Cleavage Of Receptor Research Articles

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients.

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.

BACE1 regulates expression of Clusterin in astrocytes for enhancing clearance of β-amyloid peptides

BackgroundAbnormal accumulation of amyloid beta peptide (Aβ) in the brain induces a cascade of pathological changes in Alzheimer’s disease (AD), and inhibiting BACE1, which is required for Aβ generation, is therefore being explored for the treatment of AD by reducing Aβ accumulation. As Bace1 knockout mice exhibit increased number of reactive astrocytes and AD brains have reactive astrocytes that surround amyloid plaques, we investigated the role of BACE1 in astrocytes and determined whether BACE1 regulates astrocytic functions.MethodsWe conducted unbiased single cell RNA-seq (scRNA-seq) using purified astrocytes from Bace1 KO mice and wild type control littermates. Similar scRNA-seq was also conducted using AD mice with conditional deletion of Bace1 in the adult stage (5xFAD;Bace1fl/fl;UBC-creER compared to 5xFAD;Bace1fl/fl controls). We compared the transcriptomes of astrocyte and reactive astrocyte clusters and identified several differentially expressed genes, which were further validated using Bace1 KO astrocyte cultures. Mice with astrocyte-specific Bace1 knockout in 5xFAD background were used to compare amyloid deposition. Mechanistic studies using cultured astrocytes were used to identify BACE1 substrates for changes in gene expression and signaling activity. ResultsAmong altered genes, Clusterin (Clu) and Cxcl14 were significantly upregulated and validated by measuring protein levels. Moreover, BACE1 deficiency enhanced both astrocytic Aβ uptake and degradation, and this effect was significantly attenuated by siRNA knockdown of Clu. Mechanistic study suggests that BACE1 deficiency abolishes cleavage of astrocytic insulin receptors (IR), and this may enhance expression of Clu and Cxcl14. Acutely isolated astrocytes from astrocyte-specific knockout of Bace1 mice (Bace1 fl/fl;Gfap-cre) show similar increases in CLU and IR. Furthermore, astrocyte-specific knockout of Bace1 in a 5xFAD background resulted in a significant attenuation in cortical Aβ plaque load through enhanced clearance.ConclusionTogether, our study suggests that BACE1 in astrocytes regulates expression of Clu and Cxcl14, likely via the control of insulin receptor pathway, and inhibition of astrocytic BACE1 is a potential alternative strategy for enhancing Aβ clearance.

Protease allergens as initiators-regulators of allergic inflammation.

Tremendous progress in the last few years has been made to explain how seemingly harmless environmental proteins from different origins can induce potent Th2-biased inflammatory responses. Convergent findings have shown the key roles of allergens displaying proteolytic activity in the initiation and progression of the allergic response. Through their propensity to activate IgE-independent inflammatory pathways, certain allergenic proteases are now considered as initiators for sensitization to themselves and to non-protease allergens. The protease allergens degrade junctional proteins of keratinocytes or airway epithelium to facilitate allergen delivery across the epithelial barrier and their subsequent uptake by antigen-presenting cells. Epithelial injuries mediated by these proteases together with their sensing by protease-activated receptors (PARs) elicit potent inflammatory responses resulting in the release of pro-Th2 cytokines (IL-6, IL-25, IL-1β, TSLP) and danger-associated molecular patterns (DAMPs; IL-33, ATP, uric acid). Recently, protease allergens were shown to cleave the protease sensor domain of IL-33 to produce a super-active form of the alarmin. At the same time, proteolytic cleavage of fibrinogen can trigger TLR4 signaling, and cleavage of various cell surface receptors further shape the Th2 polarization. Remarkably, the sensing of protease allergens by nociceptive neurons can represent a primary step in the development of the allergic response. The goal of this review is to highlight the multiple innate immune mechanisms triggered by protease allergens that converge to initiate the allergic response.

TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

BackgroundTREM2 is a transmembrane receptor expressed by myeloid cells and acts to regulate their immune response. TREM2 governs the response of microglia to amyloid and tau pathologies in the Alzheimer’s disease (AD) brain. TREM2 is also present in a soluble form (sTREM2), and its CSF levels fluctuate as a function of AD progression. Analysis of stroke and AD mouse models revealed that sTREM2 proteins bind to neurons, which suggests sTREM2 may act in a non-cell autonomous manner to influence neuronal function. sTREM2 arises from the proteolytic cleavage of the membrane-associated receptor. However, alternatively spliced TREM2 species lacking a transmembrane domain have been postulated to contribute to the pool of sTREM2. Thus, both the source of sTREM2 species and its actions in the brain remain unclear.MethodsThe expression of TREM2 isoforms in the AD brain was assessed through the analysis of the Accelerating Medicines Partnership for Alzheimer’s Disease Consortium transcriptomics data, as well as qPCR analysis using post-mortem samples of AD patients and of the AD mouse model 5xFAD. TREM2 cleavage and secretion were studied in vitro using HEK-293T and HMC3 cell lines. Synaptic plasticity, as evaluated by induction of LTP in hippocampal brain slices, was employed as a measure of sTREM2 actions.ResultsThree distinct TREM2 transcripts, namely ENST00000373113 (TREM2230), which encodes the full-length transmembrane receptor, and the alternatively spliced isoforms ENST00000373122 (TREM2222) and ENST00000338469 (TREM2219), are moderately increased in specific brain regions of patients with AD. We provide experimental evidence that TREM2 alternatively spliced isoforms are translated and secreted as sTREM2. Furthermore, our functional analysis reveals that all sTREM2 species inhibit LTP induction, and this effect is abolished by the GABAA receptor antagonist picrotoxin.ConclusionsTREM2 transcripts can give rise to a heterogeneous pool of sTREM2 which acts to inhibit LTP. These results provide novel insight into the generation, regulation, and function of sTREM2 which fits into the complex biology of TREM2 and its role in human health and disease. Given that sTREM2 levels are linked to AD pathogenesis and progression, our finding that sTREM2 species interfere with LTP furthers our understanding about the role of TREM2 in AD.

Granzyme B in Autoimmune Skin Disease.

Autoimmune diseases often present with cutaneous symptoms that contribute to dysfunction, disfigurement, and in many cases, reduced quality-of-life. Unfortunately, treatment options for many autoimmune skin diseases are limited. Local and systemic corticosteroids remain the current standard-of-care but are associated with significant adverse effects. Hence, there is an unmet need for novel therapies that block molecular drivers of disease in a local and/or targeted manner. Granzyme B (GzmB) is a serine protease with known cytotoxic activity and emerging extracellular functions, including the cleavage of cell-cell junctions, basement membranes, cell receptors, and other structural proteins. While minimal to absent in healthy skin, GzmB is markedly elevated in alopecia areata, interface dermatitis, pemphigoid disease, psoriasis, systemic sclerosis, and vitiligo. This review will discuss the role of GzmB in immunity, blistering, apoptosis, and barrier dysfunction in the context of autoimmune skin disease. GzmB plays a causal role in the development of pemphigoid disease and carries diagnostic and prognostic significance in cutaneous lupus erythematosus, vitiligo, and alopecia areata. Taken together, these data support GzmB as a promising therapeutic target for autoimmune skin diseases impacted by impaired barrier function, inflammation, and/or blistering.

Immunoglobulin M-degrading enzyme of Streptococcus suis (Ide Ssuis ) impairs porcine B cell signaling.

Streptococcus suis (S. suis) is an important porcine pathogen, causing severe disease like meningitis and septicemia primarily in piglets. Previous work showed that the IgM-degrading enzyme of S. suis (Ide Ssuis ) specifically cleaves soluble porcine IgM and is involved in complement evasion. The objective of this study was to investigate Ide Ssuis cleavage of the IgM B cell receptor and subsequent changes in B cell receptor mediated signaling. Flow cytometry analysis revealed cleavage of the IgM B cell receptor by recombinant (r) Ide Ssuis _homologue as well as Ide Ssuis derived from culture supernatants of S. suis serotype 2 on porcine PBMCs and mandibular lymph node cells. Point-mutated rIde Ssuis _homologue_C195S did not cleave the IgM B cell receptor. After receptor cleavage by rIde Ssuis _homologue, it took at least 20 h for mandibular lymph node cells to restore the IgM B cell receptor to levels comparable to cells previously treated with rIde Ssuis _homologue_C195S. B cell receptor mediated signaling after specific stimulation via the F(ab')2 portion was significantly inhibited by rIde Ssuis _homologue receptor cleavage in IgM+ B cells, but not in IgG+ B cells. Within IgM+ cells, CD21+ B2 cells and CD21- B1-like cells were equally impaired in their signaling capacity upon rIde Ssuis _homologue B cell receptor cleavage. In comparison, intracellular B cell receptor independent stimulation with tyrosine phosphatase inhibitor pervanadate increased signaling in all investigated B cell types. In conclusion, this study demonstrates Ide Ssuis cleavage efficacy on the IgM B cell receptor and its consequences for B cell signaling.

Matrix stiffness regulates Notch signaling activity in endothelial cells.

Notch signaling is critical for many developmental and disease-related processes. It is widely accepted that Notch has a mechanotransduction module that regulates receptor cleavage. However, the role of biomechanical properties of the cellular environment in Notch signaling in general is still poorly understood. During angiogenesis, differentiation of endothelial cells into tip and stalk cells is regulated by Notch signaling, and remodeling of the extracellular matrix occurs. We investigated the influence of substrate stiffness on the Notch signaling pathway in endothelial cells. Using stiffness-tuned polydimethylsiloxane (PDMS) substrates, we show that activity of the Notch signaling pathway inversely correlates with a physiologically relevant range of substrate stiffness (i.e. increased Notch signaling activity on softer substrates). Trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness, and integrin cell-matrix connections are both stiffness dependent and influenced by Notch signaling. We conclude that mechanotransduction of Notch activation is modulated by substrate stiffness, highlighting the role of substrate rigidity as an important cue for signaling. This might have implications in pathological situations associated with stiffening of the extracellular matrix, such as tumor growth.

Downregulation of Mucin1 in Cancer Cells is Associated with Modulation of Calcium Signalling Pathways and Alteration in Procoagulant Related Activity

Background Intracellular calcium ions play an essential role in regulating numerous physiological functions by acting as a second messenger in signal transduction pathways. Alterations in calcium signaling are also implicated in several pathological conditions including cancer. Aim The purpose of this study is to investigate whether MUC1, a well-established tumor marker, plays a role in modulating calcium signalling in cancer cells. Methods MUC1 knockdown breast cancer MCF-7 cells were used to investigate whether the overexpression of MUC1 by cancer cells has an influence on calcium signalling pathways, by determining the level of a range of calcium signalling proteins and the dynamics of intracellular calcium mobilization as well as the cellular effects induced by calcium treatment. The investigation was also extended to normal human mammary epithelial, human breast and pancreatic cancer cell lines, which express different levels of MUC1. Result MUC1 downregulation altered the aberrant expression of several calcium transporter proteins in MCF-7 cancer cells. The level of each of these calcium signaling pathways proteins was correlated to MUC1 level in normal human mammary epithelial, human breast and pancreatic cancer cell lines. Consequently, a number of proteins whose activities are calcium dependent such as p-CaMKII, and pro-coagulant proteins Va, Xa and thrombin were found to be reduced in MUC1 downregulated MCF-7 cells. In addition, the effect of thrombin on calcium mobilization and the cleavage of the thrombin receptors was also reduced in MUC1 downregulated MCF-7 cells. Conclusion The downregulation of MUC1 is capable of modulating calcium cellular effects via alteration in the expression of calcium signalling proteins and by impacting calcium induced initiation of procoagulant activity, which is capable of influencing the effect of thrombin on cancer cells. Such properties contribute to another crucial role for MUC1 within the signalling network associated with tumorigenesis.

Abstract 14933: Beta1-Adrenergic Receptor Polymorphisms Modulate Receptor Processing and Function

The beta1-adrenergic receptor (beta1AR) is a G protein-coupled receptor that modulates the sympathetic activation of the heart. The extracellular N-terminal domain of the beta1AR, that undergoes extensive post-translational modifications, is generally overlooked as a domain influencing receptor signaling and regulation. Previously, we showed that the N-terminal domain is cleaved in an activation dependent manner, and more recently, we demonstrated that O-glycosylation inhibits the N-terminal cleavage and modulates signaling of the receptor. This suggests that processing of the N-terminal domain has a complex role in the regulation of beta1AR function. Here, we investigated the three human beta1AR (hbeta1AR) N-terminal single-nucleotide polymorphisms (SNPs) that reside close to the previously identified cleavage and O-glycosylation sites. These SNPs, S49G, R31Q and A29T, have allele frequencies of ~15%, <1% and <1%, respectively. The aim was to investigate if these SNPs can modulate the O-glycosylation -mediated receptor regulation. We studied the impact of the SNPs on hbeta1AR O-glycosylation, proteolytic processing and cell surface expression using cultured cell lines and isolated neonatal rat cardiomyocytes. We find that the proteolytic cleavage of the receptor is attenuated for the T29 and Q31 variants and enhanced for the G49 variant. The attenuated cleavage of T29 and Q31 variants leads to an increase in the number of full-length receptors at the cell surface. For the Q31 variant, the attenuated cleavage is caused by the disruption of the receptor cleavage site at position R31↓L32, whereas cleavage of the T29 variant is inhibited due to an additional O-glycan found at position 29. In comparison, the existing O-glycan is eliminated at position 49 in the G49 variant. We conclude that the hbeta1AR SNPs affect receptor processing via disrupting the proteolytic cleavage site at R31↓L32 (Q31 variant) or through altered O-glycosylation (T29 and G49 variants). Since the altered glycosylation/cleavage of the beta1AR variants affects the number of functional receptors at the cell surface, it can offer new strategies for drug design and help in understanding the mechanism for the decreased cardiomyocyte beta1AR density in heart failure.

Design, synthesis, and biological evaluation of new heterocycles bearing both silicon and phosphorus as potent MMP‐2 inhibitors

AbstractMatrix metalloproteinases (MMPs) attracted medicinal chemists as they are biological targets that are involved in various pathophysiological processes such as extracellular matrix protein degradation, cell‐surface receptor release and cleavage, tumor growth, homeostatic control, and innate immunity. New heterocycles, bearing both silicon and phosphorus in their molecular structures, were successfully produced via reacting silylated isocyanates and/or isothiocyanates with appropriate phosphorus ylides. Reacting nucleophilic phosphacumulenes (2,6) with isocyanate 1 and isothiocyanate 9 yielded four‐membered ring heterocycles containing phosphorus and silicon (4 and 11) and six‐membered rings heterocycles (5, 8 and 12). Moreover, novel heterocycles containing two phosphorus atoms and one silicon atom were produced by the reaction of diphosphorane 13 with 1 or 9. On the other side, four‐membered (23, 26 and 29a) and six‐membered heterocyclic derivatives (20b and 29b) containing two phosphorus atoms in their structures were also obtained from the reaction of phosphallene ylide 13 and substrates (18, 21, 24 and 27). All the final compounds were tested for their MMP‐2 inhibitor activity showing IC50 values in the range of 0.0408–0.4985 μM. The thiolactam derivative 11 and the azaphosphetidin‐4‐one derivative 15, both bearing silicon and phosphorous, showed the highest activity with IC50 values of 0.0408 and 0.0693 μM, respectively, both are significantly higher than that of quercetin (IC50 = 0.1631 μM). Docking simulations were carried out for the most two active compounds 11 and 15 in the vicinity of the active site of MMP‐2 enzyme and both formed coordinate bonds with the zinc cation key interaction for MMP‐2 enzyme. In conclusion, compounds 11 and 15 can be considered as potential MMP‐2 inhibitors and can be subjected to further studies as antimetastatic agents.

MMP7 cleavage of amino-terminal CD95 death receptor switches signaling toward non-apoptotic pathways

CD95 is a death receptor that can promote oncogenesis through molecular mechanisms that are not fully elucidated. Although the mature CD95 membrane receptor is considered to start with the arginine at position 17 after elimination of the signal peptide, this receptor can also be cleaved by MMP7 upstream of its leucine at position 37. This post-translational modification occurs in cancer cells but also in normal cells such as peripheral blood leukocytes. The non-cleaved CD95 amino-terminal region consists in a disordered domain and its in silico reconstitution suggests that it might contribute to receptor aggregation and thereby, regulate the downstream death signaling pathways. In agreement with this molecular modeling analysis, the comparison of CD95-deficient cells reconstituted with full-length or N-terminally truncated CD95 reveals that the loss of the amino-terminal region of CD95 impairs the initial steps of the apoptotic signal while favoring the induction of pro-survival signals, including the PI3K and MAPK pathways.

Hesperidin potentially interacts with the catalytic site of gamma-secretase and modifies notch sensitive genes and cancer stemness marker expression in colon cancer cells and colonosphere

Gamma secretase (GS) produces Notch Intracellular Domain (NICD) by trans-membrane cleavage of notch receptor. The NICD enters the nucleus and activates the notch signaling pathway (NSP) by activating notch-responsive gene transcription. Hyperactivation of NSP is related to cancer aggressiveness, therapy resistance, and poor therapy outcome, and decreased overall disease-free survival in patients. Till date, none of the GS inhibitors (GSI) has been clinically approved due to their toxicity in patients. Thus in the present study, we explored the GS catalytic site binding potential of hesperidin (natural flavone glycoside) and its effect on notch responsive gene expression in HCT-116 cells. Molecular docking, MM-GBSA binding energy calculations, and molecular dynamics (MD) simulation experiments were performed to study the GS catalytic site binding potential of hesperidin. The compound showed better GS catalytic site binding potential at the active site compared to experimentally validated GSI, N-N-(3, 5-Difluorophenacetyl)-L-alanyl-S-phenylglycine t-butyl ester (DAPT) in molecular docking and MM-GBSA experiments. MD simulation results showed that hesperidin forms stable and energetically favorable complex with gamma secretase in comparison to standard inhibitor (DAPT)-GS complex. Further, in vitro experiments showed that hesperidin inhibited cell growth and sphere formation potential in HCT-116 cells. Further, hesperidin treatment altered notch responsive genes (Hes1, Hey1, and E-cad) and cancer stemness/self-renewal markers expression at transcription levels. In conclusion, hesperidin produces toxicity in HCT-116 cells and decreases colonosphere formation by inhibiting transcription of notch signaling pathway target genes and stemness markers. Communicated by Ramaswamy H. Sarma

Notch signaling functions in noncanonical juxtacrine manner in platelets to amplify thrombogenicity.

Notch signaling dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets remains elusive. Transcripts/peptides of Notch1 and Delta-like ligand (DLL)-4 were detected in platelets isolated from human blood by RT-qPCR, Western analysis and flow cytometry. Platelet aggregation, granule secretion and platelet-leukocyte interaction were analyzed by lumi-aggregometry and flow cytometry. Platelet-derived extracellular vesicles were documented with Nanoparticle Tracking Analyzer. Platelet thrombus on immobilized collagen was quantified using microfluidics platform. Intracellular calcium was monitored by fluorescence spectrophotometry. Whole blood coagulation was studied by thromboelastography. Ferric chloride-induced mouse mesenteric arteriolar thrombosis was imaged by intravital microscopy. We demonstrate expression of Notch1, its ligand DLL-4 and their respective transcripts in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated by thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to 'activated' phenotype through cleavage of Notch receptor and release of its intracellular domain (NICD), which was averted by inhibition of γ-secretase and phosphatidylinositol-3-kinase (PI3K). Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin. Our study presents compelling evidence in support of non-canonical juxtacrine Notch signaling within platelet aggregates that synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, Notch pathway can be a potential anti-platelet/anti-thrombotic therapeutic target. Research was supported by grants received by DD from JC Bose Fellowship (JCB/2017/000029), ICMR (71/4/2018-BMS/CAR), DBT (BT/PR-20645/BRB/10/1541/2016) and SERB (EMR/2015/000583). SNC, ME and VS are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively. Funders had no role in design, analysis and reporting of study.

The proprotein convertase furin regulates the development of thymic epithelial cells to ensure central immune tolerance

SummaryThe generation of mature T cells and establishment of central tolerance is predominantly orchestrated by thymic epithelial cells (TECs). Proprotein convertases are responsible for the proteolysis of proproteins into their mature bioactive counterparts. Here, we found that Furin, a member of the subtilisin/kexin-like PCs family, is highly expressed in TECs compared with other members of this family. TEC-specific deletion of Furin caused severe thymic atrophy and predominantly reduced the number of medullary TECs and thymic tuft cells, and to a less degree, cortical TECs. Furin deletion attenuated the proliferation of TECs, impaired thymopoiesis, and led to autoimmune disorders in mice. Furin promotes the development of TECs via cleavage of proIGF1 receptor and pro-Insulin receptor and the activation of downstream ERK/MAPK and Akt signaling pathways. Thus, this study uncovered the role of furin in TEC development and function and highlighted the importance of post-translational modification of immature proproteins in TEC biology.

γ-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor.

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. Here, we show that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development.

Regulation of age-associated insulin resistance by MT1-MMP-mediated cleavage of insulin receptor

Insulin sensitivity progressively declines with age. Currently, the mechanism underlying age-associated insulin resistance remains unknown. Here, we identify membrane-bound matrix metalloproteinase 14 (MT1-MMP/MMP14) as a central regulator of insulin sensitivity during ageing. Ageing promotes MMP14 activation in insulin-sensitive tissues, which cleaves Insulin Receptor to suppress insulin signaling. MT1-MMP inhibition restores Insulin Receptor expression, improving insulin sensitivity in aged mice. The cleavage of Insulin Receptor by MT1-MMP also contributes to obesity-induced insulin resistance and inhibition of MT1-MMP activities normalizes metabolic dysfunctions in diabetic mouse models. Conversely, overexpression of MT1-MMP in the liver reduces the level of Insulin Receptor, impairing hepatic insulin sensitivity in young mice. The soluble Insulin Receptor and circulating MT1-MMP are positively correlated in plasma from aged human subjects and non-human primates. Our findings provide mechanistic insights into regulation of insulin sensitivity during physiological ageing and highlight MT1-MMP as a promising target for therapeutic avenue against diabetes.

BACE-1 inhibition facilitates the transition from homeostatic microglia to DAM-1.

BACE-1 is required for generating β-amyloid (Aβ) peptides in Alzheimer’s disease (AD). Here, we report that microglial BACE-1 regulates the transition of homeostatic to stage 1 disease-associated microglia (DAM-1) signature. BACE-1 deficiency elevated levels of transcription factors including Jun, Jund, Btg2, Erg1, Junb, Fos, and Fosb in the transition signature, which transition from more homeostatic to highly phagocytic DAM-1. Consistently, similar transition-state microglia in human AD brains correlated with lowered levels of BACE-1 expression. Targeted deletion of Bace-1 in adult 5xFAD mice microglia elevated these phagocytic microglia, correlated with significant reduction in amyloid plaques without synaptic toxicity. Silencing or pharmacologically inhibiting BACE-1 in cultured microglia-derived cells shows higher phagocytic function in microglia. Mechanistic exploration suggests that abolished cleavage of IL-1R2 and Toll-like receptors via BACE-1 inhibition contributes to the enhanced signaling via the PI3K and p38 MAPK kinase pathway. Together, targeted inhibition of BACE-1 in microglia may offer AD treatment.

Identification of a Distinct Platelet Phenotype in the Elderly: ADP Hypersensitivity Coexists With Platelet PAR (Protease-Activated Receptor)-1 and PAR-4-Mediated Thrombin Resistance.

Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1β/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson’s disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

Cleavage of the selective autophagy receptor SQSTM1/p62 by the SARS-CoV-2 main protease NSP5 prevents the autophagic degradation of viral membrane proteins

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) global pandemic. Omicron, a new variant of SARS-CoV-2, has the characteristics of strong transmission and pathogenicity, short incubation period, and rapid onset progression, and has spread rapidly around the world. The high replication rate and intracellular accumulation of SARS-CoV-2 are remarkable, but the underlying molecular mechanisms remain unclear. Autophagy acts as a conservative cellular defence mechanism against invading pathogens. Here, we provide evidence that the main protease of SARS-CoV-2, NSP5, effectively cleaves the selective autophagy receptor p62. NSP5 targets p62 for cleavage at glutamic acid 354 and thus abolishes the capacity of p62 to mediate selective autophagy. It was further shown that p62 specifically interacted with ubiquitinated SARS-CoV-2 M, the viral membrane protein, to promote its autophagic degradation. In the presence of NSP5, p62-mediated autophagic degradation of the M protein was inhibited. The cleaved products of p62 also cannot facilitate the degradation of the M protein. Collectively, our findings reveal that p62 is a novel host target of SARS-CoV-2 NSP5 and suggest that selective autophagy targets viruses and potential strategies by which the virus evades autophagic clearance. Our results may provide new ideas for the development of anti-COVID-19 drugs based on autophagy and NSP5.