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1.
Nature ; 613(7943): 345-354, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599983

RESUMO

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.


Assuntos
Células-Tronco Pluripotentes Induzidas , Espaço Intracelular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Célula Única , Conjuntos de Dados como Assunto , Interfase , Forma Celular , Mitose , Polaridade Celular , Sobrevivência Celular
2.
Nat Commun ; 13(1): 7845, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36543777

RESUMO

The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.


Assuntos
Condensados Biomoleculares , Microfluídica , Espaço Intracelular , Transição de Fase
3.
Nat Commun ; 13(1): 7640, 2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36496406

RESUMO

Spatially resolved transcriptomics provides the opportunity to investigate the gene expression profiles and the spatial context of cells in naive state, but at low transcript detection sensitivity or with limited gene throughput. Comprehensive annotating of cell types in spatially resolved transcriptomics to understand biological processes at the single cell level remains challenging. Here we propose Spatial-ID, a supervision-based cell typing method, that combines the existing knowledge of reference single-cell RNA-seq data and the spatial information of spatially resolved transcriptomics data. We present a series of benchmarking analyses on publicly available spatially resolved transcriptomics datasets, that demonstrate the superiority of Spatial-ID compared with state-of-the-art methods. Besides, we apply Spatial-ID on a self-collected mouse brain hemisphere dataset measured by Stereo-seq, that shows the scalability of Spatial-ID to three-dimensional large field tissues with subcellular spatial resolution.


Assuntos
Perfilação da Expressão Gênica , Análise de Célula Única , Camundongos , Animais , Análise de Célula Única/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma , Espaço Intracelular , Aprendizado de Máquina
4.
Biomolecules ; 12(10)2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36291650

RESUMO

The discovery of intrinsically disordered proteins (IDPs) that do not have an ordered structure and nevertheless perform essential functions has opened a new era in the understanding of cellular compartmentalization. It threw the bridge from the mostly mechanistic model of the organization of the living matter to the idea of highly dynamic and functional "soft matter". This paradigm is based on the notion of the major role of liquid-liquid phase separation (LLPS) of biopolymers in the spatial-temporal organization of intracellular space. The LLPS leads to the formation of self-assembled membrane-less organelles (MLOs). MLOs are multicomponent and multifunctional biological condensates, highly dynamic in structure and composition, that allow them to fine-tune the regulation of various intracellular processes. IDPs play a central role in the assembly and functioning of MLOs. The LLPS importance for the regulation of chemical reactions inside the cell is clearly illustrated by the reorganization of the intracellular space during stress response. As a reaction to various types of stresses, stress-induced MLOs appear in the cell, enabling the preservation of the genetic and protein material during unfavourable conditions. In addition, stress causes structural, functional, and compositional changes in the MLOs permanently present inside the cells. In this review, we describe the assembly of stress-induced MLOs and the stress-induced modification of existing MLOs in eukaryotes, yeasts, and prokaryotes in response to various stress factors.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Organelas/química , Eucariotos/metabolismo , Espaço Intracelular/metabolismo
5.
Nature ; 609(7928): 815-821, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36071159

RESUMO

Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.


Assuntos
Lisossomos , Fosfatidilinositóis , Transdução de Sinais , Proteínas Relacionadas à Autofagia/metabolismo , Transporte Biológico , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Oxisteróis/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteômica , Receptores de Esteroides/metabolismo
6.
Biochem Biophys Res Commun ; 630: 125-132, 2022 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-36155058

RESUMO

The shortest dystrophins, Dp71 and Dp40, are transcribed from the DMD gene through an internal promoter located in intron 62. These proteins are the main product of the DMD gene in the nervous system and have been involved in various functions related to cellular differentiation and proliferation as well as other cellular processes. Dp71 mRNA undergoes alternative splicing that results in different Dp71 protein isoforms. The subcellular localization of some of these isoforms in the PC12 cell line has been previously reported, and a differential subcellular distribution was observed, which suggests a particular role for each isoform. With the aim of obtaining information on their function, this study identified factors involved in the nuclear transport of Dp71 and Dp40 isoforms in the PC12 cell line. Cell cultures were treated with specific nuclear import/export inhibitors to determine the Dp71 isoform transport routes. The results showed that all isoforms of Dp71 and Dp40 included in the analysis have the ability to enter the cell nucleus through α/ß importin, and the main route of nuclear export for Dp71 isoforms is through the exportin CRM1, which is not the case for Dp40.


Assuntos
Distrofina , beta Carioferinas , Transporte Ativo do Núcleo Celular , Animais , Distrofina/genética , Distrofina/metabolismo , Espaço Intracelular , Carioferinas/metabolismo , Células PC12 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , beta Carioferinas/metabolismo
7.
Biochim Biophys Acta Mol Basis Dis ; 1868(11): 166519, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35964875

RESUMO

BACKGROUND: Community-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. OBJECTIVE: To obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. METHODS: Monocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-10. RESULTS: 50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1ß and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. CONCLUSION: These results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.


Assuntos
Monócitos , Pneumonia , Citocinas/metabolismo , Humanos , Interleucina-10/metabolismo , Espaço Intracelular , Lipopolissacarídeos/farmacologia , Monócitos/metabolismo , Pneumonia/metabolismo , Piruvato Quinase/metabolismo , Ácido Pirúvico/metabolismo , Ácidos Tricarboxílicos , Fator de Necrose Tumoral alfa/metabolismo
8.
Sci Rep ; 12(1): 14087, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982220

RESUMO

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models-with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.


Assuntos
Interferon gama , Neoplasias , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Espaço Intracelular/metabolismo , Masculino , Camundongos , Ligação Proteica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Biomolecules ; 12(7)2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35883463

RESUMO

Liquid-liquid phase separation (LLPS) of biomolecules has emerged as a new paradigm in cell biology, and the process is one proposed mechanism for the formation of membraneless organelles (MLOs). Bacterial cells have only recently drawn strong interest in terms of studies on both liquid-to-liquid and liquid-to-solid phase transitions. It seems that these processes drive the formation of prokaryotic cellular condensates that resemble eukaryotic MLOs. In this review, we present an overview of the key microbial biomolecules that undergo LLPS, as well as the formation and organization of biomacromolecular condensates within the intracellular space. We also discuss the current challenges in investigating bacterial biomacromolecular condensates. Additionally, we highlight a summary of recent knowledge about the participation of bacterial biomolecules in a phase transition and provide some new in silico analyses that can be helpful for further investigations.


Assuntos
Eucariotos , Organelas , Espaço Intracelular , Transição de Fase
10.
J Mater Chem B ; 10(31): 6038-6048, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35894777

RESUMO

As a nonreducing disaccharide, trehalose can be used as a biocompatible cryoprotectant for solvent-free cell cryopreservation, but the membrane-impermeability limits its cryoprotective efficiency. Herein, a series of aromatic monoamines with a 1-4 methylene spacer were grafted onto γ-poly(glutamic acid) (γ-PGA) for promoting intracellular trehalose uptake in human red blood cells (hRBCs) via membrane perturbation. The self-assembled nanoparticles of the obtained amphiphilic γ-PGA could be adsorbed on the cell membrane by the hydrophobic interaction to disturb the lipid arrangement and increase the membrane permeability of trehalose under hypertonic conditions. Results suggested that the intracellular trehalose could be enhanced progressively with the methylene spacer length, significantly increasing to 75.1 ± 0.7 mM by incubating hRBCs in 0.8 M trehalose containing phenylbutylamine-grafted γ-PGA at 4 °C for 24 h. Meanwhile, the other three polymers exhibited membrane stabilization in addition to improved intracellular trehalose, maintaining the membrane integrity during cryopreservation to achieve high cryosurvival. Molecular dynamics simulation further confirmed that defects could be formed by interaction of the above four amphiphilic polymers on the modeled phospholipid bilayer. It was believed that glycerol-free cryopreservation of human cells could be realized by using trehalose as the biocompatible cryoprotectant, and membrane stabilization can be a compensatory approach to membrane perturbation during impermeable biomolecule delivery.


Assuntos
Criopreservação , Trealose , Sobrevivência Celular , Criopreservação/métodos , Crioprotetores/química , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Eritrócitos/metabolismo , Humanos , Espaço Intracelular , Ácido Poliglutâmico/análogos & derivados , Polímeros/metabolismo , Trealose/química
11.
Cell Mol Life Sci ; 79(8): 420, 2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35833994

RESUMO

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.


Assuntos
IMP Desidrogenase , Espaço Intracelular , Polímeros , Compartimento Celular/fisiologia , Humanos , IMP Desidrogenase/metabolismo , Espaço Intracelular/metabolismo , Polímeros/metabolismo
12.
Nature ; 606(7915): 769-775, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35676476

RESUMO

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.


Assuntos
Imunidade Materno-Adquirida , Imunoglobulina G , Espaço Intracelular , Listeria monocytogenes , Mães , Gravidez , Acetilesterase , Animais , Animais Recém-Nascidos , Linfócitos B , Feminino , Imunidade Materno-Adquirida/imunologia , Imunoglobulina G/imunologia , Interleucina-10/biossíntese , Espaço Intracelular/imunologia , Espaço Intracelular/microbiologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/prevenção & controle , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Gravidez/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T
13.
Int J Mol Sci ; 23(12)2022 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-35742860

RESUMO

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins.


Assuntos
Neoplasias Colorretais , Proteômica , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA , Fatores de Transcrição Forkhead , Galectina 4 , Humanos , Espaço Intracelular/metabolismo , Proteômica/métodos , Recombinases , Fatores de Transcrição
14.
Elife ; 112022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35670565

RESUMO

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.


Assuntos
Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Membrana Celular , Escherichia coli , Lactoferrina , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Espaço Intracelular/química , Espaço Intracelular/microbiologia , Lactoferrina/metabolismo , Lactoferrina/farmacologia , Fatores de Tempo
15.
J Med Genet ; 59(12): 1139-1149, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35701104

RESUMO

BACKGROUND: The SCN5A variant is a common cause of familial dilated cardiomyopathy (DCM). We previously reported a SCN5A variant (c.674G>A), located in the high-risk S4 segment of domain I (DI-S4) region in patients with idiopathic DCM and R225Q knockin (p.R225Q) mice carrying the c.674G>A variant exhibited prolonged baseline PR intervals without DCM phenotypes. In this study, we explored the association and mechanism between R225Q variant and DCM phenotype. METHODS: Prevalence of DI-S4 variant was compared between patients with idiopathic DCM and the control participants. R225Q knockin and wild-type (WT) mice were subjected to doxorubicin (DOX), D-galactose (D-gal) or D-gal combined with DOX. RESULTS: Clinical data suggested that the prevalence of DI-S4 variant was higher in DCM group than in the control group (4/90 (4.4%) vs 3/1339 (0.2%), p<0.001). Cardiomyocytes from R225Q knockin mice treated with D-gal and DOX exhibited more significant hypertrophic phenotype and weaker contraction/dilation function and an increased level of apoptosis as compared with WT mice. Mechanistically, we found that R225Q variant could increase intracellular pH and further induce the activation of the WNT/ß-catenin pathway as well as the overexpression of pro-hypertrophic and pro-apoptotic targets. WNT-C59 inhibitor improved cardiac function in the R225Q knockin mice treated with D-gal and DOX. CONCLUSION: Our results suggest that R225Q variant is associated with increased susceptibility to DCM. Ageing could enhance this process via activating WNT/ß-catenin signaling in response to increased intracellular pH. Antagonising the WNT/ß-catenin pathway might be a potential therapeutic strategy for mitigating R225Q variant-related DCM pathogenesis.


Assuntos
Cardiomiopatia Dilatada , Animais , Humanos , Camundongos , beta Catenina , Cardiomiopatia Dilatada/genética , Doxorrubicina , Concentração de Íons de Hidrogênio , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Via de Sinalização Wnt , Espaço Intracelular/metabolismo
16.
J Virol ; 96(14): e0066522, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35762760

RESUMO

Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.


Assuntos
Infecções por Astroviridae , Proteínas do Capsídeo , Mamastrovirus , Precursores de Proteínas , Infecções por Astroviridae/virologia , Células CACO-2 , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Humanos , Espaço Intracelular/virologia , Mamastrovirus/fisiologia , Precursores de Proteínas/metabolismo , Tripsina/metabolismo
17.
Microbiol Spectr ; 10(3): e0150522, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35608352

RESUMO

Human papillomaviruses (HPVs) consist of two capsid proteins: major capsid protein L1 and minor capsid protein L2. The L2 protein has been shown to be involved in intracellular trafficking events that lead to the deposition of the viral DNA into the nucleus. In this study, we investigate the role of HPV16 L2 residues 43-DQILQ-47 during intracellular trafficking in human keratinocytes. We demonstrate that the highly conserved amino acids aspartic acid, isoleucine, and leucine are involved with the intracellular trafficking of the virus. Amino acid substitution of the isoleucine and leucine residues with alanine residues results in a significant decrease in infectivity of the pseudovirions without any changes to the binding or internalization of the virus. The pseudovirions containing these substitutions exhibit an altered trafficking pattern and do not deposit the viral pseudogenome into the nucleus. Instead, these mutated pseudovirions display a lack of interaction with syntaxin 18, an ER SNARE protein, are unable to progress past the endoplasmic reticulum (ER) and are redirected to the lysosomes. The results of this study help to elucidate the role and potential involvement of the 43-DQILQ-47 sequence during intracellular trafficking, specifically during trafficking beyond the ER. IMPORTANCE High-risk types of human papillomaviruses (HPVs), such as HPV16, are highly associated with cervical, anogenital, and oropharyngeal cancers. The minor capsid protein L2 is essential for the intracellular trafficking of the viral DNA to the nucleus. This study investigates the role of amino acid residues 43-DQILQ-47 of the HPV16 L2 protein in the intracellular trafficking of the virus. Understanding how the virus traffics through the cell is a key factor in the development of additional preventative antiviral therapies. This study illustrates, through modification of the 43-DQILQ-47 sequence in pseudovirions, the importance of the 43-DQILQ-47 sequence in the trafficking of the virus beyond the endoplasmic reticulum.


Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Alphapapillomavirus/genética , Alphapapillomavirus/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , DNA Viral/genética , Retículo Endoplasmático/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Humanos , Espaço Intracelular/metabolismo , Isoleucina/metabolismo , Leucina/metabolismo , Papillomaviridae/genética , Transporte Proteico
18.
Cell Mol Life Sci ; 79(5): 251, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35445278

RESUMO

At the turn of the twenty-first century, fundamental changes took place in the understanding of the structure and function of proteins and then in the appreciation of the intracellular space organization. A rather mechanistic model of the organization of living matter, where the function of proteins is determined by their rigid globular structure, and the intracellular processes occur in rigidly determined compartments, was replaced by an idea that highly dynamic and multifunctional "soft matter" lies at the heart of all living things. According this "new view", the most important role in the spatio-temporal organization of the intracellular space is played by liquid-liquid phase transitions of biopolymers. These self-organizing cellular compartments are open dynamic systems existing at the edge of chaos. They are characterized by the exceptional structural and compositional dynamics, and their multicomponent nature and polyfunctionality provide means for the finely tuned regulation of various intracellular processes. Changes in the external conditions can cause a disruption of the biogenesis of these cellular bodies leading to the irreversible aggregation of their constituent proteins, followed by the transition to a gel-like state and the emergence of amyloid fibrils. This work represents a historical overview of changes in our understanding of the intracellular space compartmentalization. It also reflects methodological breakthroughs that led to a change in paradigms in this area of science and discusses modern ideas about the organization of the intracellular space. It is emphasized here that the membrane-less organelles have to combine a certain resistance to the changes in their environment and, at the same time, show high sensitivity to the external signals, which ensures the normal functioning of the cell.


Assuntos
Organelas , Proteínas , Espaço Intracelular/metabolismo , Organelas/metabolismo , Proteínas/metabolismo
19.
FASEB J ; 36(5): e22274, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35416331

RESUMO

Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6 secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6 levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38 MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.


Assuntos
Lipoproteínas HDL , Fosfatidilserinas , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Espaço Intracelular/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Camundongos , Fosfatidilserinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Sci Rep ; 12(1): 4383, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35288628

RESUMO

Intracellular delivery of nanomaterials into the cells of interest has enabled cell manipulation for numerous applications ranging from cell-based therapies to biomedical research. To date, different carriers or membrane poration-based techniques have been developed to load nanomaterials to the cell interior. These biotools have shown promise to surpass the membrane barrier and provide access to the intracellular space followed by passive diffusion of exogenous cargoes. However, most of them suffer from inconsistent delivery, cytotoxicity, and expensive protocols, somewhat limiting their utility in a variety of delivery applications. Here, by leveraging the benefits of microengineered porous membranes with a suitable porosity, we demonstrated an efficient intracellular loading of diverse nanomaterials to different cell types based on inducing mechanical disruption to the cell membrane. In this work, for the first time, we used ultra-thin silicon nitride (SiN) filter membranes with uniform micropores smaller than the cell diameter to load impermeable nanomaterials into adherent and non-adherent cell types. The delivery performance using SiN microsieves has been validated through the loading of functional nanomaterials from a few nanometers to hundreds of nanometers into mammalian cells with minimal undesired impacts. Besides the high delivery efficiency and improved cell viability, this simple and low-cost approach offers less clogging and higher throughput (107 cell min-1). Therefore, it yields to the efficient introduction of exogenous nanomaterials into the large population of cells, illustrating the potential of these microengineered filters to be widely used in the microfiltroporation (MFP) setup.


Assuntos
Nanoestruturas , Animais , Membrana Celular/metabolismo , Sobrevivência Celular , Espaço Intracelular , Mamíferos
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