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1.
Nat Commun ; 15(1): 4061, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38744897

RESUMO

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.


Assuntos
Senescência Celular , Citosol , Dano ao DNA , Comunicação Parácrina , Telômero , Senescência Celular/genética , Animais , Telômero/metabolismo , Telômero/genética , Camundongos , Citosol/metabolismo , DNA/metabolismo , Transcrição Gênica , Camundongos Knockout , Humanos , Vesículas Extracelulares/metabolismo , Instabilidade Genômica , Envelhecimento/genética , Envelhecimento/metabolismo , Estresse Oxidativo , Camundongos Endogâmicos C57BL
2.
Sci Adv ; 10(18): eadl6082, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38701207

RESUMO

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Núcleo Celular , Citosol , Proteína com Valosina , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Humanos , Citosol/metabolismo , Núcleo Celular/metabolismo , Mutação , Transporte Ativo do Núcleo Celular , Dano ao DNA , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Transporte Proteico , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Reparo do DNA , Autofagia , Ligação Proteica , Células HEK293
3.
Proc Natl Acad Sci U S A ; 121(21): e2400740121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38743629

RESUMO

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.


Assuntos
Citosol , Glutarredoxinas , Glutationa , Proteínas Ferro-Enxofre , Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Citosol/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Glutationa/metabolismo , Mitocôndrias/metabolismo , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Mitocondriais/metabolismo
4.
Methods Mol Biol ; 2801: 189-197, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38578422

RESUMO

The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.


Assuntos
Cálcio , Conexinas , Animais , Humanos , Conexinas/genética , Conexinas/metabolismo , Células HeLa , Cálcio/metabolismo , Citosol/metabolismo , Membrana Celular/metabolismo , Mamíferos/metabolismo
5.
Biochem Biophys Res Commun ; 710: 149826, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38581946

RESUMO

Cytosolic peptide:N-glycanase (NGLY1, PNGase) is an enzyme that cleaves N-glycans from misfolded glycoproteins. In 2012, a human genetic disorder, NGLY1 deficiency, was first reported to be caused by mutations of the NGLY1 gene. Since then, there has been rapid progresses on NGLY1 biology, and gene therapy has been proposed as a promising therapeutic option for NGLY1 deficiency. While a plasma/urine biomarker has also been developed for this disease, detection of NGLY1 activity could be another viable option for early diagnosis of NGLY1 deficiency. Thus far, several in vitro and in cellulo NGLY1 assays have been reported, but those assay systems have several issues that must be addressed in order to develop an assay system compatible for routine clinical examination. Here, we show a facile, highly sensitive in vitro assay system that could be used to detect NGLY1 activity by utilizing its sequence editing function, i.e. conversion of glycosylated Asn into Asp, followed by a detection of newly generated epitope (HA)-tag by anti-HA antibody. Using this ELISA-based assay, we detected endogenous NGLY1 activity in as little as 2 µg of crude extract, which is the equivalent of 5 × 103 cells. Our system also detects NGLY1 activity from cells with compromised NGLY1 activity, such as iPS cells from patient samples. This assay system could be applied in future clinical examinations to achieve an early diagnosis of NGLY1 deficiency.


Assuntos
Defeitos Congênitos da Glicosilação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Humanos , Citosol/metabolismo , Glicosilação , Glicoproteínas/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética
6.
Proc Natl Acad Sci U S A ; 121(15): e2313004121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38564631

RESUMO

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.


Assuntos
Polifosfatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Polifosfatos/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo
7.
Int J Mol Sci ; 25(8)2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38673839

RESUMO

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.


Assuntos
Sinalização do Cálcio , Cálcio , Neutrófilos , Fagocitose , Fagossomos , Humanos , Cálcio/metabolismo , Fagossomos/metabolismo , Neutrófilos/metabolismo , Citosol/metabolismo , Membrana Celular/metabolismo
8.
Redox Biol ; 72: 103141, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38599017

RESUMO

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.


Assuntos
Arabidopsis , Citosol , Glutationa , Peróxido de Hidrogênio , Oxirredução , Peróxido de Hidrogênio/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Glutationa/metabolismo , Citosol/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Peroxirredoxinas/metabolismo , Peroxirredoxinas/genética , Glutarredoxinas/metabolismo , Glutarredoxinas/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Dissulfeto de Glutationa/metabolismo , NADP/metabolismo
9.
PLoS Biol ; 22(4): e3002597, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38684033

RESUMO

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.


Assuntos
Proteínas de Ligação ao Cálcio , Citosol , Flagelina , Interações Hospedeiro-Patógeno , Inflamassomos , Salmonella typhimurium , Sistemas de Secreção Tipo III , Citosol/metabolismo , Citosol/microbiologia , Animais , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Inflamassomos/metabolismo , Camundongos , Flagelina/metabolismo , Proteína Inibidora de Apoptose Neuronal/metabolismo , Proteína Inibidora de Apoptose Neuronal/genética , Células Epiteliais/microbiologia , Células Epiteliais/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Camundongos Endogâmicos C57BL , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Análise de Célula Única/métodos , Infecções por Salmonella/microbiologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo
10.
Eur Biophys J ; 53(4): 183-192, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38647542

RESUMO

The sensitivity of cytosol water's microwave dielectric (MD) response to D-glucose uptake in Red Blood Cells (RBCs) allows the detailed study of cellular mechanisms as a function of controlled exposures to glucose and other related analytes like electrolytes. However, the underlying mechanism behind the sensitivity to glucose exposure remains a topic of debate. In this research, we utilize MDS within the frequency range of 0.5-40 GHz to explore how ionic redistributions within the cell impact the microwave dielectric characteristics associated with D-glucose uptake in RBC suspensions. Specifically, we compare glucose uptake in RBCs exposed to the physiological concentration of Ca2+ vs. Ca-free conditions. We also investigate the potential involvement of Na+/K+ redistribution in glucose-mediated dielectric response by studying RBCs treated with a specific Na+/K+ pump inhibitor, ouabain. We present some insights into the MD response of cytosol water when exposed to Ca2+ in the absence of D-glucose. The findings from this study confirm that ion-induced alterations in bound/bulk water balance do not affect the MD response of cytosol water during glucose uptake.


Assuntos
Citosol , Eritrócitos , Glucose , Micro-Ondas , Água , Citosol/metabolismo , Glucose/metabolismo , Água/metabolismo , Eritrócitos/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/citologia , Cálcio/metabolismo , Humanos , Transporte Biológico , Íons/metabolismo , Ouabaína/farmacologia , Sódio/metabolismo
11.
ACS Infect Dis ; 10(5): 1602-1611, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38592927

RESUMO

Antibiotic resistance is an alarming public health concern that affects millions of individuals across the globe each year. A major challenge in the development of effective antibiotics lies in their limited ability to permeate cells, noting that numerous susceptible antibiotic targets reside within the bacterial cytosol. Consequently, improving the cellular permeability is often a key consideration during antibiotic development, underscoring the need for reliable methods to assess the permeability of molecules across cellular membranes. Currently, methods used to measure permeability often fail to discriminate between the arrival within the cytoplasm and the overall association of molecules with the cell. Additionally, these techniques typically possess throughput limitations. In this work, we describe a luciferase-based assay designed for assessing the permeability of molecules in the cytosolic compartment of Gram-negative bacteria. Our findings demonstrate a robust system that can elucidate the kinetics of intracellular antibiotic accumulation in live bacterial cells in real time.


Assuntos
Antibacterianos , Citosol , Escherichia coli , Medições Luminescentes , Antibacterianos/farmacologia , Escherichia coli/metabolismo , Escherichia coli/genética , Citosol/metabolismo , Citosol/química , Testes de Sensibilidade Microbiana , Permeabilidade da Membrana Celular
12.
Plant Physiol Biochem ; 210: 108631, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38657550

RESUMO

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Germinação , Glutamato-Amônia Ligase , Sementes , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/enzimologia , Sementes/crescimento & desenvolvimento , Sementes/genética , Sementes/enzimologia , Germinação/genética , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosol/enzimologia , Citosol/metabolismo , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas , Isoenzimas/genética , Isoenzimas/metabolismo
13.
J Hazard Mater ; 471: 134270, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38640676

RESUMO

Alachlor, a widely used chloroacetanilide herbicide for controlling annual grasses in crops, has been reported to rapidly trigger protein denaturation and aggregation in the eukaryotic model organism Saccharomyces cerevisiae. Therefore, this study aimed to uncover cellular mechanisms involved in preventing alachlor-induced proteotoxicity. The findings reveal that the ubiquitin-proteasome system (UPS) plays a crucial role in eliminating alachlor-denatured proteins by tagging them with polyubiquitin for subsequent proteasomal degradation. Exposure to alachlor rapidly induced an inhibition of proteasome activity by 90 % within 30 min. The molecular docking analysis suggests that this inhibition likely results from the binding of alachlor to ß subunits within the catalytic core of the proteasome. Notably, our data suggest that nascent proteins in the endoplasmic reticulum (ER) are the primary targets of alachlor. Consequently, the unfolded protein response (UPR), responsible for coping with aberrant proteins in the ER, becomes activated within 1 h of alachlor treatment, leading to the splicing of HAC1 mRNA into the active transcription activator Hac1p and the upregulation of UPR gene expression. These findings underscore the critical roles of the protein quality control systems UPS and UPR in mitigating alachlor-induced proteotoxicity by degrading alachlor-denatured proteins and enhancing the protein folding capacity of the ER.


Assuntos
Acetamidas , Retículo Endoplasmático , Herbicidas , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Resposta a Proteínas não Dobradas , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Acetamidas/farmacologia , Acetamidas/toxicidade , Herbicidas/toxicidade , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Citosol/metabolismo , Citosol/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estresse Proteotóxico
14.
Basic Clin Pharmacol Toxicol ; 134(6): 846-857, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38664998

RESUMO

Fungal anthraquinones dermocybin and dermorubin are attractive alternatives for synthetic dyes but their metabolism is largely unknown. We conducted a qualitative in vitro study to identify their metabolism using human liver microsomes and cytosol, as well as recombinant human cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes. Additionally, liver microsomal and cytosolic fractions from rat, mouse and pig were used. Following incubations of the biocolourants with the enzymes in the presence of nicotinamide adenine dinucleotide phosphate, UDP-glucuronic acid, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or S-adenosyl methionine (SAM) to enable CYP oxidation, glucuronidation, sulfonation or methylation, we observed several oxidation and conjugation metabolites for dermocybin but none for dermorubin. Human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A7 catalysed dermocybin oxidation. The formation of dermocybin glucuronides was catalysed by human UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10 and 2B15. Human SULT1B1, 1C2 and 2A1 sulfonated dermocybin. Dermocybin oxidation was faster than conjugation in human liver microsomes. Species differences were seen in dermocybin glucuronidation between human, rat, mouse and pig. In conclusion, many CYP and conjugation enzymes metabolized dermocybin, whereas dermorubin was not metabolized in human liver fractions in vitro. The results indicate that dermocybin would be metabolized in humans in vivo.


Assuntos
Antraquinonas , Sistema Enzimático do Citocromo P-450 , Glucuronosiltransferase , Microssomos Hepáticos , Microssomos Hepáticos/metabolismo , Humanos , Animais , Ratos , Camundongos , Suínos , Glucuronosiltransferase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Antraquinonas/metabolismo , Masculino , Proteínas Recombinantes/metabolismo , Fígado/metabolismo , Fígado/enzimologia , Citosol/metabolismo , Oxirredução , Glucuronídeos/metabolismo
15.
Biomolecules ; 14(4)2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38672417

RESUMO

Silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene and a consequent lack of FMR protein (FMRP) synthesis are associated with fragile X syndrome, one of the most common inherited intellectual disabilities. FMRP is a multifunctional protein that is involved in many cellular functions in almost all subcellular compartments under both normal and cellular stress conditions in neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and it is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes the recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.


Assuntos
Nucléolo Celular , Citosol , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Humanos , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Nucléolo Celular/metabolismo , Citosol/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/genética , Animais , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Processamento de Proteína Pós-Traducional
16.
Methods Mol Biol ; 2760: 21-34, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468080

RESUMO

As the field of plant synthetic biology continues to grow, Agrobacterium-mediated transient expression has become an essential method to rapidly test pathway candidate genes in a combinatorial fashion. This is especially important when elucidating and engineering more complex pathways to produce commercially relevant chemicals like many terpenoids, a widely diverse class of natural products of often industrial relevance. Agrobacterium-mediated transient expression has facilitated multiplex expression of recombinant and modified enzymes, including synthetic biology approaches to compartmentalize the biosynthesis of terpenoids subcellularly. Here, we describe methods on how to deploy Agrobacterium-mediated transient expression in Nicotiana benthamiana to rapidly develop terpenoid pathways and compartmentalize terpenoid biosynthesis within plastids, the cytosol, or at the surface of lipid droplets.


Assuntos
Agrobacterium , Terpenos , Terpenos/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Plantas/metabolismo , Nicotiana/genética , Citosol/metabolismo
17.
PLoS Biol ; 22(3): e3002526, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38427703

RESUMO

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in Caenorhabditis elegans has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic cells. We have tagged endogenous transcripts with MS2 hairpins in the 3' untranslated region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs for dlg-1 fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, and dlg-1 RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.


Assuntos
Caenorhabditis elegans , RNA , Animais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citosol/metabolismo
18.
Proc Natl Acad Sci U S A ; 121(10): e2319491121, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38427601

RESUMO

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here, we directly compare an identical association reaction, the binding of complementary DNA strands, in solution and on supported membranes. The measured rate constants show that for a 10-µm-radius spherical cell, association is 22- to 33-fold faster at the membrane than in the cytoplasm. The kinetic advantage depends on cell size and is essentially negligible for typical ~1 µm prokaryotic cells. The rate enhancement is attributable to a combination of higher encounter rates in two dimensions and a higher reaction probability per encounter.


Assuntos
Transdução de Sinais , Citoplasma/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Membranas , Cinética
19.
Methods Mol Biol ; 2776: 289-302, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38502512

RESUMO

Excluding the few dozen proteins encoded by the chloroplast and mitochondrial genomes, the majority of plant cell proteins are synthesized by cytosolic ribosomes. Most of these nuclear-encoded proteins are then targeted to specific cell compartments thanks to localization signals present in their amino acid sequence. These signals can be specific amino acid sequences known as transit peptides, or post-translational modifications, ability to interact with specific proteins or other more complex regulatory processes. Furthermore, in eukaryotic cells, protein synthesis can be regulated so that certain proteins are synthesized close to their destination site, thus enabling local protein synthesis in specific compartments of the cell. Previous studies have revealed that such locally translating cytosolic ribosomes are present in the vicinity of mitochondria and emerging views suggest that localized translation near chloroplasts could also occur. However, in higher plants, very little information is available on molecular mechanisms controlling these processes and there is a need to characterize cytosolic ribosomes associated with organelles membranes. To this goal, this protocol describes the purification of higher plant chloroplast and mitochondria and the organelle-associated cytosolic ribosomes.


Assuntos
Cloroplastos , Ribossomos , Citosol/metabolismo , Cloroplastos/metabolismo , Ribossomos/metabolismo , Plantas/metabolismo , Proteínas de Plantas/metabolismo , Mitocôndrias/metabolismo
20.
Biochim Biophys Acta Gen Subj ; 1868(6): 130603, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38521470

RESUMO

BACKGROUND: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. METHODS: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. RESULTS: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. CONCLUSIONS: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. GENERAL SIGNIFICANCE: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.


Assuntos
Citosol , Glutationa , Mitocôndrias , Oxirredução , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Glutationa/metabolismo , Mitocôndrias/metabolismo , Citosol/metabolismo , Desenvolvimento Embrionário , Dissulfeto de Glutationa/metabolismo , Embrião não Mamífero/metabolismo
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