The probable role and therapeutic potential of the PI3K/AKT signaling pathway in SARS-CoV-2 induced coagulopathy

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is associated with a high mortality rate. The majority of deaths in this disease are caused by ARDS (acute respiratory distress syndrome) followed by cytokine storm and coagulation complications. Although alterations in the level of the number of coagulation factors have been detected in samples from COVID-19 patients, the direct molecular mechanism which has been involved in this pathologic process has not been explored yet. The PI3K/AKT signaling pathway is an intracellular pathway which plays a central role in cell survival. Also, in recent years the association between this pathway and coagulopathies has been well clarified. Therefore, based on the evidence on over-activity of the PI3K/AKT signaling pathway in SARS-CoV-2 infection, in the current review, the probable role of this cellular pathway as a therapeutic target for the prevention of coagulation complications in patients with COVID-19 is discussed.

Interleukin-10 in the Vasculature: Pathophysiological Implications

Interleukin-10 (IL-10) is an important immunomodulatory cytokine, initially characterized as an anti-inflammatory agent released by immune cells during infectious and inflammatory processes. IL-10 exhibits biological functions that extend to the regulation of different intracellular signaling pathways directly associated with vascular function. This cytokine plays a vital role in vascular tone regulation through the change of important proteins involved in vasoconstriction and vasodilation. Numerous investigations covered here have shown that therapeutic strategies inducing IL-10 result in anti-inflammatory, anti-hypertrophic, antihyperplastic, anti-apoptotic and antihypertensive effects. This non-systematic review summarizes the modulating effects mediated by IL-10 in vascular tissue, particularly on vascular tone, and the intracellular pathway induced by this cytokine. We also highlight the advances in IL-10 manipulation as a therapeutic target in different cardiovascular pathophysiologies, including the physiological implications in animals and humans. Finally, the review illustrates current and potential future perspectives of the potential use of IL-10 in clinical trials, based on the clinical evidence.

Fibroblast Growth Factor 23 (FGF23) leads to endolysosomal routing of the renal phosphate co-transporters NaPi-IIa and NaPi-IIc in vivo

The sodium-dependent phosphate co-transporters NaPi-IIa and NaPi-IIc located at the brush border membrane of renal proximal tubules are regulated by numerous factors, including fibroblast growth factor 23 (FGF23). FGF23 downregulates NaPi-IIa and NaPi-IIc abundance after activating a signaling pathway involving phosphorylation of the extracellular signal-regulated protein kinase (phospho-ERK1/2). FGF23 also downregulates the expression of renal 1-α-hydroxylase (Cyp27b1) and upregulates 24-hydroxylase (Cyp24a1), thus reducing plasma calcitriol levels. Here, we examined the time course of the FGF23-induced internalization of NaPi-IIa and NaPi-IIc and their intracellular pathway towards degradation in vivo. Mice were injected intraperitoneally with recombinant human FGF23 (rh-FGF23) in the absence (biochemical analysis) or presence (immunohistochemistry) of leupeptin, an inhibitor of lysosomal proteases. Phosphorylation of ERK1/2 was enhanced 60 minutes after rh-FGF23 administration, and increased phosphorylation was still detected 480 minutes post-injection. Co-localization of phospho-ERK1/2 with NaPi-IIa was seen at 60, 120 and partly at 480 minutes. The abundance of both co-transporters was reduced 240 minutes after rh-FGF23 administration, with a further reduction at 480 minutes. NaPi-IIa and NaPi-IIc were found to co-localize with clathrin and early endosomal antigen 1 (EEA1) as early as 120 minutes after rh-FGF23 injection. Both co-transporters partially co-localized with cathepsin B and Lamp1, markers of lysosomes, 120 minutes after rh-FGF23 injection. Thus, NaPi-IIa and NaPi-IIc are internalized within 2 hours upon rh-FGF23 injection. Both co-transporters share the pathway of clathrin-mediated endocytosis that leads first to early endosomes, finally resulting in trafficking towards the lysosome as early as 120 minutes after rh-FGF23 administration.

Ebulin l Is Internalized in Cells by Both Clathrin-Dependent and -Independent Mechanisms and Does Not Require Clathrin or Dynamin for Intoxication

Ebulin l is an A-B toxin, and despite the presence of a B chain, this toxin displays much less toxicity to cells than the potent A-B toxin ricin. Here, we studied the binding, mechanisms of endocytosis, and intracellular pathway followed by ebulin l and compared it with ricin. COS-1 cells and HeLa cells with inducible synthesis of a mutant dynamin (K44A) were used in this study. The transport of these toxins was measured using radioactively or fluorescently labeled toxins. The data show that ebulin l binds to cells to a lesser extent than ricin. Moreover, the expression of mutant dynamin does not affect the endocytosis, degradation, or toxicity of ebulin l. However, the inhibition of clathrin-coated pit formation by acidification of the cytosol reduced ebulin l endocytosis but not toxicity. Remarkably, unlike ricin, ebulin l is not transported through the Golgi apparatus to intoxicate the cells and ebulin l induces apoptosis as the predominant cell death mechanism. Therefore, after binding to cells, ebulin l is taken up by clathrin-dependent and -independent endocytosis into the endosomal/lysosomal system, but there is no apparent role for clathrin and dynamin in productive intracellular routing leading to intoxication.

An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC

Possible mechanisms of action of cannabidiol in the epilepsies: a review

Background: Cannabidiol (CBD) is a compound of Cannabis Sativa plant that has been studied since the 1970s for its effectiveness in the treatment of refractory epilepsies. With the discovery of the endocannabinoid system, most recent studies have been dedicated to elucidating its mechanisms of action. Objective: To review scientific articles in order to enlightening the antiepileptic cannabidiol’s mechanisms of action. Methods: Literature review on both PubMed and Google Scholar searching for the terms: “epilepsy”, “cannabidiol” and “mechanism of action”. Results: We found that cannabidiol has a lot of mechanisms of action which can explain its effectiveness, among which stand out: endocannabinoid system facilitation, by inhibition of recaption and hydrolysis of anandamide as well as by the facilitation of its synthesis and release. These processes must result in the indirect activation of CB1 and CB2 receptors. Furthermore, CBD promotes the activation of mTOR and PI3K proteins intracellular pathway, with subsequent reduction of glutamatergic release. Conclusions: The general hypothesis is that cannabidiol has antiepileptic effectiveness, even in cases of refractory epilepsies, precisely for showing several mechanisms of action. We emphasize, however, the necessity of more researches in this area for further enlightenment of theses possible mechanisms of action and the applicability in the treatment of epilepsies.

Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway

Primary cilia form by two pathways: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium forms inside the cell. Here we identify the GTPase Rab34 as a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and paralog Rab36. Together, these findings reveal a new context-specific molecular requirement for ciliary membrane biogenesis.