Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder of the brain, clinically characterised by cognitive deficits that gradually worsen over time. There is, at present, no established cure, or disease-modifying treatments for AD. As life expectancy increases globally, the number of individuals suffering from the disease is projected to increase substantially. Cumulative evidence indicates that AD neuropathological process is initiated several years, if not decades, before clinical signs are evident in patients, and diagnosis made. While several imaging, cognitive, CSF and blood-based biomarkers have been proposed for the early detection of AD; their sensitivity and specificity in the symptomatic stages is highly variable and it is difficult to justify their use in even earlier, pre-clinical stages of the disease. Research has identified potentially measurable functional, structural, metabolic and vascular changes in the retina during early stages of AD. Retina offers a distinctively accessible insight into brain pathology and current and developing ophthalmic technologies have provided us with the possibility of detecting and characterising subtle, disease-related changes. Recent human and animal model studies have further provided mechanistic insights into the biochemical pathways that are altered in the retina in disease, including amyloid and tau deposition. This information coupled with advances in molecular imaging has allowed attempts to monitor biochemical changes and protein aggregation pathology in the retina in AD. This review summarises the existing knowledge that informs our understanding of the impact of AD on the retina and highlights some of the gaps that need to be addressed. Future research will integrate molecular imaging innovation with functional and structural changes to enhance our knowledge of the AD pathophysiological mechanisms and establish the utility of monitoring retinal changes as a potential biomarker for AD.

Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence–dependent endocytotic process. Both clathrin- and caveolae/lipid raft–mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 ؊/؊ studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling in a tissue-specific manner. In this study, we made a novel finding that Grb14 inhibits the activity of PTP1B, the major negative regulator of insulin receptor (IR) signaling, in a phosphorylationregulated manner. Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for interaction with PTP1B, resulting in the competitive inhibition of PTP1B activity. We also found that rhodopsin-regulated Src kinase activation in retina leads to the phosphorylation of Grb14. Further, ablation of Grb14 resulted in significantly elevated retinal PTP1B activity in vivo. PTP1B is known to be regulated by oxidation, glutathionylation, phosphorylation, and SUMOlyation, and our study for the first time demonstrates the inhibition of PTP1B activity in vivo by protein molecule Grb14 in a tissue-specific manner.

5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A 4 to leukotriene C 4 (LTC 4 ), suggesting a role for the activity of LTC 4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP-and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.inflammation | eicosanoid | oxylipin L eukotrienes (LTs) are lipid mediators of inflammation with roles in both normal host defense and pathophysiological conditions (1). 5-Lipoxygenase (5LO) is a key enzyme in LT biosynthesis, and its regulation is a determinant of LT formation (2). In addition, 5LO is involved in biosynthesis of lipoxins and resolvin E1 (3). Cellular 5LO activity depends on several cofactors that regulate LT biosynthesis and prevent LT production in unstimulated situations. Increased intracellular Ca 2+ levels activate and translocate 5LO to the perinuclear region, where it accesses its substrate arachidonic acid (AA) released by cytosolic phospholipase A 2 (cPLA 2 ) (1). Cellular formation of LTs from endogenous AA depends on the small membrane protein 5LO-activating protein (FLAP) (4). FLAP has been suggested to function as a membrane anchor scaffold for 5LO to facilitate the transfer of AA to 5LO (5,6). In the cell, 5LO is subject to phosphorylation. MAP kinase pathways can activate 5LO during cell stress (7), whereas phosphorylation by protein kinase A inhibits cellular 5LO activity (8).5LO has two domains, an N-terminal C 2 -like regulatory domain with a β-sandwich structure and a catalytic C-terminal domain composed predominantly of α-helices that harbors the prosthetic iron (9). C...

Resolution of acute inflammation is an active process coordinated by proresolving lipid mediators (SPMs) such as lipoxins (LXs) and resolvins (Rvs), which are formed by the concerted action of 2 lipoxygenases (LOs). Because the exact molecular mechanisms of SPM biosynthesis are not completely understood, we aimed to investigate LX and D-type Rv formation in human leukocytes and HEK293T cells overexpressing leukotriene (LT) pathway enzymes. Activity assays in precursor (15-hydroxyeicosatetraenoic acids, 17-HDoHE)-treated granulocytes [polymorphonuclear leukocytes (PMNLs)] showed a strict dependence of LXA 4 /RvD 1 biosynthesis on cell integrity, and incubation with recombinant human 5-LO did not lead to LX or Rv formation. Pharmacologic inhibition of 5-LO activating protein (FLAP) by MK-886 inhibited LXA 4 /RvD 1 biosynthesis in precursor-treated PMNLs (drug concentration causing 50% inhibition ∼0.3/0.2 mM), as did knockdown of the enzyme in MM6 cells, and precursor-treated HEK293T overexpressing 5-LO produced high amounts of LXA 4 only in the presence of FLAP. In addition, inhibition of cytosolic phospholipase A 2a (cPLA 2a ) interfered with LXA 4 / RvD 1 formation from exogenous precursors in PMNLs. Furthermore, inhibition of the LT synthases LTA 4 hydrolase and LTC 4 synthase in PMNL/platelet coincubations augmented LXA 4 levels. These findings show that several enzymes known to be involved in the biosynthesis of proinflammatory LTs, such as FLAP and cPLA 2a , also contribute to LX and Rv formation.-

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.

Background Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. Methods For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CLpro) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. Results Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CLpro enzyme, which regulates viral replication machinery. Conclusions Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.