Academic literature on the topic 'Endosomal signalling'

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor–receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor–receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor–receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered.

GPCR (G-protein-coupled receptor) signalling at the plasma membrane is under tight control. In the case of neuropeptides such as SP (substance P), plasma membrane signalling is regulated by cell-surface endopeptidases (e.g. neprilysin) that degrade extracellular neuropeptides, and receptor interaction with β-arrestins, which uncouple receptors from heterotrimeric G-proteins and mediate receptor endocytosis. By recruiting GPCRs, kinases and phosphatases to endocytosed GPCRs, β-arrestins assemble signalosomes that can mediate a second wave of signalling by internalized receptors. Endosomal peptidases, such as ECE-1 (endothelin-converting enzyme-1), can degrade SP in acidified endosomes, which destabilizes signalosomes and allows receptors, freed from β-arrestins, to recycle and resensitize. By disassembling signalosomes, ECE-1 terminates β-arrestin-mediated endosomal signalling. These mechanisms have been studied in model cell systems, and the relative importance of plasma membrane and endosomal signalling to complex pathophysiological processes, such as inflammation, pain and proliferation, is unclear. However, deletion or inhibition of metalloendopeptidases that control neuropeptide signalling at the plasma membrane and in endosomes has marked effects on inflammation. Neprilysin deletion exacerbates inflammation because of diminished degradation of pro-inflammatory SP. Conversely, inhibition of ECE-1 attenuates inflammation by preventing receptor recycling/resensitization, which is required for sustained pro-inflammatory signals from the plasma membrane. β-Arrestin deletion also affects inflammation because of the involvement of β-arrestins in pro-inflammatory signalling and migration of inflammatory cells. Knowledge of GPCR signalling in specific subcellular locations provides insights into pathophysiological processes, and can provide new opportunities for therapy. Selective targeting of β-arrestin-mediated endosomal signalling or of mechanisms of receptor recycling/resensitization may offer more effective and selective treatments than global targeting of cell-surface signalling.

Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to ILVs (intraluminal vesicles) of endosomes before degradation in the lysosome. Sorting of endocytosed EGFR on to ILVs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. EGFR signalling is also subject to down-regulation through receptor dephosphorylation by the ER (endoplasmic reticulum)-localized PTP1B (protein tyrosine phosphatase 1B). PTP1B on the cytoplasmic face of the ER interacts with endocytosed EGFR via direct membrane contacts sites between the ER and endosomes. In the present paper, we review the relationship between ER–endosome membrane contact sites and ILV formation, and their potential role in the regulation of EGFR sorting on to ILVs, through PTP1B-mediated dephosphorylation of both EGFR and components of the ESCRT machinery.

The Kes1 OSBP (oxysterol-binding protein) is a key regulator of membrane trafficking through the TGN (trans-Golgi network) and endosomal membranes. We demonstrated recently that Kes1 acts as a sterol-regulated rheostat for TGN/endosomal phosphatidylinositol 4-phosphate signalling. Kes1 utilizes its dual lipid-binding activities to integrate endosomal lipid metabolism with TORC1 (target of rapamycin complex 1)-dependent proliferative pathways and transcriptional control of nutrient signalling.

ObjectivesHydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL).MethodsFor in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis.ResultsHCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1β and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation.ConclusionsWe describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.

Signalling through phosphoinositide lipids is essential for regulating many cellular processes, including endosomal trafficking. A number of intracellular pathogens have found ways to subvert host trafficking pathways via exploitation of endosomal phosphoinositides. This review will discuss how pathogens such as bacteria, viruses, and eukaryotic parasites depend on endosomal phosphoinositides for infection as well as the mechanisms through which some are able to actively manipulate these signalling lipids to facilitate invasion, survival, replication, and immune evasion.

The ESCRT (endosomal sorting complex required for transport) machinery consists of four protein complexes that mediate sorting of ubiquitinated membrane proteins into the intraluminal vesicles of multivesicular endosomes, thereby targeting them for degradation in lysosomes. In the present paper, we review how ESCRT-mediated receptor down-regulation affects signalling downstream of Notch and growth factor receptors, and how ESCRTs may control cell proliferation, survival and cytoskeletal functions and contribute to tumour suppression.

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.

La maladie rénale chronique (MRC) se caractérise par des lésions rénales aboutissant à une perte fonctionnelle des néphrons. Une activation du récepteur transmembranaire Epidermal Growth Factor Receptor (EGFR) par le Transforming Growth Factor alpha (TGF-a) est responsable de l'hyperprolifération tubulaire observée. Dans ce contexte, il a été montré que la Lipocaline 2 (Lcn2) médie l'effet mitogène de l'EGFR mais les mécanismes intracellulaires sous-jacents sont méconnus. Lcn2 est une protéine de 25 kDa dont l'expression est induite dans différentes conditions pathologiques dont l'inflammation, le cancer et l'atteinte rénale. Mon travail de thèse s'est intéressé à comprendre les mécanismes intracellulaires par lesquels Lcn2 pouvait moduler la voie de l'EGFR. Je montre dans une lignée cellulaire rénale que Lcn2 augmente l'adressage de l'EGFR à la membrane plasmatique aussi bien en condition de repos qu'après stimulation par du TGF-a. Ceci permet de potentialiser l'activation de l'EGFR et de ses cascades de phosphorylation intracellulaires. L'absence de Lcn2 entraînant une dégradation lysosomale de l'EGFR après activation par le TGF-a. Mes travaux indiquent que Lcn2 peut se trouver dans le cytosol où elle interagit avec le domaine intracellulaire de l'EGFR au niveau des endosomes tardifs. Nous confirmons dans un modèle murin de MRC que l'invalidation du gène Lcn2 abolit le renforcement membranaire de l'EGFR observé dans des souris sauvages. De façon intéressante, j'observe une phosphorylation en Tyrosine de Lcn2 induite par l'activation de l'EGFR. Je démontre aussi que l'état de phosphorylation de Lcn2 module son interaction avec le récepteur ainsi que sa capacité à induire son recyclage membranaire. D'autre part, une étude de l'interactome de Lcn2 me permet de proposer de nouvelles pistes d'étude concernant le rôle de Lcn2. En effet, je mets en avant des partenaires potentiels de Lcn2 en dehors de la voie de sécrétion, comme des protéines du cytosquelette ou des vésicules de transport intracellulaire. Par l'étude de voies potentielles, je montre d'une part l'implication de Lcn2 dans la mise en place des adhésions focales pouvant expliquer son rôle dans la migration cellulaire. Et d'autre part je propose que l'expression de Lcn2 participe à la régulation de l'élongation du cil primaire
Chronic Kidney Disease (CKD) is characterized by a functional deterioration of kidney parenchyma. Transforming Growth Factor alpha (TGF-a) dependent activation of Epidermal Growth Factor Receptor (EGFR) participates to CKD onset and progression. In this context, it has been previously shown that Lipocalin 2 (Lcn2) mediates EGFR mitogenic effect, but the underlying intracellular mechanisms on EGFR pathway regulation by Lcn2 are yet to be discovered. Lcn2 is a 25 kDa protein that is expressed in many pathological conditions as in inflammation, cancer or kidney damage. During my thesis I wanted to better understand the intracellular mechanisms by which Lcn2 can regulate EGFR pathway. First I establish, in an in vitro model of kidney epithelial cells, that Lcn2 enhances EGFR recycling to the plasma membrane in steady state as well as after its activation by TGF-a. This allows for an increased activation of EGFR and its downstream targets. Conversely, Lcn2 inhibition induced a lysosomal degradation of TGF-a-stimulated EGFR. In order to gain insight into the mechanisms by which Lcn2 regulates EGFR trafficking, I demonstrate that a cytosolic fraction of Lcn2 binds the intracellular domain of activated EGFR during its journey in the late endosomes. In agreement, using a murine experimental model of CKD, I confirmed EGFR abundance decrease at tubular cell membrane in Lcn2 knock-out mice. Interestingly, I demonstrate that EGFR activation by TGF-a induces Lcn2 phosphorylation on Tyrosine residues. Furthermore, I establish that Lcn2 phosphorylation enhances the interaction between EGFR and Lcn2 and that it is necessary for inducing EGFR recycling to the plasma membrane. Moreover, an interactome analysis of Lcn2 protein interaction network unraveled new pathways in which Lcn2 could be involved. Indeed, putative partners of Lcn2 identified in this analysis are components of the cytoskeletal compartment and of the vesicular transport machineries. This interactome analysis allowed me to show that Lcn2 could be implicated in phenomena such as focal adhesion assembly, which could be linked to Lcn2 role on cell migration, or primary cilia length, an important actor in CKD

Disrupted pancreatic β-cell function is a key event in the pathogenesis of diabetes mellitus, a metabolic disorder resulting in elevated blood sugar levels. β-cells are responsible for the secretion of insulin, which promotes the uptake of blood glucose into peripheral tissue. Additionally, autocrine insulin signalling contributes to the maintenance of properly functioning β-cells. Upon insulin binding, the insulin receptor tyrosine kinase is activated and recruits insulin receptor substrates to its intracellular domain. These substrates can activate two major signalling branches, the Akt branch and the Ras/Erk branch. Both signalling branches are suggested to be involved in the maintenance of β-cell function and survival. Interestingly, results from experiments in adipose-like cell lines demonstrate, that endocytic vesicles can act as signalling hubs potentially directing insulin receptor signals between the Erk and Akt branches. Endosomes have also been suggested as organelles that are capable of buffering the rapid influx of calcium into β-cells following glucose stimulation thus avoiding calcium-induced β-cell death. These findings highlight endosomes as important organelles involved in the maintenance of β-cell function. This thesis examines the role of endosomes in autocrine insulin signalling and their involvement in calcium homeostasis. To observe the impact of endocytosis on autocrine insulin signalling, a novel fluorescent protein-labeled insulin receptor construct was developed and validated, revealing that tyrosine-phosphorylated caveolin-1 (Cav1) participates in insulin receptor internalization in β-cells. Remarkably, this process was found to bias insulin signalling towards the Erk branch in vitro and in vivo. As a functional consequence, reduction of Cav1 activity inhibited Erk signalling and was associated with increased β-cell apoptosis and decreased β-cell mass in mice lacking Cav1. The role of endosomes in β-cell calcium buffering was elucidated by creating a genetically encoded calcium sensor specifically localized to the lumen of endosomes and estimating calcium levels in defined endosome sub-populations. Indeed, endosomes accumulate calcium during glucose stimulation. Together, this work highlights endosomes as hubs for autocrine insulin signalling and contributors to the calcium homeostasis in the glucose response of β-cells.
Medicine, Faculty of
Graduate

In December 2019, a new infectious respiratory disease emerged in Wuhan, Hubei province, China. A novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2), shows features that were similar to that SARS-CoV. Soon it was believed to have caused a new lung disease later on known as COVID-19. Originally, the susceptible index patient was asymptomatic and later was confirmed as COVID positive with fever, cough, and sore throat-like symptoms. Later the index patient symptoms rapidly severed along with a high respiratory rate. The severe acute respiratory syndrome coronavirus (SARS-CoV) is associated with lung injury, while acute respiratory distress syndrome may result in a pulmonary failure resulting in mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) soon captured the world's attention due to its capacity to widespread fatality leading to the failure of the healthcare system across the globe. It was also revleaed by the researchers that both SARS-CoV-2 and SARS-CoV exhibited the same features while making their entry into the host cells. They made use of host angiotensin-converting enzyme II (ACE2) for their entry into the host. This enzyme is present on the host cell surface, especially in epithelial cells of respiratory organs like lungs and small intestine in humans. From the accumulated existing published data, it is obvious that the SARS-CoV-2 employs two ways for making its entry into the host cells: one path is initiated by transmembrane protease serine 2 (TMPRSS2) that lies on the surface of the cell while the other is mediated by angiotensin converting enzyme II (ACE2) endosomal pathway. On the other hand, Cholesterol and sphingolipid-rich lipid raft, and micro-domains in the plasma membrane that are used as several physiological signalling pathways, are also involved in virus entry. This chapter aims at briefly evaluating the pathogenesis of SARS-CoV and new anti viral drugs against the disease.