Mitochondria-associated membranes (MAMs) are essential communication subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. We previously demonstrated that, upon macroautophagy/autophagy induction, AMBRA1 is recruited to the BECN1 complex and relocalizes to MAMs, where it regulates autophagy by interacting with raft-like components. ERLIN1 is an endoplasmic reticulum lipid raft protein of the prohibitin family. However, little is known about its association with the MAM interface and its involvement in autophagic initiation. In this study, we investigated ERLIN1 association with MAM raft-like microdomains and its interaction with AMBRA1 in the regulation of the autophagic process. We show that ERLIN1 interacts with AMBRA1 at MAM raft-like microdomains, which represents an essential condition for autophagosome formation upon nutrient starvation, as demonstrated by knocking down ERLIN1 gene expression. Moreover, this interaction depends on the "integrity" of key molecules, such as ganglioside GD3 and MFN2. Indeed, knocking down ST8SIA1/GD3-synthase or MFN2 expression impairs AMBRA1-ERLIN1 interaction at the MAM level and hinders autophagy. In conclusion, AMBRA1-ERLIN1 interaction within MAM raft-like microdomains appears to be pivotal in promoting the formation of autophagosomes. Abbreviations: ACSL4/ACS4: acyl-CoA synthetase long chain family member 4; ACTB/β-actin: actin beta;

Lipid rafts are functional membrane microdomains containing sphingolipids, including gangliosides, and cholesterol. These regions are characterized by highly ordered and tightly packed lipid molecules. Several studies revealed that lipid rafts are involved in life cycle of different viruses, including coronaviruses. Among these recently emerged the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The main receptor for SARS-CoV-2 is represented by the angiotensin-converting enzyme-2 (ACE-2), although it also binds to sialic acids linked to host cell surface gangliosides. A new type of ganglioside-binding domain within the N-terminal portion of the SARS-CoV-2 spike protein was identified. Lipid rafts provide a suitable platform able to concentrate ACE-2 receptor on host cell membranes where they may interact with the spike protein on viral envelope. This review is focused on selective targeting lipid rafts components as a strategy against coronavirus. Indeed, cholesterol-binding agents, including statins or methyl-β-cyclodextrin (MβCD), can affect cholesterol, causing disruption of lipid rafts, consequently impairing coronavirus adhesion and binding. Moreover, these compounds can block downstream key molecules in virus infectivity, reducing the levels of proinflammatory molecules [tumor necrosis factor alpha (TNF-α), interleukin (IL)-6], and/or affecting the autophagic process involved in both viral replication and clearance. Furthermore, cyclodextrins can assemble into complexes with various drugs to form host–guest inclusions and may be used as pharmaceutical excipients of antiviral compounds, such as lopinavir and remdesivir, by improving bioavailability and solubility. In conclusion, the role of lipid rafts-affecting drugs in the process of coronavirus entry into the host cells prompts to introduce a new potential task in the pharmacological approach against coronavirus.

Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity. It is well known that in these patients thrombosis may be the result of a hypercoagulable state related to anti-β2glycoprotein I (β2-GPI) antibodies. Moreover, platelets may play a role in thrombotic manifestations by binding of anti-β2-GPI antibodies. Platelets express tissue factor (TF), the major initiator of the clotting cascade, after activation. We primarily analyzed whether anti-β2-GPI antibodies may trigger a signal transduction pathway leading to TF expression in human platelets. Platelets from healthy donors were incubated with affinity purified anti-β2-GPI antibodies for different times. Platelet lysates were analyzed for phospho-interleukin-1 receptor-associated kinase 1 (IRAK), phospho-p65 nuclear factor kappaB (NF-κB) and TF by Western blot. IRAK phosphorylation was observed as early as 10 min of anti-β2-GPI treatment, with consequent NF-κB activation, whereas TF expression, detectable at 45 min, was significantly increased after 4 h of anti-β2-GPI treatment. Virtually no activation was observed following treatment with control immunoglobulin IgG. We then analyzed TF expression in platelets from 20 APS patients and 20 healthy donors. We observed a significant increase of TF in APS patients versus control subjects (P < 0·0001). This work demonstrates that anti-β2-GPI antibodies may trigger in vitro a signal transduction pathway in human platelets, which involves IRAK phosphorylation and NF-κB activation, followed by TF expression. Furthermore, ex vivo, platelets of APS patients showed a significantly increased expression of TF. These findings support the view that platelets may play a role in the pathogenesis of APS, with consequent release of different procoagulant mediators, including TF.

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, pregnancy morbidity in the presence of circulating “anti-phospholipid antibodies” (aPL). One of the main target antigens of aPL is β2-glycoprotein I (β2-GPI). APS may occur as a primary syndrome or associated with Systemic Lupus Erythematosus (SLE). High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different type of cells during activation and/or cell death and may act as a proinflammatory mediator through ligation to its receptors, including RAGE. There is accumulating evidence that HMGB1 contributes to the pathogenesis of inflammatory and autoimmune diseases, especially SLE. In a previous study we demonstrated increased serum levels of HMGB1 in both primary and secondary APS patients. In this work we analyzed: (i) in vitro whether anti-β2-GPI antibodies from APS patients may induce both a HMGB1 cellular relocation by activation of its putative receptor RAGE in platelets and monocytes and, (ii) ex vivo, serum levels of HMGB1/soluble RAGE (sRAGE) in APS patients and their possible correlation with clinical manifestations. Platelets and monocytes from healthy donors were incubated with affinity purified anti-β2-GPI antibodies. HMGB1 and RAGE expression were analyzed by Western Blot. Sera from 60 consecutive APS patients (primary or secondary), diagnosed according to the Sydney Classification Criteria, were enrolled. As a control, 30 matched healthy subjects were studied. Serum levels of HMGB1 and sRAGE were analyzed by Western Blot. In vitro results showed that anti-β2-GPI antibodies were able to induce RAGE activation and HMGB1 cellular relocation in both monocytes and platelets. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with healthy subjects (p<0.0001). Interestingly, APS patients with spontaneous recurrent abortion showed significantly higher levels of sRAGE; moreover, in APS patients a direct correlation between serum levels of HMGB1 and disease duration was detected. Our observations suggest that anti-β2-GPI antibodies may trigger RAGE activation and HMGB1 cellular relocation during APS. Monitoring these molecules serum levels may represent an useful tool to evaluate the pathogenesis and risk stratification of clinical manifestations in APS.

Antiphospholipid Syndrome (APS) is an autoimmune disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity, associated with circulating antiphospholipid antibodies (aPL). In some cases, patients with a clinical profile indicative of APS (thrombosis, recurrent miscarriages or fetal loss), who are persistently negative for conventional laboratory diagnostic criteria, are classified as “seronegative” APS patients (SN-APS). Several findings suggest that aPL, which target phospholipids and/or phospholipid binding proteins, mainly β-glycoprotein I (β-GPI), may contribute to thrombotic diathesis by interfering with hemostasis. Despite the strong association between aPL and thrombosis, the exact pathogenic mechanisms underlying thrombotic events and pregnancy morbidity in APS have not yet been fully elucidated and multiple mechanisms may be involved. Furthermore, in many SN-APS patients, it is possible to demonstrate the presence of unconventional aPL (“non-criteria” aPL) or to detect aPL with alternative laboratory methods. These findings allowed the scientists to study the pathogenic mechanism of SN-APS. This review is focused on the evidence showing that these antibodies may play a functional role in the signal transduction pathway(s) leading to thrombosis and pregnancy morbidity in SN-APS. A better comprehension of the molecular mechanisms triggered by aPL may drive development of potential therapeutic strategies in APS patients.