Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31.186
Filtrar
1.
J Cell Sci ; 137(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38841902

RESUMO

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.


Assuntos
Citoplasma , Homeostase , RNA Mensageiro , Grânulos de Estresse , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Citoplasma/metabolismo , Capuzes de RNA/metabolismo , Arsenitos/farmacologia , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/genética , Compostos de Sódio/farmacologia , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Grânulos Citoplasmáticos/metabolismo , Estabilidade de RNA , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Nucleotidiltransferases
2.
Nat Commun ; 15(1): 4986, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862544

RESUMO

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.


Assuntos
Membrana Celular , Adesões Focais , Talina , Vinculina , Talina/metabolismo , Talina/química , Adesões Focais/metabolismo , Membrana Celular/metabolismo , Vinculina/metabolismo , Vinculina/química , Humanos , Animais , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Integrinas/metabolismo , Integrinas/química , Citoplasma/metabolismo , Ligação Proteica , Separação de Fases
3.
Neuromolecular Med ; 26(1): 23, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861223

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving its mislocalisation to the cytoplasm and the formation of misfolded inclusions, are present in almost all ALS cases (97%), and ~ 50% cases of the related condition, frontotemporal dementia (FTD), highlighting its importance in neurodegeneration. Previous studies have shown that endoplasmic reticulum protein 57 (ERp57), a member of the protein disulphide isomerase (PDI) family of redox chaperones, is protective against ALS-linked mutant superoxide dismutase (SOD1) in neuronal cells and transgenic SOD1G93A mouse models. However, it remains unclear whether ERp57 is protective against pathological TDP-43 in ALS. Here, we demonstrate that ERp57 is protective against key features of TDP-43 pathology in neuronal cells. ERp57 inhibited the mislocalisation of TDP-43M337V from the nucleus to the cytoplasm. In addition, ERp57 inhibited the number of inclusions formed by ALS-associated variant TDP-43M337V and reduced the size of these inclusions. ERp57 was also protective against ER stress and induction of apoptosis. Furthermore, ERp57 modulated the steady-state expression levels of TDP-43. This study therefore demonstrates a novel mechanism of action of ERp57 in ALS. It also implies that ERp57 may have potential as a novel therapeutic target to prevent the TDP-43 pathology associated with neurodegeneration.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Corpos de Inclusão , Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/genética , Animais , Camundongos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Superóxido Dismutase-1/genética , Mutação
4.
Curr Opin Cell Biol ; 88: 102374, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38824902

RESUMO

Intracellular organization is a highly regulated homeostatic state maintained to ensure eukaryotic cells' correct and efficient functioning. Thanks to decades of research, vast knowledge of the proteins involved in intracellular transport and organization has been acquired. However, how these influence and potentially regulate the intracellular mechanical properties of the cell is largely unknown. There is a deep knowledge gap between the understanding of cortical mechanics, which is accessible by a series of experimental tools, and the intracellular situation that has been largely neglected due to the difficulty of performing intracellular mechanics measurements. Recently, tools required for such quantitative and localized analysis of intracellular mechanics have been introduced. Here, we review how these approaches and the resulting viscoelastic models lead the way to a full mechanical description of the cytoplasm, which is instrumental for a quantitative characterization of the intracellular life of cells.


Assuntos
Pinças Ópticas , Humanos , Animais , Citoplasma/metabolismo , Reologia , Fenômenos Biomecânicos
5.
Redox Biol ; 73: 103212, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38838552

RESUMO

The dynamic regulation of mitochondria through fission and fusion is essential for maintaining cellular homeostasis. In this study, we discovered a role of coactivator-associated arginine methyltransferase 1 (CARM1) in mitochondrial dynamics. CARM1 methylates specific residues (R403 and R634) on dynamin-related protein 1 (DRP1). Methylated DRP1 interacts with mitochondrial fission factor (Mff) and forms self-assembly on the outer mitochondrial membrane, thereby triggering fission, reducing oxygen consumption, and increasing reactive oxygen species (ROS) production. This sets in motion a feedback loop that facilitates the translocation of CARM1 from the nucleus to the cytoplasm, enhancing DRP1 methylation and ROS production through mitochondrial fragmentation. Consequently, ROS reinforces the CARM1-DRP1-ROS axis, resulting in cellular senescence. Depletion of CARM1 or DRP1 impedes cellular senescence by reducing ROS accumulation. The uncovering of the above-described mechanism fills a missing piece in the vicious cycle of ROS-induced senescence and contributes to a better understanding of the aging process.


Assuntos
Senescência Celular , Citoplasma , Dinaminas , Dinâmica Mitocondrial , Proteína-Arginina N-Metiltransferases , Espécies Reativas de Oxigênio , Dinaminas/metabolismo , Dinaminas/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Humanos , Espécies Reativas de Oxigênio/metabolismo , Metilação , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Membrana
6.
Cell Mol Life Sci ; 81(1): 253, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38852108

RESUMO

Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.


Assuntos
Citoplasma , Proteína Semelhante a ELAV 1 , Inflamação , Poli(ADP-Ribose) Polimerase-1 , RNA Mensageiro , Proteínas Quinases p38 Ativadas por Mitógeno , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Citoplasma/metabolismo , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação , Regulação da Expressão Gênica , Animais , Poli ADP Ribosilação/genética , Células HEK293 , Núcleo Celular/metabolismo , Camundongos
7.
Proc Natl Acad Sci U S A ; 121(26): e2405553121, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38889144

RESUMO

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested that changes in the biophysical properties of the cytoplasm play a key role in cellular homeostasis and adaptation. However, it still remains unclear how cells control their cytoplasmic properties in response to environmental cues. Here, we used fission yeast spores as a model system of dormant cells to elucidate the mechanisms underlying regulation of the cytoplasmic properties. By tracking fluorescent tracer particles, we found that particle mobility decreased in spores compared to vegetative cells and rapidly increased at the onset of dormancy breaking upon glucose addition. This cytoplasmic fluidization depended on glucose-sensing via the cyclic adenosine monophosphate-protein kinase A pathway. PKA activation led to trehalose degradation through trehalase Ntp1, thereby increasing particle mobility as the amount of trehalose decreased. In contrast, the rapid cytoplasmic fluidization did not require de novo protein synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, the measurement of diffusion coefficients with tracer particles of different sizes suggests that the spore cytoplasm impedes the movement of larger protein complexes (40 to 150 nm) such as ribosomes, while allowing free diffusion of smaller molecules (~3 nm) such as second messengers and signaling proteins. Our experiments have thus uncovered a series of signaling events that enable cells to quickly fluidize the cytoplasm at the onset of dormancy breaking.


Assuntos
Citoplasma , Schizosaccharomyces , Esporos Fúngicos , Trealose , Esporos Fúngicos/metabolismo , Esporos Fúngicos/fisiologia , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologia , Citoplasma/metabolismo , Trealose/metabolismo , Glucose/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais
8.
Microb Cell Fact ; 23(1): 170, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38867249

RESUMO

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.


Assuntos
Bacillus subtilis , Peso Molecular , Paenibacillus , beta-Galactosidase , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Paenibacillus/enzimologia , Paenibacillus/genética , Citoplasma/metabolismo , Regiões Promotoras Genéticas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Sinais Direcionadores de Proteínas
9.
Elife ; 122024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38896469

RESUMO

While inhomogeneous diffusivity has been identified as a ubiquitous feature of the cellular interior, its implications for particle mobility and concentration at different length scales remain largely unexplored. In this work, we use agent-based simulations of diffusion to investigate how heterogeneous diffusivity affects the movement and concentration of diffusing particles. We propose that a nonequilibrium mode of membrane-less compartmentalization arising from the convergence of diffusive trajectories into low-diffusive sinks, which we call 'diffusive lensing,' is relevant for living systems. Our work highlights the phenomenon of diffusive lensing as a potentially key driver of mesoscale dynamics in the cytoplasm, with possible far-reaching implications for biochemical processes.


Assuntos
Citoplasma , Difusão , Transporte Biológico , Citoplasma/metabolismo , Modelos Biológicos , Compartimento Celular , Simulação por Computador
10.
Math Biosci ; 373: 109204, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38710441

RESUMO

We introduce a biologically detailed, stochastic model of gene expression describing the multiple rate-limiting steps of transcription, nuclear pre-mRNA processing, nuclear mRNA export, cytoplasmic mRNA degradation and translation of mRNA into protein. The processes in sub-cellular compartments are described by an arbitrary number of processing stages, thus accounting for a significantly finer molecular description of gene expression than conventional models such as the telegraph, two-stage and three-stage models of gene expression. We use two distinct tools, queueing theory and model reduction using the slow-scale linear-noise approximation, to derive exact or approximate analytic expressions for the moments or distributions of nuclear mRNA, cytoplasmic mRNA and protein fluctuations, as well as lower bounds for their Fano factors in steady-state conditions. We use these to study the phase diagram of the stochastic model; in particular we derive parametric conditions determining three types of transitions in the properties of mRNA fluctuations: from sub-Poissonian to super-Poissonian noise, from high noise in the nucleus to high noise in the cytoplasm, and from a monotonic increase to a monotonic decrease of the Fano factor with the number of processing stages. In contrast, protein fluctuations are always super-Poissonian and show weak dependence on the number of mRNA processing stages. Our results delineate the region of parameter space where conventional models give qualitatively incorrect results and provide insight into how the number of processing stages, e.g. the number of rate-limiting steps in initiation, splicing and mRNA degradation, shape stochastic gene expression by modulation of molecular memory.


Assuntos
Modelos Genéticos , RNA Mensageiro , Processos Estocásticos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regulação da Expressão Gênica , Núcleo Celular/metabolismo , Núcleo Celular/genética , Citoplasma/metabolismo , Expressão Gênica , Biossíntese de Proteínas/genética , Transcrição Gênica
11.
Mol Brain ; 17(1): 28, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38790036

RESUMO

The aggregated alpha-synuclein (αsyn) in oligodendrocytes (OLGs) is one of the pathological hallmarks in multiple system atrophy (MSA). We have previously reported that αsyn accumulates not only in neurons but also in OLGs long after the administration of αsyn preformed fibrils (PFFs) in mice. However, detailed spatial and temporal analysis of oligodendroglial αsyn aggregates was technically difficult due to the background neuronal αsyn aggregates. The aim of this study is to create a novel mouse that easily enables sensitive and specific detection of αsyn aggregates in OLGs and the comparable analysis of the cellular tropism of αsyn aggregates in MSA brains. To this end, we generated transgenic (Tg) mice expressing human αsyn-green fluorescent protein (GFP) fusion proteins in OLGs under the control of the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter (CNP-SNCAGFP Tg mice). Injection of αsyn PFFs in these mice induced distinct GFP-positive aggregates in the processes of OLGs as early as one month post-inoculation (mpi), and their number and size increased in a centripetal manner. Moreover, MSA-brain homogenates (BH) induced significantly more oligodendroglial αsyn aggregates than neuronal αsyn aggregates compared to DLB-BH in CNP-SNCAGFP Tg mice, suggestive of their potential tropism of αsyn seeds for OLGs. In conclusion, CNP-SNCAGFP Tg mice are useful for studying the development and tropism of αsyn aggregates in OLGs and could contribute to the development of therapeutics targeting αsyn aggregates in OLGs.


Assuntos
Modelos Animais de Doenças , Corpos de Inclusão , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas , Oligodendroglia , Agregados Proteicos , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Atrofia de Múltiplos Sistemas/patologia , Atrofia de Múltiplos Sistemas/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Humanos , Proteínas de Fluorescência Verde/metabolismo , Citoplasma/metabolismo , Camundongos , Encéfalo/patologia , Encéfalo/metabolismo , Agregação Patológica de Proteínas/metabolismo
12.
Planta ; 260(1): 6, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38780795

RESUMO

MAIN CONCLUSION: TaAGL66, a MADS-box transcription factor highly expressed in fertile anthers of KTM3315A, regulates anther and/or pollen development, as well as male fertility in wheat with Aegilops kotschyi cytoplasm. Male sterility, as a string of sophisticated biological processes in higher plants, is commonly regulated by transcription factors (TFs). Among them, MADS-box TFs are mainly participated in the processes of floral organ formation and pollen development, which are tightly related to male sterility, but they have been little studied in the reproductive development in wheat. In our study, TaAGL66, a gene that was specifically expressed in spikes and highly expressed in fertile anthers, was identified by RNA sequencing and the expression profiles data of these genes, and qRT-PCR analyses, which was localized to the nucleus. Silencing of TaAGL66 under fertility condition in KTM3315A, a thermo-sensitive male sterile line with Ae. kotschyi cytoplasm, displayed severe fertility reduction, abnormal anther dehiscence, defective pollen development, decreased viability, and low seed-setting. It can be concluded that TaAGL66 plays an important role in wheat pollen development in the presence of Ae. kotschyi cytoplasm, providing new insights into the utilization of male sterility.


Assuntos
Aegilops , Citoplasma , Fertilidade , Regulação da Expressão Gênica de Plantas , Infertilidade das Plantas , Proteínas de Plantas , Pólen , Triticum , Triticum/genética , Triticum/crescimento & desenvolvimento , Triticum/fisiologia , Citoplasma/metabolismo , Citoplasma/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Aegilops/genética , Infertilidade das Plantas/genética , Fertilidade/genética , Flores/genética , Flores/crescimento & desenvolvimento , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Genes de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Reprod Biol Endocrinol ; 22(1): 55, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38745305

RESUMO

The role of cytoplasmic fragmentation in human embryo development and reproductive potential is widely recognized, albeit without standard definition nor agreed upon implication. While fragmentation is best understood to be a natural process across species, the origin of fragmentation remains incompletely understood and likely multifactorial. Several factors including embryo culture condition, gamete quality, aneuploidy, and abnormal cytokinesis seem to have important role in the etiology of cytoplasmic fragmentation. Fragmentation reduces the volume of cytoplasm and depletes embryo of essential organelles and regulatory proteins, compromising the developmental potential of the embryo. While it has been shown that degree of fragmentation and embryo implantation potential are inversely proportional, the degree, pattern, and distribution of fragmentation as it relates to pregnancy outcome is debated in the literature. This review highlights some of the challenges in analysis of fragmentation, while revealing trends in our evolving knowledge of how fragmentation may relate to functional development of the human embryos, implantation, and pregnancy outcome.


Assuntos
Citoplasma , Desenvolvimento Embrionário , Resultado da Gravidez , Humanos , Feminino , Gravidez , Desenvolvimento Embrionário/fisiologia , Citoplasma/metabolismo , Citoplasma/fisiologia , Implantação do Embrião/fisiologia
14.
Biochem Biophys Res Commun ; 718: 149981, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38735134

RESUMO

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.


Assuntos
Endossomos , PTEN Fosfo-Hidrolase , Fosfatidilinositóis , Vacúolos , Vacúolos/metabolismo , Vacúolos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Humanos , Fosfatidilinositóis/metabolismo , Animais , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Camundongos , Morfolinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética , Citoplasma/metabolismo , Células HeLa , Aminopiridinas , Compostos Heterocíclicos com 3 Anéis
15.
Nat Commun ; 15(1): 3901, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724505

RESUMO

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.


Assuntos
Citoplasma , Inibidor de NF-kappaB alfa , NF-kappa B , Proteínas Tirosina Quinases , Fator de Transcrição RelA , Animais , Fosforilação , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Camundongos , Fator de Transcrição RelA/metabolismo , Humanos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , NF-kappa B/metabolismo , Citoplasma/metabolismo , Proteólise , Núcleo Celular/metabolismo , Replicação Viral , Células HEK293 , Transdução de Sinais , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Serina-Treonina Quinases
17.
J Biotechnol ; 390: 62-70, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38761885

RESUMO

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.


Assuntos
Escherichia coli , Oxirredução , Dobramento de Proteína , Proteínas Recombinantes , Albumina Sérica Humana , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Citoplasma/metabolismo , Receptores Fc/metabolismo , Receptores Fc/química , Antígenos de Histocompatibilidade Classe I/metabolismo
18.
ACS Infect Dis ; 10(6): 2047-2062, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38811007

RESUMO

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.


Assuntos
Núcleo Celular , Vírus da Dengue , Sinais de Localização Nuclear , Sorogrupo , Proteínas não Estruturais Virais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Núcleo Celular/metabolismo , Humanos , Citoplasma/metabolismo , Replicação Viral , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/química , Animais , Dengue/virologia , Transporte Proteico
19.
Biochem Pharmacol ; 225: 116251, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38701867

RESUMO

Hepatocellular carcinoma (HCC) is the main histological subtype of primary liver cancer and remains one of the most common solid malignancies globally. Ferroptosis was recently defined as an iron-catalyzed form of regulated necrosis. Because cancer cells exhibit higher iron requirements than noncancer cells, treatment with ferroptosis-inducing compounds may be a feasible strategy for cancer therapy. However, cancer cells develop acquired resistance to evade ferroptosis, and the mechanisms responsible for ferroptosis resistance are not fully clarified. In the current study, we reported that DDX39B was downregulated during sorafenib-induced ferroptosis in a dose- and time-dependent manner. Exogenous introduction of DDX39B ensured the survival of HCC cells upon exposure to sorafenib, while the opposite phenomenon was observed in DDX39B-silenced HCC cells. Mechanistically, we demonstrated that DDX39B increased GPX4 levels by promoting the splicing and cytoplasmic translocation of GPX4 pre-mRNA, which was sufficient to detoxify sorafenib-triggered excess lipid ROS production, lipid peroxidation accumulation, ferrous iron levels, and mitochondrial damage. Inhibition of DDX39B ATPase activity by CCT018159 repressed the splicing and cytoplasmic export of GPX4 pre-mRNA and synergistically assisted sorafenib-induced ferroptotic cell death in HCC cells. Taken together, our data uncover a novel role for DDX39B in ferroptosis resistance by modulating the maturation of GPX4 mRNA via a posttranscriptional approach and suggest that DDX39B inhibition may be a promising therapeutic strategy to enhance the sensitivity and vulnerability of HCC cells to sorafenib.


Assuntos
Antineoplásicos , Carcinoma Hepatocelular , RNA Helicases DEAD-box , Ferroptose , Neoplasias Hepáticas , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Precursores de RNA , Sorafenibe , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Sorafenibe/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Precursores de RNA/metabolismo , Precursores de RNA/genética , Antineoplásicos/farmacologia , Animais , Camundongos , Splicing de RNA/efeitos dos fármacos , Camundongos Nus , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Camundongos Endogâmicos BALB C , Masculino , Citoplasma/metabolismo , Citoplasma/efeitos dos fármacos
20.
Colloids Surf B Biointerfaces ; 240: 113984, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38795588

RESUMO

Developing the delivery systems with high therapeutic efficacy and low side effects is of great interest and significance for anticancer therapy. Compared to the high cost in synthesizing new chemotherapeutic drugs, exploring the anticancer potentials of existing chemicals is more convenient and efficient. Sodium bicarbonate (BC), a simple inorganic salt, has shown its tumor inhibition capacity via regulating the acidity of tumor microenvironment. However, the effects of intracytoplasmic BC on tumor growth and the potentials of BC to serve as an anticancer agent are still unknown. Herein, we developed a BC-loaded cationic liposome system (BC-CLP) to deliver BC into the cytosol of cancer cells. The in vitro studies showed that the BC-CLP containing 1% BC (w/v) had a size of 112.9 nm and a zeta potential of 19.1 mV, which reduced the viability of the model cancer cells (human oral squamous cell carcinoma HSC-3 cells) to 13.7%. In contrast, the neutral BC-LP caused less than 50% viability reduction. We further found that BC-CLP released BC directly into cytoplasm via membrane fusion pathway rather than endocytosis, leading to the remarkable increase of cytosolic pH, which may contribute to the anticancer effect of BC-CLP. Our findings indicate that BC-CLP is a potential system for high-efficiency cancer therapy without causing drug-related side effects or resistance.


Assuntos
Antineoplásicos , Cátions , Sobrevivência Celular , Lipossomos , Bicarbonato de Sódio , Lipossomos/química , Humanos , Bicarbonato de Sódio/química , Bicarbonato de Sódio/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Cátions/química , Sobrevivência Celular/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Sistemas de Liberação de Medicamentos , Tamanho da Partícula , Ensaios de Seleção de Medicamentos Antitumorais , Citoplasma/metabolismo , Citoplasma/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...