Meningococcus Hijacks a ß2-adrenoceptor/ß-Arrestin pathway to cross brain microvasculature endothelium.

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased ß2-adrenoceptor/ß-arrestin signaling pathway in endothelial cells, which ultimately traps ß-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by ß-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas ß-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of ß-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.

Pharmacological chaperone-rescued cystic fibrosis CFTR-F508del mutant overcomes PRAF2-gated access to endoplasmic reticulum exit sites.

The endoplasmic reticulum exit of some polytopic plasma membrane proteins (PMPs) is controlled by arginin-based retention motifs. PRAF2, a gatekeeper which recognizes these motifs, was shown to retain the GABAB-receptor GB1 subunit in the ER. We report that PRAF2 can interact on a stoichiometric basis with both wild type and mutant F508del Cystic Fibrosis (CF) Transmembrane Conductance Regulator (CFTR), preventing the access of newly synthesized cargo to ER exit sites. Because of its lower abundance, compared to wild-type CFTR, CFTR-F508del recruitment into COPII vesicles is suppressed by the ER-resident PRAF2. We also demonstrate that some pharmacological chaperones that efficiently rescue CFTR-F508del loss of function in CF patients target CFTR-F508del retention by PRAF2 operating with various mechanisms. Our findings open new therapeutic perspectives for diseases caused by the impaired cell surface trafficking of mutant PMPs, which contain RXR-based retention motifs that might be recognized by PRAF2.

Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABAB receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5's interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER.

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters.

Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.

Protective reactive thymus hyperplasia in COVID-19 acute respiratory distress syndrome.

BACKGROUND: Patients with COVID-19 (COVID) may develop acute respiratory distress syndrome with or without sepsis, coagulopathy and visceral damage. While chest CT scans are routinely performed in the initial assessment of patients with severe pulmonary forms, thymus involvement and reactivation have not been investigated so far. METHODS: In this observational study, we systematically scored the enlargement of the thymus and the lung involvement, using CT scans, in all adult patients admitted to the ICU for COVID or any other cause (control group) at one centre between March and April 2020. Initial biological investigations included nasal detection of SARS-CoV-2 ribonucleic acid by polymerase chain reaction (PCR). In a subgroup of 24 patients with different degrees of pulmonary involvement and thymus hypertrophy, plasma cytokine concentrations were measured and the export of mature T cells from the thymus was estimated simultaneously by PCR quantification of T cell receptor excision circles (TRECs). RESULTS: Eighty-seven patients were studied: 50 COVID patients and 37 controls. Non-atrophic or enlarged thymus was more commonly observed in COVID patients than in controls (66% vs. 24%, p < 0.0001). Thymus enlargement in COVID patients was associated with more extensive lung injury score on CT scans (4 [3-5] vs. 2 [1.5-4], p = 0.01), but a lower mortality rate (8.6% vs. 41.2%, p < 0.001). Other factors associated with mortality were age, lymphopaenia, high CRP and co-morbidities. COVID patients had higher concentrations of IL-7 (6.00 [3.72-9.25] vs. 2.17 [1.76-4.4] pg/mL; p = 0.04) and higher thymic production of new lymphocytes (sj/ßTREC ratio = 2.88 [1.98-4.51] vs. 0.23 [0.15-0.60]; p = 0.004). Thymic production was also correlated with the CT scan thymic score (r = 0.38, p = 0.03) and inversely correlated with the number of lymphocytes (r = 0.56, p = 0.007). CONCLUSION: In COVID patients, thymus enlargement was frequent and associated with increased T lymphocyte production, which appears to be a beneficial adaptation to virus-induced lymphopaenia. The lack of thymic activity/reactivation in older SARS-CoV-2 infected patients could contribute to a worse prognosis.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-ß-arrestin complex to the plasma membrane, ß-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient ß-arrestin and by a specific inhibitor of ß-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of ß-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

Endoplasmic reticulum stress induces G2 cell-cycle arrest via mRNA translation of the p53 isoform p53/47.

p53 downstream pathways control G1 and G2 cell-cycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3sigma and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.

Receptor sequestration in response to ß-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression.

MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and ß-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with ß-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with ß-arrestin, implicating this scaffolding protein in the receptor's subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking ß-arrestins combined with in vitro kinase assays revealed that ß-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator.

CXCR7 participates in CXCL12-induced CD34+ cell cycling through ß-arrestin-dependent Akt activation.

In addition to its well-known effect on migration and homing of hematopoietic stem/progenitor cells (HSPCs), CXCL12 chemokine also exhibits a cell cycle and survival-promoting factor for human CD34(+) HSPCs. CXCR4 was suggested to be responsible for CXCL12-induced biological effects until the recent discovery of its second receptor, CXCR7. Until now, the participation of CXCR7 in CXCL12-induced HSPC cycling and survival is unknown. We show here that CXCL12 was capable of binding CXCR7 despite its scarce expression at CD34(+) cell surface. Blocking CXCR7 inhibited CXCL12-induced Akt activation as well as the percentage of CD34(+) cells in cycle, colony formation, and survival, demonstrating its participation in CXCL12-induced functional effects in HSPCs. At steady state, CXCR7 and ß-arrestin2 co-localized near the plasma membrane of CD34(+) cells. After CXCL12 treatment, ß-arrestin2 translocated to the nucleus, and this required both CXCR7 and CXCR4. Silencing ß-arrestin expression decreased CXCL12-induced Akt activation in CD34(+) cells. Our results demonstrate for the first time the role of CXCR7, complementary to that played by CXCR4, in the control of HSPC cycling, survival, and colony formation induced by CXCL12. We also provide evidence for the involvement of ß-arrestins as signaling hubs downstream of both CXCL12 receptors in primary human HSPCs.

Targeting spare CC chemokine receptor 5 (CCR5) as a principle to inhibit HIV-1 entry.

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of ß-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.

Distinct functional outputs of PTEN signalling are controlled by dynamic association with ß-arrestins.

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins ß-arrestins (ß-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, ß-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, ß-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. ß-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration.

Neisseria meningitidis colonization of the brain endothelium and cerebrospinal fluid invasion.

The brain and meningeal spaces are protected from bacterial invasion by the blood-brain barrier, formed by specialized endothelial cells and tight intercellular junctional complexes. However, once in the bloodstream, Neisseria meningitidis crosses this barrier in about 60% of the cases. This highlights the particular efficacy with which N. meningitidis targets the brain vascular cell wall. The first step of central nervous system invasion is the direct interaction between bacteria and endothelial cells. This step is mediated by the type IV pili, which induce a remodelling of the endothelial monolayer, leading to the opening of the intercellular space. In this review, strategies used by the bacteria to survive in the bloodstream, to colonize the brain vasculature and to cross the blood-brain barrier will be discussed.

Arrestins in host-pathogen interactions.

In the context of host-pathogen interaction, host cell receptors and signaling pathways are essential for both invading pathogens, which exploit them for their own profit, and the defending organism, which activates early mechanism of defense, known as innate immunity, to block the aggression. Because of their central role as scaffolding proteins downstream of activated receptors, ß-arrestins are involved in multiple signaling pathways activated in host cells by pathogens. Some of these pathways participate in the innate immunity and the inflammatory response. Other ß-arrestin-dependent pathways are actually hijacked by microbes and toxins to penetrate into host cells and spread in the organism.

Regulated GPCR trafficking to the plasma membrane: general issues and the CCR5 chemokine receptor example.

The regulated export of nascent G protein coupled receptors (GPCRs) from intracellular stores is an emerging concept with important implications in cell biology and pharmacology. This phenomenon requires a complex network of interactions between GPCRs with either chaperones and escort proteins or gatekeepers, which are respectively involved in the progression of GPCRs along the biosynthetic pathway to the plasma membrane or in their retention in intracellular compartments. The regulated export of GPCRs is also controlled by external stimuli and might represent an adaptive mechanism to specific physiological constraints, such as the sustained activation of the CCR5 chemokine receptor in the context of chemotaxis.

Genetically determined thymic function affects strength and duration of immune response in COVID patients with pneumonia.

Thymic activation improves the outcome of COVID-19 patients with severe pneumonia. The rs2204985 genetic polymorphism within the TCRA-TCRD locus, which affects thymic output in healthy individuals, was found here to modify SARS-CoV-2-specific immunity and disease severity in COVID-19 patients with severe pneumonia. Forty patients with severe COVID-19 pneumonia were investigated. The GG genotype at the rs2204985 locus was associated, independently of age and sex, with stronger and long-lasting anti-SARS-CoV-2 helper and cytotoxic T cell responses 6 months after recovery. The GG genotype was also associated with less severe lung involvement, higher thymic production, and higher counts of blood naïve T lymphocytes, including recent thymic emigrants, and a larger population of activated stem cell memory CD4+ T cells. Overall, GG patients developed a more robust and sustained immunity to SARS-CoV-2. Polymorphism at rs2204985 locus should be considered as an additional predictive marker of anti-SARS-CoV-2 immune response.

Mechanical Activation of the ß2-Adrenergic Receptor by Meningococcus: A Historical and Future Perspective Analysis of How a Bacterial Probe Can Reveal Signalling Pathways in Endothelial Cells, and a Unique Mode of Receptor Activation Involving Its N-Terminal Glycan Chains.

More than 12 years have passed since the seminal observation that meningococcus, a pathogen causing epidemic meningitis in humans, occasionally associated with infectious vasculitis and septic shock, can promote the translocation of ß-arrestins to the cell surface beneath bacterial colonies. The cellular receptor used by the pathogen to induce signalling in host cells and allowing it to open endothelial cell junctions and reach meninges was unknown. The involvement of ß-arrestins, which are scaffolding proteins regulating G protein coupled receptor signalling and function, incited us to specifically investigate this class of receptors. In this perspective article we will summarize the events leading to the discovery that the ß2-adrenergic receptor is the receptor that initiates the signalling cascades induced by meningococcus in host cells. This receptor, however, cannot mediate cell infection on its own. It needs to be pre-associated with an "early" adhesion receptor, CD147, within a hetero-oligomeric complex, stabilized by the cytoskeletal protein α-actinin 4. It then required several years to understand how the pathogen actually activates the signalling receptor. Once bound to the N-terminal glycans of the ß2-adrenergic receptor, meningococcus provides a mechanical stimulation that induces the biased activation of ß-arrestin-mediated signalling pathways. This activating mechanical stimulus can be reproduced in the absence of any pathogen by applying equivalent forces on receptor glycans. Mechanical activation of the ß2-adrenergic receptor might have a physiological role in signalling events promoted in the context of cell-to-cell interaction.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface.

The association of CD4, a glycoprotein involved in T-cell development and antigen recognition, and CC chemokine receptor 5 (CCR5), a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for HIV. We observed that the majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relatively low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration-dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell-surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cellular distribution of CXCR4, the other HIV coreceptor. These results reveal a previously unappreciated role of CD4, which contributes to regulating CCR5 export to the plasma membrane.

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

CXCR4-CCR5: a couple modulating T cell functions.

Chemokines and their receptors direct leukocyte migration among blood, lymph and tissues. Evidence has recently accumulated that, besides their chemotactic functions, chemokine receptors are highly versatile players that fine tune immune responses. During human T cell activation by antigen-presenting cells, the chemokine receptors CCR5 and CXCR4 are recruited into the immunological synapse, where they deliver costimulatory signals. However, the molecular mechanisms allowing signaling versatility of chemokine receptors are unknown. Here, we describe the functional interaction between CXCR4 and CCR5 to exert specific biological functions and modulate T lymphocyte responses. We demonstrate that simultaneous expression and cooperation between CCR5 and CXCR4 are required for chemokine-induced T cell costimulation at the immunological synapse. In addition, we provide evidence for a physical association of the two receptors in a signaling complex that activates distinct T cell functions. We suggest that cooperation between receptors represents one key strategy for the functional plasticity of chemokines.

The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.