RESUMO

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

Assuntos

RESUMO

Perfluoroalkyl substances are man-made chemicals with ample consumer and industrial applications. They are widely used and are resistant to environmental and metabolic degradation. Several studies have evaluated the effects of Perfluorohexane sulfonate on reproduction. However, there are few reports exploring the cell and molecular mechanisms of its toxicity in the ovary. The aim of this study was to investigate the effects of PFHxS exposure on the estrous cycle, ovulation rate, and the underlying mechanisms of action in female mice in vivo. The animals received a single sub-lethal dose of PFHxS (25.1 mg/kg, 62.5 mg/kg) or vehicle and were stimulated to obtain immature cumulus cell-oocyte complexes (COCs) from the ovaries, or superovulated to develop mature COCs. To evaluate oocyte physiology, Gap-junction intercellular communication (GJIC) was analyzed in immature COCs and calcium homeostasis was evaluated in mature oocytes. PFHxS exposure prolonged the estrous cycle and decreased ovulation rate in female mice. Connexins, Cx43 and Cx37, were downregulated and GJIC was impaired in immature COCs, providing a possible mechanism for the alterations in the estrous cycle and ovulation. No morphological abnormalities were observed in the mature PFHxS-exposed oocytes, but calcium homeostasis was affected. This effect is probably due, at least partially, to deregulation of the endoplasmic reticulum calcium modulator, Stim1. These mechanisms of ovarian injury could explain the reported correlation among PFHxS levels and subfertility in women undergoing fertility treatments.

Assuntos

RESUMO

Extracellular vesicles (EVs) are naturally released membrane vesicles that act as carriers of proteins and RNAs for intercellular communication. With various biomolecules and specific ligands, EV has represented a novel form of information transfer, which possesses extremely outstanding efficiency and specificity compared to the classical signal transduction. In addition, EV has extended the concept of signal transduction to intercellular aspect by working as the collection of extracellular information. Therefore, the functions of EVs have been extensively characterized and EVs exhibit an exciting prospect for clinical applications. However, the biogenesis of EVs and, in particular, the regulation of this process by extracellular signals, which are essential to conduct further studies and support optimal utility, remain unclear. Here, we review the current understanding of the biogenesis of EVs, focus on the regulation of this process by extracellular signals and discuss their therapeutic value.

Assuntos

RESUMO

Extracellular vesicles (EVs) have been identified as active components in cellular communication, which are easily altered both morphologically and chemically by the cellular environment and metabolic state of the body. Due to this sensitivity to the conditions of the cellular microenvironment, EVs have been found to be associated with disease conditions, including those associated with obesity and undernutrition. The sensitivity that EVs show to changes in the cellular microenvironment could be a reflection of early cellular alterations related to conditions of malnutrition, which could eventually be used in the routine monitoring and control of diseases or complications associated with it. However, little is known about the influence of malnutrition alone; that is, without the influence of additional diseases on the heterogeneity and specific content of EVs. To date, studies in "apparently healthy" obese patients show that there are changes in the size, quantity, and content of EVs, as well as correlations with some metabolic parameters (glucose, insulin, and serum lipids) in comparison with non-obese individuals. In light of these changes, a direct participation of EVs in the development of metabolic and cardiovascular complications in obese subjects is thought to exist. However, the mechanisms through which this process might occur are not yet fully understood. The evidence on EVs in conditions of undernutrition is limited, but it suggests that EVs play a role in the maintenance of homeostasis and muscle repair. A better understanding of how EVs participate in or promote cellular signaling in malnutrition conditions could help in the development of new strategies to treat them and their comorbidities.

Assuntos

RESUMO

Extracellular vesicles (EVs) are naturally released membrane vesicles that act as carriers of proteins and RNAs for intercellular communication. With various biomolecules and specific ligands, EV has represented a novel form of information transfer, which possesses extremely outstanding efficiency and specificity compared to the classical signal transduction. In addition, EV has extended the concept of signal transduction to intercellular aspect by working as the collection of extracellular information. Therefore, the functions of EVs have been extensively characterized and EVs exhibit an exciting prospect for clinical applications. However, the biogenesis of EVs and, in particular, the regulation of this process by extracellular signals, which are essential to conduct further studies and support optimal utility, remain unclear. Here, we review the current understanding of the biogenesis of EVs, focus on the regulation of this process by extracellular signals and discuss their therapeutic value.

Assuntos

RESUMO

Previous studies indicate that the activity of hypothalamic POMC neurons can be regulated by glucose via intracellular mechanisms, but its regulation by lactate is poorly understood. In addition to its energetic role, lactate acts as a signaling molecule. In this study, we evaluated the function and location of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1). We used a conditional genetic approach to label POMC neurons and evaluated their sensitivity to lactate using patch-clamp recordings. L-Lactate and 3-chloro-5-hydroxybenzoic acid (3Cl-HBA), HCAR1 specific agonist depolarized POMC neurons and the increase in excitability was abolished by pertussis toxin (PTX), indicating the involvement of Gαi/o-protein-coupled receptors. In addition, the depolarization of a subset of POMC neurons was sensitive to α-cyano-4-hydroxycinnamate (4-CIN), a lactate transporter blocker, suggesting that the depolarization induced by L-lactate can also occur by direct intracellular action. Surprisingly, HCAR1 was not detected in POMC neurons, but instead localized in astrocytes. These results suggest a new lactate-mediated mechanism for astrocyte-neuron intercellular communication.

Assuntos

RESUMO

The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host-pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.

Assuntos

Assuntos

RESUMO

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.

Assuntos

RESUMO

Early compositional studies of fungal EVs revealed a complex combination of biomolecules, including proteins, lipids, glycans, polysaccharides, nucleic acid and pigments, indicating that these compartments could be involved with multiple functions. Curiously, some of the activities attributed to fungal EVs were already attested experimentally and are implicated with contrasting effects in vitro and in vivo. For instance, the presence of virulence factors is correlated with increased pathogenic potential. Indeed, the administration to hosts of EVs along with some fungal pathogens seems to help the disease development. However, it has been clearly shown that immunization of insects and mice with fungal EVs can protect these animals against a subsequent infection. Fungal EVs not only influence the host response, as concluded from the observation that these compartments also work as messengers between fungal organisms. In this context, despite their size characterization, other physical properties of EVs are poorly known. For instance, their stability and half-life under physiological conditions can be a crucial parameter determining their long-distance effects. In this review, we will discuss the paradoxical and still unexploited functions and properties of fungal EVs that could be determinant for their biological functions.

Assuntos