal approach toward high-fidelity imaging of intracellular miRNA by designing a multifunctional nanoprobe that integrates MnO2 nanosheet-mediated intracellular delivery and activation by a fat mass and obesity-associated protein (FTO)-switched positive feedback. MnO2 nanosheets facilitate nanoprobe delivery and intracellular DNAzyme cofactors are released upon glutathione-triggered reduction. Meanwhile, an m6A-caged DNAzyme probe could be bioorthogonally activated by intracellular FTO to eliminate potential off-target activation. Therefore, the activated DNAzyme probe and substrate probe could recognize miRNA to perform cascade signal amplification in the initiation of the release of Mn2+ from MnO2 nanosheets. This strategy realized high-fidelity imaging of intracellular aberrant miRNA within tumor cells with a satisfactory detection limit of 9.7 pM, paving the way to facilitate clinical tumor diagnosis and prognosis monitoring.


Assuntos
DNA Catalítico , MicroRNAs , Neoplasias , Humanos , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Diagnóstico por Imagem , DNA Catalítico/genética , Compostos de Manganês , MicroRNAs/genética , Neoplasias/patologia , Óxidos , Espaço Intracelular/genética
16.
J Biol Chem ; 299(5): 104628, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36963491

RESUMO

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H+ ions are transported in exchange for Ca2+ and Mn2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+, Mn2+ or H+ gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H+ ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H+ transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Magnésio/metabolismo , Cátions/metabolismo , Prótons , Lactococcus lactis/genética , Membranas Intracelulares/metabolismo , Espaço Intracelular/química , Espaço Intracelular/metabolismo
17.
ACS Appl Mater Interfaces ; 15(8): 10398-10413, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36795046

RESUMO

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.


Assuntos
Neoplasias Colorretais , Neoplasias , Animais , Camundongos , Carcinogênese , Proliferação de Células , Neoplasias Colorretais/patologia , Micelas , Mutação , Polímeros/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/farmacologia , Espaço Intracelular
18.
Int J Mol Sci ; 24(3)2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36768740

RESUMO

Cytochrome P450 (CYP) 1B1 is a heme-containing monooxygenase found mainly in extrahepatic tissues, including the retina. CYP1B1 substrates include exogenous aromatic hydrocarbons, such as dioxins, and endogenous bioactive compounds, including 17ß-estradiol (E2) and arachidonic acid. The endogenous compounds and their metabolites are mediators of various cellular and physiological processes, suggesting that CYP1B1 activity is likely important in maintaining proper cellular and tissue functions. We previously demonstrated that lack of CYP1B1 expression and activity are associated with increased levels of reactive oxygen species and oxidative stress in the retinal vasculature and vascular cells, including retinal endothelial cells (ECs). However, the detailed mechanism(s) of how CYP1B1 activity modulates redox homeostasis remained unknown. We hypothesized that CYP1B1 metabolism of E2 affects bone morphogenic protein 6 (BMP6)-hepcidin-mediated iron homeostasis and lipid peroxidation impacting cellular redox state. Here, we demonstrate retinal EC prepared from Cyp1b1-deficient (Cyp1b1-/-) mice exhibits increased estrogen receptor-α (ERα) activity and expresses higher levels of BMP6. BMP6 is an inducer of the iron-regulatory hormone hepcidin in the endothelium. Increased hepcidin expression in Cyp1b1-/- retinal EC resulted in decreased levels of the iron exporter protein ferroportin and, as a result, increased intracellular iron accumulation. Removal of excess iron or antagonism of ERα in Cyp1b1-/- retinal EC was sufficient to mitigate increased lipid peroxidation and reduce oxidative stress. Suppression of lipid peroxidation and antagonism of ERα also restored ischemia-mediated retinal neovascularization in Cyp1b1-/- mice. Thus, CYP1B1 expression in retinal EC is important in the regulation of intracellular iron levels, with a significant impact on ocular redox homeostasis and oxidative stress through modulation of the ERα/BMP6/hepcidin axis.


Assuntos
Receptor alfa de Estrogênio , Hepcidinas , Animais , Camundongos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Receptor alfa de Estrogênio/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Ferro/metabolismo , Estresse Oxidativo/fisiologia , Retina/metabolismo , Espaço Intracelular/metabolismo
19.
Autophagy ; 19(10): 2811-2813, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-36779581

RESUMO

Previously considered as an exclusive extracellular bacterium, Staphylococcus aureus has been shown to be able to invade many cells in vitro and in humans. Once inside the host cell, both cytosolic and endosome-associated S. aureus strongly induce macroautophagy/autophagy. Whether autophagy fosters S. aureus intracellular survival or clearance remains unclear. The YAP1-TEAD axis regulates the expression of target genes controlling the cell fate (e.g., proliferation, migration, cell cycle …). Growing evidence indicates that YAP1-TEAD also regulates autophagy and lysosomal pathways. Recently we showed that the YAP1-TEAD axis promotes autophagy and lysosome biogenesis to restrict S. aureus intracellular replication. We also discovered that the C3 exoenzyme-like EDIN-B toxin produced by the pathogenic S. aureus ST80 strain inhibits YAP1 nuclear translocation resulting in a strong increase of intracellular S. aureus burden.


Assuntos
Autofagia , Espaço Intracelular , Staphylococcus aureus , Fatores de Transcrição de Domínio TEA , Humanos , Autofagia/imunologia , Células HEK293 , Espaço Intracelular/microbiologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Fatores de Transcrição de Domínio TEA/metabolismo , Técnicas In Vitro
20.
Biochem Biophys Res Commun ; 649: 16-24, 2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36739695

RESUMO

Bone morphogenetic protein 2 (BMP2)-inducible kinase (BMP2K) is induced by the cytokine BMP2, which is also implicated in the production of bone differentiation. In addition to regulating bone differentiation, BMP2K is implicated in a variety of cancers. Therefore, understanding the variables that determine where in the cell this kinase functions may help in understanding malignancies linked to BMP2K. However, the mechanisms regulating the subcellular localization of BMP2K are mainly unknown. By liquid-liquid phase separation (LLPS), BMP2K forms droplets in the cytoplasm, but how the droplets are regulated remains unclear. The reason why BMP2K localizes to the cytoplasm irrespective of having a nuclear localization signal (NLS) is also unknown. Here we show the element that controls BMP2K's LLPS and cytoplasmic localization. A glutamine-rich area is necessary for BMP2K phase separation, and droplet formation is controlled by hyperosmolarity. Cytoplasmic localization of BMP2K is managed by inhibition of NLS function through phosphorylation of Ser-1010 and by a newly found cytoplasmic localization region that antagonizes the NLS. These results will provide an important biochemical foundation for the advancement of BMP2K-related cell biology, structural biology, and pathophysiology.


Assuntos
Proteína Morfogenética Óssea 2 , Sinais de Localização Nuclear , Transporte Ativo do Núcleo Celular , Proteína Morfogenética Óssea 2/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sinais de Localização Nuclear/metabolismo , Fosforilação , Espaço Intracelular/metabolismo
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