RESUMO
Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.
Assuntos
Lisossomos , Transdução de Sinais , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes , Fenômenos Fisiológicos CelularesRESUMO
Processes such as cell migration, phagocytosis, endocytosis, and exocytosis refer to the intense exchange of information between the internal and external environment in the cells, known as vesicular trafficking. In eukaryotic cells, these essential cellular crosstalks are controlled by Rab GTPases proteins through diverse adaptor proteins like SNAREs complex, coat proteins, phospholipids, kinases, phosphatases, molecular motors, actin, or tubulin cytoskeleton, among others, all necessary for appropriate mobilization of vesicles and distribution of molecules. Considering these molecular events, Rab GTPases are critical components in specific biological processes of immune cells, and many reports refer primarily to macrophages; therefore, in this review, we address specific functions in immune cells, concretely in the mechanism by which the GTPase contributes in dendritic cells (DCs) and, T/B lymphocytes.
Assuntos
Linfócitos T , Proteínas rab de Ligação ao GTP , Humanos , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linfócitos T/metabolismo , Linfócitos T/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/imunologiaRESUMO
Zika virus (ZIKV) infections have been associated with severe clinical outcomes, which may include neurological manifestations, especially in newborns with intrauterine infection. However, licensed vaccines and specific antiviral agents are not yet available. Therefore, a safe and low-cost therapy is required, especially for pregnant women. In this regard, metformin, an FDA-approved drug used to treat gestational diabetes, has previously exhibited an anti-ZIKA effect in vitro in HUVEC cells by activating AMPK. In this study, we evaluated metformin treatment during ZIKV infection in vitro in a JEG3-permissive trophoblast cell line. Our results demonstrate that metformin affects viral replication and protein synthesis and reverses cytoskeletal changes promoted by ZIKV infection. In addition, it reduces lipid droplet formation, which is associated with lipogenic activation of infection. Taken together, our results indicate that metformin has potential as an antiviral agent against ZIKV infection in vitro in trophoblast cells.
Assuntos
Metformina , Infecção por Zika virus , Zika virus , Recém-Nascido , Gravidez , Feminino , Humanos , Infecção por Zika virus/tratamento farmacológico , Linhagem Celular Tumoral , Trofoblastos , Antivirais/farmacologia , Metformina/farmacologiaRESUMO
Coronavirus disease (COVID-19) is an infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can be asymptomatic or present with multiple organ dysfunction. Many infected individuals have chronic alterations associated with neuropsychiatric, endocrine, gastrointestinal, and musculoskeletal symptoms, even several months after disease onset, developing long-COVID or post-acute COVID-19 syndrome (PACS). Microbiota dysbiosis contributes to the onset and progression of many viral diseases, including COVID-19 and post-COVID-19 manifestations, which could serve as potential diagnostic and prognostic biomarkers. This review aimed to discuss the most recent findings on gut microbiota dysbiosis and its relationship with the sequelae of PACS. Elucidating these mechanisms could help develop personalized and non-invasive clinical strategies to identify individuals at a higher risk of experiencing severe disease progression or complications associated with PACS. Moreover, the review highlights the importance of targeting the gut microbiota composition to avoid dysbiosis and to develop possible prophylactic and therapeutic measures against COVID-19 and PACS in future studies.
Assuntos
COVID-19 , Microbiota , Humanos , Síndrome de COVID-19 Pós-Aguda , Disbiose/complicações , COVID-19/complicações , SARS-CoV-2RESUMO
The current obesity pandemic has been expanding in both developing and developed countries. This suggests that the factors contributing to this condition need to be reconsidered since some new factors are arising as etiological causes of this disease. Moreover, recent clinical and experimental findings have shown an association between the progress of obesity and some infections, and the functions of adipose tissues, which involve cell metabolism and adipokine release, among others. Furthermore, it has recently been reported that adipocytes could either be reservoirs for these pathogens or play an active role in this process. In addition, there is abundant evidence indicating that during obesity, the immune system is exacerbated, suggesting an increased susceptibility of the patient to the development of several forms of illness or death. Thus, there could be a relationship between infection as a trigger for an increase in adipose cells and the impact on the metabolism that contributes to the development of obesity. In this review, we describe the findings concerning the role of adipose tissue as a mediator in the immune response as well as the possible role of adipocytes as infection targets, with both roles constituting a possible cause of obesity.
Assuntos
Adipócitos , Tecido Adiposo , Adipócitos/metabolismo , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Humanos , Imunidade , Obesidade/etiologiaRESUMO
Lipopolysaccharide (LPS)-responsive beige-like anchor (LRBA) protein was initially described as a monogenetic cause for common variable immune deficiency, a syndrome characterized by low levels of B cells, defects in memory B cell differentiation and hypogammaglobulinaemia. LRBA was identified as an LPS up-regulated gene in B cells, macrophages and T cells. LRBA weighs 320 kDa and has 2863 amino acids. Its sequence contains multiple domains, suggesting that LRBA can act as a scaffolding protein. It contains two putative binding sites for cAMP-dependent kinase (PKA) regulatory subunits, suggesting this protein can act as A-kinase anchor protein (AKAP); however, physical interactions involving LRBA and PKA have not been demonstrated to date, and functional roles for such interactions are unexplored. In this work, we investigated physical interactions involving LRBA with regulatory subunits of PKA in human B cell lines and primary human B cells. PKA is a holoenzyme composed of two regulatory subunits, which can be RIα, RIß, RIIα or RIIß, and two catalytic subunits, Cα or Cß. We co-immunoprecipitated LRBA using Ramos B cell lymphoma cells and observed that LRBA interacts with RIIß. Interestingly, St-Ht31, an inhibitory peptide that disrupts AKAP interactions with regulatory subunits, reduced the amount of interacting protein. Furthermore, in primary human B cells, LRBA was induced after CD40L and IL-4 stimulation, and under such activation, we found that LRBA interacts with RIIα and RIIß, suggesting that LRBA acts as an AKAP and binds RII subunits. Interestingly, we also identified that LRBA interacts with activation-induced cytidine deaminase in primary B cells, suggesting that it is involved in B cell function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ligante de CD40/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/química , Humanos , Interleucina-4/metabolismo , Ativação Linfocitária/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte ProteicoRESUMO
Myosins comprise a family of motor proteins whose role in muscle contraction and motility in a large range of eukaryotic cells has been widely studied. Although these proteins have been characterized extensively and much is known about their function in different cellular compartments, little is known about these molecules in hematopoietic cells. Myosins expressed by cells from the immune response are involved in maintaining plasma membrane tension, moving and secreting vesicles, endo- and exocytotic processes, and promoting the adhesion and motility of cells. Herein, we summarize our current understanding of class I myosins in B cells, with an emphasis on the emerging roles of these molecular motors in immune functions.
Assuntos
Linfócitos B/fisiologia , Miosina Tipo I/metabolismo , Animais , Apresentação de Antígeno/imunologia , Movimento Celular , Humanos , Sinapses Imunológicas/fisiologia , Vesículas Transportadoras/metabolismoRESUMO
Myosin 1g (Myo1g) is a hematopoietic-specific myosin expressed mainly by lymphocytes. Here, we report the localization of Myo1g in B-cell membrane compartments such as lipid rafts, microvilli, and membrane extensions formed during spreading. By using Myo1g-deficient mouse B cells, we detected abnormalities in the adhesion ability and chemokine-induced directed migration of these lymphocytes. We also assessed a role for Myo1g in phagocytosis and exocytosis processes, as these were also irregular in Myo1g-deficient B cells. Taken together, our results show that Myo1g acts as a main regulator of different membrane/cytoskeleton-dependent processes in B lymphocytes.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Movimento Celular/genética , Movimento Celular/imunologia , Citoesqueleto/metabolismo , Endocitose/fisiologia , Exocitose/fisiologia , Miosinas/genética , Animais , Adesão Celular/genética , Células Cultivadas , Feminino , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Miosinas/metabolismo , Transporte ProteicoRESUMO
To better understand the significant variability displayed by influenza viruses, we need to be aware not only of its genetic characteristics, but also of the effect this genetic makeup has on proteins associated with viral replication and antigenicity. The origin of such diversity is due first and foremost to its segmented genome that allows segment reassortment (antigenic shift) and second to the error prone viral polymerase (antigenic drift) responsible of copying the genes enclosed in these segments. These two combined mechanisms confer a genetic plasticity that often leads to the emergence of new influenza viruses in nature.
Assuntos
Farmacorresistência Viral/genética , Variação Genética , Orthomyxoviridae/genética , Tropismo Viral , Animais , Variação Antigênica , Genoma Viral , Humanos , Alphainfluenzavirus/efeitos dos fármacos , Alphainfluenzavirus/genética , Betainfluenzavirus/genética , Gammainfluenzavirus/genética , Mutação , Replicação Viral , Eliminação de Partículas ViraisRESUMO
B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.
Assuntos
Linfócitos B/metabolismo , Membrana Celular/metabolismo , Movimento Celular/genética , Endocitose/genética , Tetraspaninas/genética , Linfócitos B/ultraestrutura , Sistemas CRISPR-Cas , Adesão Celular/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica , Fagocitose/genética , Estresse Mecânico , Tetraspaninas/metabolismoRESUMO
Salmonella enterica serovar Typhimurium (S. Typhimurium) changes the structure of its lipopolysaccharide (LPS) in response to the environment. The two main LPS variants found in S. Typhimurium correspond to LPS with a hepta-acylated lipid A (LPS 430) and LPS with modified phosphate groups on its lipid A (LPS 435). We have previously shown that these modified LPS have a lower capacity than wild type (WT) LPS to induce the production of pro-inflammatory cytokines in mice. Nevertheless, it is not know if LPS 430 and LPS 435 could also subvert the innate immune responses in human cells. In this study, we found that LPS 430 and LPS 435 were less efficient than WT LPS to induce the production of pro-inflammatory cytokines by human monocytes, in addition we found a decreased dimerization of the TLR4/MD-2 complex in response to LPS 430, suggesting that structurally modified LPS are sensed differently than WT LPS by this receptor; however, LPS 430 and 435 induced similar activation of the transcription factors NF-κB p65, IRF3, p38 and ERK1/2 than WT LPS. Microarray analysis of LPS 430- and LPS 435-activated monocytes revealed a gene transcription profile with differences only in the expression levels of microRNA genes compared to the profile induced by WT LPS, suggesting that the lipid A modifications present in LPS 430 and LPS 435 have a moderate effect on the activation of the human TLR4/MD-2 complex. Our results are relevant to understand LPS modulation of immune responses and this knowledge could be useful for the development of novel adjuvants and immunomodulators.
Assuntos
Citocinas/imunologia , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/imunologia , Monócitos/imunologia , Salmonella typhimurium/imunologia , Receptor 4 Toll-Like/imunologia , Acilação/imunologia , Dimerização , Humanos , Inflamação/microbiologia , Lipídeo A/imunologia , Monócitos/microbiologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Transdução de Sinais/imunologia , Fatores de Transcrição/imunologia , Transcrição Gênica/imunologiaRESUMO
The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.
Assuntos
Dessecação , Pseudomonas putida/fisiologia , Contagem de Colônia Microbiana , Proteínas de Fluorescência Verde/metabolismo , Umidade , Microscopia de Fluorescência , Oligonucleotídeos , Raízes de Plantas/microbiologia , RNA Ribossômico/metabolismo , RNA Ribossômico 16S/metabolismo , Rizosfera , Temperatura , Trealose , Zea mays/microbiologiaRESUMO
Cell migration and adhesion are critical for immune system function and involve many proteins, which must be continuously transported and recycled in the cell. Recycling of adhesion molecules requires the participation of several proteins, including actin, tubulin, and GTPases, and of membrane components such as sphingolipids and cholesterol. However, roles of actin motor proteins in adhesion molecule recycling are poorly understood. In this study, we identified myosin 1g as one of the important motor proteins that drives recycling of the adhesion protein CD44 in B lymphocytes. We demonstrate that the lack of Myo1g decreases the cell-surface levels of CD44 and of the lipid raft surrogate GM1. In cells depleted of Myo1g, the recycling of CD44 was delayed, the delay seems to be caused at the level of formation of recycling complex and entry into recycling endosomes. Moreover, a defective lipid raft recycling in Myo1g-deficient cells had an impact both on the capping of CD44 and on cell migration. Both processes required the transportation of lipid rafts to the cell surface to deliver signaling components. Furthermore, the extramembrane was essential for cell expansion and remodeling of the plasma membrane topology. Therefore, Myo1g is important during the recycling of lipid rafts to the membrane and to the accompanied proteins that regulate plasma membrane plasticity. Thus, Myosin 1g contributes to cell adhesion and cell migration through CD44 recycling in B lymphocytes.
RESUMO
Antigen processing for presentation by major histocompatibility complex class II (MHCII) molecules requires the latter to travel through the endocytic pathway together with its chaperons: the invariant chain (Ii) and DM. Nevertheless, the nature of the compartments where MHCII molecules travel to acquire peptides lacks definition regarding molecules involved in intracellular vesicular trafficking, such as Rab small GTPases. We aimed to define which Rab proteins are present during the intracellular transport of MHCII, DM, and Ii through the endocytic pathway on their route to the cell surface during dendritic cell (DC) maturation. We examined, by means of three-color confocal microscopy, the association of MHCII, DM, and Ii with Rab5, Rab7, Rab9, and Rab11 during the maturation of bone marrow-derived or spleen DC in response to LPS as an inflammatory stimulus. Prior to the stage of immature DC, MHCII migrated from diffuse small cytoplasmic vesicles, predominantly Rab5+Rab7- and Rab5+Rab7+ into a pericentriolar Rab5+Rab7+Rab9+ cluster, with Rab11+ areas. As DC reached the mature phenotype, MHCII left the pericentriolar endocytic compartments toward the cell surface in Rab11+ and Rab9+Rab11+ vesicles. The invariant chain and MHCII transport pathways were not identical. DM and MHCII appeared to arrive to pericentriolar endocytic compartments of immature DC through partially different routes. The association of MHCII molecules with distinct Rab GTPases during DC maturation suggests that after leaving the biosynthetic pathway, MHCII sequentially traffic from typical early endosomes to multivesicular late endosomes to finally arrive at the cell surface in Rab11+ recycling-type endosomes. In immature DCs, DM encounters transiently MHCII in the Rab5+Rab7+Rab9+ compartments, to remain there in mature DC.
RESUMO
Two patients with a severe leukocyte adhesion deficiency type 1 (LAD-1) phenotype were analyzed by flow cytometry and functional assays to demonstrate the improper adhesive and phagocytic responses of their leukocytes. A single homozygous defect that involves a missense mutation (c.817G>A) that encodes for a G273R substitution in CD18 was identified in both patients. The adhesion and phagocytosis assays demonstrated the inability of patients' leukocytes to perform these functions. Expression of the LFA-1 (CD11a/CD18) on the co-transfected HEK 293 cells with the mutated form of CD18 was not detected. Finally, both patients have been treated with immunoglobulin as an adjunctive therapy with positive results. We propose that intravenous immunoglobulin treatment is safe and efficacious in LAD-1 patients before hematopoietic stem cell transplantation and helpful in controlling severe infections. Subcutaneous immunoglobulin appeared to help wound healing in refractory ulcers in these patients.