ADAM10 and ADAM17 promote SARS‐CoV‐2 cell entry and spike protein‐mediated lung cell fusion

The severe‐acute‐respiratory‐syndrome‐coronavirus‐2 (SARS‐CoV‐2) is the causative agent of COVID‐19, but host cell factors contributing to COVID‐19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS‐CoV‐2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID‐19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS‐CoV‐2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease‐targeted inhibitors severely impair lung cell infection by the SARS‐CoV‐2 variants of concern alpha, beta, delta, and omicron and also reduce SARS‐CoV‐2 infection of primary human lung cells in a TMPRSS2 protease‐independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.     

circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

Renal ischaemia‐reperfusion (RI/R) injury is one major pathological state of acute kidney injury (AKI) with a mortality rate ranking 50% to 80%. MiR‐144‐5p acts as a molecular trigger in various diseases. We presumed that miR‐144‐5p might be involved RI/R injury progression. We found that RI/R injury decreased miR‐144‐5p expression in rat models. MiR‐144‐5p downregulation promoted cell apoptosis rate and activated Wnt/β‐catenin signal in RI/R injury rats. By performing bioinformatic analysis, RIP, RNA pull‐down, luciferase reporter experiments, we found that circ‐AKT3 sponged to miR‐144‐5p and decreased its expression in RI/R injury rats. Moreover, we found that circ‐AKT3 promoted cell apoptosis rate and activated Wnt/β‐catenin signal, and miR‐144‐5p mimic reversed the promotive effect of circ‐AKT3 in rat models. We also found that circ‐AKT3 increased the oxidative stress level in rat models. In conclusion, our study suggests that the circAKT3 is involved RI/R injury progression through regulating miR‐144‐5p/Wnt/β‐catenin pathway and oxidative stress.     

PGC‐1α protects from myocardial ischaemia‐reperfusion injury by regulating mitonuclear communication

The recovery of blood supply after a period of myocardial ischaemia does not restore the heart function and instead results in a serious dysfunction called myocardial ischaemia‐reperfusion injury (IRI), which involves several complex pathophysiological processes. Mitochondria have a wide range of functions in maintaining the cellular energy supply, cell signalling and programmed cell death. When mitochondrial function is insufficient or disordered, it may have adverse effects on myocardial ischaemia‐reperfusion and therefore mitochondrial dysfunction caused by oxidative stress a core molecular mechanism of IRI. Peroxisome proliferator‐activated receptor gamma co‐activator 1α (PGC‐1α) is an important antioxidant molecule found in mitochondria. However, its role in IRI has not yet been systematically summarized. In this review, we speculate the role of PGC‐1α as a key regulator of mitonuclear communication, which may interacts with nuclear factor, erythroid 2 like ‐1 and ‐2 (NRF‐1/2) to inhibit mitochondrial oxidative stress, promote the clearance of damaged mitochondria, enhance mitochondrial biogenesis, and reduce the burden of IRI.     

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki‐67

Ki‐67 is a chromatin‐associated protein with a dynamic distribution pattern throughout the cell cycle and is thought to be involved in chromatin organization. The lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA‐DamID mapping in human cell lines, we find that Ki‐67 associates with large genomic domains that overlap mostly with late‐replicating regions. Early in interphase, when Ki‐67 is present in pre‐nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki‐67 is confined to nucleoli, it shows a striking shift toward small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki‐67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki‐67 does not detectably control chromatin‐chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late‐replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki‐67 in heterochromatin organization.     

Adenosine triphosphate energy‐independently controls protein homeostasis with unique structure and diverse mechanisms

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases.     

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic β‐cells for protection

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic β‐cells. ER stress or ROS causes c‐Jun N‐terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi‐stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual‐luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in β‐cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p‐JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome‐dependent manner. We found that NR4A1 increased the expression of cbl‐b (an E3 ligase); knocking down cbl‐b expression increased MKK4 and p‐JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl‐b promoter by physical association. We further confirmed that cbl‐b expression in β‐cells was reduced in NR4A1‐knockout mice compared with WT mice. NR4A1 down‐regulates JNK activation by ER stress or ROS in β‐cells via enhancing cbl‐b expression.     

Mitochondrial uncoupling protein‐2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age‐associated lung fibrosis

Mitochondrial dysfunction has been associated with age‐related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein‐2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro‐oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro‐fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age‐related diseases associated with impaired tissue regeneration and organ fibrosis.     

Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans‐4‐hydroxy‐L‐proline

Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD⁺‐dependent oxidations of L‐glutamate‐γ‐semialdehyde to L‐glutamate (proline metabolism), and 4‐hydroxy‐L‐glutamate‐γ‐semialdehyde to 4‐erythro‐hydroxy‐L‐glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4‐hydroxyproline using steady‐state kinetics and X‐ray crystallography. Trans‐4‐hydroxy‐L‐proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L‐proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L‐stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans‐4‐hydroxy‐L‐proline and trans‐4‐hydroxy‐D‐proline. The structures suggest that the 10‐fold greater preference for the L‐stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans‐4‐hydroxy‐L‐proline. In contrast, the amine group of the D‐stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans‐4‐hydroxy‐L‐proline, analogous to the known inhibition of proline catabolism by L‐proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans‐4‐hydroxy‐L‐proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.     

A multifaceted cellular damage repair and prevention pathway promotes high‐level tolerance to β‐lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two‐component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high‐level β‐lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β‐lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β‐lactams. We propose that VxrAB reduces antibiotic‐induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β‐lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism''s ability to counteract diverse antibiotic‐induced stresses promotes high‐level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.     

CircSAMD4A aggravates H/R‐induced cardiomyocyte apoptosis and inflammatory response by sponging miR‐138‐5p

Hypoxia/reoxygenation (H/R)‐induced myocardial cell injury is the main cause of acute myocardial infarction (AMI). Many proofs show that circular RNA plays an important role in the development of AMI. The purpose of this study was to investigate the role of circSAMD4A in H/R‐induced myocardial injury. The levels of circular SAMD4A (circSAMD4A) were detected in the heart tissues of AMI mice and H/R‐induced H9C2 cells, and the circSAMD4A was suppressed in AMI mice and H/R‐induced H9C2 cells to investigate its’ function in AMI. The levels of circSAMD4A and miR‐138‐5p were detected by real‐time quantitative PCR, and MTT assay was used to detect cell viability. TUNEL analysis and Annexin V‐FITC were used to determine apoptosis. The expression of Bcl‐2 and Bax proteins was detected by Western blot. IL‐1β, TNF‐α and IL‐6 were detected by ELISA kits. The study found that the levels of circSAMD4A were up‐regulated after H/R induction and inhibition of circSAMD4A expression would reduce the H/R‐induced apoptosis and inflammation. MiR‐138‐5p was down‐regulated in H/R‐induced H9C2 cells. circSAMD4A was a targeted regulator of miR‐138‐5p. CircSAMD4A inhibited the expression of miR‐138‐5p to promote H/R‐induced myocardial cell injury in vitro and vivo. In conclusion, CircSAMD4A can sponge miR‐138‐5p to promote H/R‐induced apoptosis and inflammatory response.     

3,6'‐disinapoyl sucrose attenuates Aβ1‐42‐ induced neurotoxicity in Caenorhabditis elegans by enhancing antioxidation and regulating autophagy

The aggregation of β‐amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer''s disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6''‐Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti‐depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ_1‐42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg‐laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ_1‐42 transgenic C. elegans. Gene analysis showed that DISS could up‐regulate the mRNA expression of sod‐3, gst‐4, daf‐16, bec‐1 and lgg‐1, while down‐regulate the mRNA expression of daf‐2 and daf‐15 in Aβ_1‐42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ_1‐42‐induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.     

Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

Antigen‐specific B‐cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B‐cell endosomal trafficking pathways and their specific roles in B‐cell responses have not been systematically investigated. Here, we report high‐throughput identification of genes regulating B‐cell receptor (BCR)‐mediated antigen internalization using genome‐wide functional screens. We show that antigen internalization depends both on constitutive, clathrin‐mediated endocytosis and on antigen‐induced, clathrin‐independent endocytosis mediated by endophilin A2. Although endophilin A2‐mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B‐cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2‐deficient mice show defects in GC B‐cell responses and production of high‐affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin‐independent intracellular trafficking in GC B‐cell clonal expansion and antibody responses.     

A circular intronic RNA ciPVT1 delays endothelial cell senescence by regulating the miR‐24‐3p/CDK4/pRb axis

Circular RNAs (circRNAs) have been established to be involved in numerous processes in the human genome, but their function in vascular aging remains largely unknown. In this study, we aimed to characterize and analyze the function of a circular intronic RNA, ciPVT1, in endothelial cell senescence. We observed significant downregulation of ciPVT1 in senescent endothelial cells. In proliferating endothelial cells, ciPVT1 knockdown induced a premature senescence‐like phenotype, inhibited proliferation, and led to an impairment in angiogenesis. An in vivo angiogenic plug assay revealed that ciPVT1 silencing significantly inhibited endothelial tube formation and decreased hemoglobin content. Conversely, overexpression of ciPVT1 in old endothelial cells delayed senescence, promoted proliferation, and increased angiogenic activity. Mechanistic studies revealed that ciPVT1 can sponge miR‐24‐3p to upregulate the expression of CDK4, resulting in enhanced Rb phosphorylation. Moreover, enforced expression of ciPVT1 reversed the senescence induction effect of miR‐24‐3p in endothelial cells. In summary, the present study reveals a pivotal role for ciPVT1 in regulating endothelial cell senescence and may have important implications in the search of strategies to counteract the development of age‐associated vascular pathologies.     

Vitamin D and COVID‐19: A review on the role of vitamin D in preventing and reducing the severity of COVID‐19 infection

Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) is a pathogenic coronavirus causing COVID‐19 infection. The interaction between the SARS‐CoV‐2 spike protein and the human receptor angiotensin‐converting enzyme 2, both of which contain several cysteine residues, is impacted by the disulfide‐thiol balance in the host cell. The host cell redox status is affected by oxidative stress due to the imbalance between the reactive oxygen/nitrogen species and antioxidants. Recent studies have shown that Vitamin D supplementation could reduce oxidative stress. It has also been proposed that vitamin D at physiological concentration has preventive effects on many viral infections, including COVID‐19. However, the molecular‐level picture of the interplay of vitamin D deficiency, oxidative stress, and the severity of COVID‐19 has remained unclear. Herein, we present a thorough review focusing on the possible molecular mechanism by which vitamin D could alter host cell redox status and block viral entry, thereby preventing COVID‐19 infection or reducing the severity of the disease.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.