CRISPR screening uncovers nucleolar RPL22 as a heterochromatin destabilizer and senescence driver.

Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process.

Dynamic intracellular exchange of nanomaterials' protein corona perturbs proteostasis and remodels cell metabolism.

The nanomaterial­protein "corona" is a dynamic entity providing a synthetic­natural interface mediating cellular uptake and subcellular distribution of nanomaterials in biological systems. As nanomaterials are central to the safe-by-design of future nanomedicines and the practice of nanosafety, understanding and delineating the biological and toxicological signatures of the ubiquitous nanomaterial­protein corona are precursors to the continued development of nano­bio science and engineering. However, despite well over a decade of extensive research, the dynamics of intracellular release or exchange of the blood protein corona from nanomaterials following their cellular internalization remains unclear, and the biological footprints of the nanoparticle­protein corona traversing cellular compartments are even less well understood. To address this crucial bottleneck, the current work screened evolution of the intracellular protein corona along the endocytotic pathway from blood via lysosomes to cytoplasm in cancer cells. Intercellular proteins, including pyruvate kinase M2 (PKM2), and chaperones, displaced some of the initially adsorbed blood proteins from the nanoparticle surface, which perturbed proteostasis and subsequently incited chaperone-mediated autophagy (CMA) to disrupt the key cellular metabolism pathway, including glycolysis and lipid metabolism. Since proteostasis is key to the sustainability of cell function, its collapse and the resulting CMA overdrive spell subsequent cell death and aging. Our findings shed light on the consequences of the transport of extracellular proteins by nanoparticles on cell metabolism.

Mitochondrial elongation factor 4 modulates energy metabolism and promotes breast cancer metastasis by orchestration of mitochondrial translation.

To cope with the requirements of energy and building blocks for rapid proliferation, cancer cells reprogram their metabolic pathways profoundly, especially in oxygen- and nutrients-deficient tumor microenvironments. However, functional mitochondria and mitochondria-dependent oxidative phosphorylation are still necessary for the tumorigenesis and metastasis of cancer cells. We show here that mitochondrial elongation factor 4 (mtEF4) is commonly upregulated in breast tumors compared to adjacent non-cancerous tissues, and is relevant to tumor progression and poor prognosis. Down regulation of mtEF4 in breast cancer cells impairs the assembly of mitochondrial respiration complexes, decreases mitochondrial respiration, reduces ATP production, attenuates the formation of lamellipodia, and suppresses cell motility in vitro and cancer metastasis in vivo. On the contrary, upregulation of mtEF4 elevates the mitochondrial oxidative phosphorylation, which contributes to the migratory capacities of breast cancer cells. mtEF4 also increases the potential of glycolysis, probably via an AMPK-related mechanism. In summary, we provide direct evidences that the aberrantly upregulated mtEF4 contributes to the metastasis of breast cancer by coordinating metabolic pathways.

Detection of tBid Oligomerization and Membrane Permeabilization by Graphene-Based Single-Molecule Surface-Induced Fluorescence Attenuation.

The permeabilization of organelle membranes by BCL-2 family proteins is a pivotal step during the regulation of apoptosis; the underlying mechanisms remain unclear. Based on the fluorescence attenuation by graphene oxide, we developed a single-molecule imaging method termed surface-induced fluorescence attenuation (smSIFA), which enabled us to track both vertical and lateral kinetics of singly labeled BCL-2 family protein tBid during membrane permeabilization. We found that tBid monomers lie shallowly on the lipid bilayer, where they self-assemble to form oligomers. During the initiation phase of self-assembly, the two central hydrophobic helices (α6 and α7) of tBid insert halfway into the phospholipid core, while the other helices remain on the surface. In oligomerized tBid clusters, α6 and α7 prefer to float up, and the other helices may sink to the bottom of the membrane and cause the formation of transient two-dimensional, micelle-like pore structures, which are responsible for the permeabilization of membranes and the induction of apoptosis. Our results shed light on the understanding of tBid-induced apoptosis, and this nanotechnology-based smSIFA approach could be used to dissect the kinetic interaction between membrane protein and lipid bilayer at the single-molecule level with subnanometer precision.

Genetically Encoded FapR-NLuc as a Biosensor to Determine Malonyl-CoA in Situ at Subcellular Scales.

Malonyl-CoA is one of the key metabolic intermediates in fatty acid metabolism as well as a key player in protein post-translational modifications. Detection of malonyl-CoA in live cells is challenging because of the lack of effective measuring tools. Here we developed a genetically encoded biosensor, FapR-NLuc, by combining a malonyl-CoA responsive bacterial transcriptional factor, FapR, with an engineered luciferase, NanoLuciferase (NLuc). FapR-NLuc specifically responds to malonyl-CoA and enables the rapid detection of malonyl-CoA at the micromolar level. More importantly, it is reflective of the fluctuations of malonyl-CoA in live cells. Upon being targeted to subcellular compartments, this biosensor can detect the changes of malonyl-CoA in situ within organelles. Thus, FapR-NLuc can potentially be used as a tool to study the kinetics of malonyl-CoA in live cells, which will shed light on the underlying mechanisms of malonyl-CoA-mediated biological processes.

Tissue distribution, subcellular localization, and enzymatic activity analysis of human SIRT5 isoforms.

SIRT5 is one of the seven mammalian sirtuins which are NAD+-dependent deacylases. In human beings, SIRT5 gene encodes for four SIRT5 protein isoforms, namely SIRT5iso1, SIRT5iso2, SIRT5iso3, and SIRT5iso4. Previous studies have focused mostly on SIRT5iso1. Characteristics regarding localization, activity and tissue distribution of the other three SIRT5 isoforms remain unclear. In the present study, we characterized these properties of these SIRT5 isoforms. We found that SIRT5iso1-3 were mitochondria-localized, while SIRT5iso4 localized mainly in cytoplasm. SIRT5iso2-4 had little deacylase activity comparing with SIRT5iso1. Although cDNAs of all SIRT5 isoforms were readily detected in multiply tissues according to EST database, proteins of SIRT5iso2-4 were seldom observed in human cell lines. Altogether, we dissected the four isoforms of human SIRT5 protein.

Highly Fluorescent Chiral N-S-Doped Carbon Dots from Cysteine: Affecting Cellular Energy Metabolism.

Cysteine-based chiral optically active carbon dots (CDs) and their effects on cellular energy metabolism, which is vital for essential cellular functions, have been barely reported. A green and effective synthesis strategy for chiral N-S-doped CDs (fluorescence quantum yield ca. 41.26 %) based on hydrothermal treatment of l- or d-cysteine at as low as 60 °C has been developed. This suggested that cysteine was instable in aqueous solutions and acts as a warning for high-temperature synthesis of nanomaterials using cysteine as stabilizer. Human bladder cancer T24 cells treated with l-CDs showed up-regulated glycolysis, while d-CDs had no similar effects. In contrast, no disturbance to the basal mitochondrial aerobic respiration of T24 cells was caused by either chiral CD.

Lysine malonylation is elevated in type 2 diabetic mouse models and enriched in metabolic associated proteins.

Protein lysine malonylation, a newly identified protein post-translational modification (PTM), has been proved to be evolutionarily conserved and is present in both eukaryotic and prokaryotic cells. However, its potential roles associated with human diseases remain largely unknown. In the present study, we observed an elevated lysine malonylation in a screening of seven lysine acylations in liver tissues of db/db mice, which is a typical model of type 2 diabetes. We also detected an elevated lysine malonylation in ob/ob mice, which is another model of type 2 diabetes. We then performed affinity enrichment coupled with proteomic analysis on liver tissues of both wild-type (wt) and db/db mice and identified a total of 573 malonylated lysine sites from 268 proteins. There were more malonylated lysine sites and proteins in db/db than in wt mice. Five proteins with elevated malonylation were verified by immunoprecipitation coupled with Western blot analysis. Bioinformatic analysis of the proteomic results revealed the enrichment of malonylated proteins in metabolic pathways, especially those involved in glucose and fatty acid metabolism. In addition, the biological role of lysine malonylation was validated in an enzyme of the glycolysis pathway. Together, our findings support a potential role of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future.

Prediction of Protein Lysine Acylation by Integrating Primary Sequence Information with Multiple Functional Features.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic methods have been widely used to identify lysine acylation proteins. However, these experimental approaches often fail to detect proteins that are in low abundance or absent in specific biological samples. To circumvent these problems, we developed a computational method to predict lysine acylation, including acetylation, malonylation, succinylation, and glutarylation. The prediction algorithm integrated flanking primary sequence determinants and evolutionary conservation of acylated lysine as well as multiple protein functional annotation features including gene ontology, conserved domains, and protein-protein interactions. The inclusion of functional annotation features increases predictive power oversimple sequence considerations for four of the acylation species evaluated. For example, the Matthews correlation coefficient (MCC) for the prediction of malonylation increased from 0.26 to 0.73. The performance of prediction was validated against an independent data set for malonylation. Likewise, when tested with independent data sets, the algorithm displayed improved sensitivity and specificity over existing methods. Experimental validation by Western blot experiments and LC-MS/MS detection further attested to the performance of prediction. We then applied our algorithm on to the mouse proteome and reported the global-scale prediction of lysine acetylation, malonylation, succinylation, and glutarylation, which should serve as a valuable resource for future functional studies.

ESeroS-GS Protects Neuronal Cells from Oxidative Stress by Stabilizing Lysosomes.

γ-l-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-l-cysteinylglycine sodium salt (ESeroS-GS) is a water-soluble derivative of α-tocopherol (vitamin E). We reported previously that ESeroS-GS can act as an anti-inflammatory agent and can induce cell death in breast cancer cells. However, the potential antioxidant capacities of ESeroS-GS remain elusive. Here, we measured its scavenging effects on free radicals and evaluated its protective effects on neuronal cells against oxidative stress. The results indicated that ESeroS-GS effectively scavenged both 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonate free radicals (ABTS(•+)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and attenuated H2O2-induced neuronal cell death. H2O2 treatment induced lysosomal membrane permeabilization rapidly, and caused the redistribution of lysosomal proteases, which were responsible for the neuronal cell death. ESeroS-GS abolished the interaction between tBid and the lysosomal membranes, blocked the translocation of tBid to the lysosomal membranes, decreased its oligomerization within the membrane circumstances, prevented the lysosomal membrane permeabilization, and thus attenuated the neuronal cell death. These data suggest that ESeroS-GS protected the neuronal cells from oxidative stress by stabilizing lysosomal membranes, and thus might act as a novel neuroprotector for neuronal diseases associated with oxidative stress.

U-shaped Association Between Weight-Adjusted-Waist Index and Arterial Stiffness Among Adult Hypertensive Patients: A Population-Based Study in the United States.

The weight-adjusted-waist index (WWI) is an innovative measure of obesity that appears to surpass body mass index (BMI) in assessing lean body mass and fat mass. This study aimed to evaluate the relationship between WWI and AS in hypertensive adults in the United States. The study included 9753 adults diagnosed with hypertension from the National Health and Nutrition Examination Survey (NHANES), which spanned the years 2007-2016. WWI was calculated by dividing waist circumference (in cm) by the square root of body weight (in kg), and arterial stiffness (represented by estimated pulse wave velocity [ePWV]) was analyzed as the outcome. Weighted multiple linear regression and smooth curve fitting were used to test for linear and nonlinear associations. Threshold effects were determined using a two-part linear regression model. Additionally, subgroup analyses and interaction tests were conducted to gain a more in-depth understanding of the observed associations. The mean WWI of the participants was 11.32 ± 0.76. After multivariable adjustment, WWI showed a significant nonlinear association with ePWV, with a U-shaped association observed between the two. Specifically, WWI below the threshold of 10.23 was negatively associated with arterial stiffness (ß = -0.39, 95% CI: -0.54 to -0.25), while WWI above the threshold of 10.23 was positively associated with arterial stiffness (ß = 0.04, 95% CI: 0.01-0.07). To conclude, the present findings imply that maintaining WWI within an optimal range could reduce AS in hypertensive individuals and potentially decrease cardiovascular risk. However, this observation needs to be confirmed in large clinical trials.

Efficacy and safety of radiofrequency ablation and laparoscopic adrenalectomy for primary aldosteronism: a meta­analysis.

OBJECTIVE: To compare the efficacy (including blood pressure, medication reduction, serum potassium, and clinical success) and safety parameters (including operative time, length of hospital stay, blood loss, hypertension crisis rate, and complication rate) of radiofrequency ablation (RFA) and laparoscopic adrenalectomy (LA) in the treatment of primary aldosteronism (PA). METHODS: Literature search was performed on PubMed, EMBASE, The Cochrane Library (Issue 8, 2023), Web of Science, China National Knowledge Infrastructure, and Wanfang from inception to August 2023. Study selection, data extraction, and risk of bias assessment were performed by two independent reviewers. Quality assessment was conducted using the Newcastle-Ottawa scale. The Stata 12.0 software was used for statistical analyses. Pooled odds ratios (OR) with corresponding 95% confidence interval (CI) were calculated for categorical outcomes, while mean difference (MD) with corresponding 95% CI were calculated for continuous outcomes. RESULTS: A total of 5 studies involving 204 patients (LA, n = 127; and RAF, n = 77) were included. LA had better diastolic blood pressure control than RFA (WMD = 5.19; 95% CI 0.96-9.43); however, the RFA demonstrated better shorter operative time (WMD = - 57.99; 95% CI - 116.54 to 0.57), and shorter length of hospital stay (OR - 1.6; 95% CI - 2.37 to - 0.83) compared to LA. All remaining parameters were comparable between the interventions. CONCLUSION: While grossly comparable in efficacy as treatment options for PA, RFA may allow for shorter operative time and hospital stay, less intraoperative blood loss, and lower hospitalization costs. However, LA has better diastolic blood pressure control. Even so, we still need larger prospective studies, specifically with comparative hypertension response (short and long term) and number of post-procedural antihypertensive medication requirement.

Structural and biochemical insights into the mechanism of the Gabija bacterial immunity system.

The Gabija system is a newly discovered bacterial immune system that consists of GajA and GajB. Here we report the cryo-EM structure of the Gabija complex from Bacillus cereus VD045 at 3.6 Å, which provides the direct evidence of interactions between GajA and GajB. The Gabija complex is an octameric ring structure with four GajA and four GajB. GajA is an OLD nucleases family protein, while GajB belongs to the SF1 helicases. The Gabija complex has sequence-specific DNA nuclease activity and prefers circular rather than linear DNA as substrate, its activity is more sensitive to concentrations change of nucleotides compared to GajA alone. Our data suggest a mechanism of Gabija immunity: the nuclease activity of Gabija complex is inhibited under physiological conditions, while it is activated by depletion of NTP and dNTP upon the replication and transcription of invading phages and cleave the circular DNA to prevent phage DNA replication.

TrxR1 and GPx2 are potently induced by isothiocyanates and selenium, and mutually cooperate to protect Caco-2 cells against free radical-mediated cell death.

Currently, there is significant interest in the field of diet-gene interactions and the mechanisms by which food compounds regulate gene expression to modify cancer susceptibility. From a nutrition perspective, two key components potentially exert cancer chemopreventive effects: isothiocyanates (ITCs), present in cruciferous vegetables, and selenium (Se) which, as selenocysteine, is an integral part of selenoproteins. However, the role of these compounds in the expression of key selenoenzymes once the cancer process has been initiated still needs elucidation. Therefore, this investigation examined the effect of two forms of selenium, selenium-methylselenocysteine and sodium selenite, both individually and in combination with two ITCs, sulforaphane or iberin, on the expression of the two selenoenzymes, thioredoxin reductase 1 (TrxR1) and gastrointestinal glutathione peroxidase (GPx2), which are targets of ITCs, in Caco-2 cells. Co-treatment with both ITCs and Se induced expression of TrxR1 and GPx2 more than either compound alone. Moreover, pre-treatment of cells with ITC+Se enhanced cytoprotection against H(2)O(2)-induced cell death through a ROS-dependent mechanism. Furthermore, a single and double knockdown of TrxR1 and/or GPx2 suggested that both selenoproteins were responsible for protecting against H(2)O(2)-induced cell death. Together, these data shed new light on the mechanism of interactions between ITC and Se in which translational expression of the enhanced transcripts by the former is dependent on an adequate Se supply, resulting in a cooperative antioxidant protective effect against cell death.

Hydrogen peroxide impairs autophagic flux in a cell model of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, but the pathogenesis of NAFLD is not fully clear. The aim of this study was to determine whether autophagy plays a role in the pathogenesis of NAFLD. We found that the levels of autophagy were elevated in hepatoma cells upon exposure to free fatty acids, as confirmed by the increase in the number of autophagosomes. However, exposure of hepatoma cells to H2O2 and TNF-α, two typical "second hit" factors, increased the initiation of autophagy but inhibited the autophagic flux. The inhibition of autophagy sensitized cells to pro-apoptotic stimuli. Taken together, our results suggest that autophagy acts as a protective mechanism in the pathogenesis of NAFLD and that impairment of autophagy might induce more severe lesions of the liver. These findings will be a benefit to the understanding of the pathogenesis of NAFLD and might suggest a strategy for the prevention and cure of NAFLD.

Phosphatidic acid: from biophysical properties to diverse functions.

Phosphatidic acid (PA), the simplest phospholipid, acts as a key metabolic intermediate and second messenger that impacts diverse cellular and physiological processes across species ranging from microbes to plants and mammals. The cellular levels of PA dynamically change in response to stimuli, and multiple enzymatic reactions can mediate its production and degradation. PA acts as a signalling molecule and regulates various cellular processes via its effects on membrane tethering, enzymatic activities of target proteins, and vesicular trafficking. Because of its unique physicochemical properties compared to other phospholipids, PA has emerged as a class of new lipid mediators influencing membrane structure, dynamics, and protein interactions. This review summarizes the biosynthesis, dynamics, and cellular functions and properties of PA.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis.

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts.

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1ß, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/ß, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Selective targeting of gold nanorods at the mitochondria of cancer cells: implications for cancer therapy.

We have observed that Au nanorods (NRs) have distinct effects on cell viability via killing cancer cells while posing negligible impact on normal cells and mesenchymal stem cells. Obvious differences in cellular uptake, intracellular trafficking, and susceptibility of lysosome to Au NRs by different types of cells resulted in selective accumulation of Au NRs in the mitochondria of cancer cells. Their long-term retention decreased mitochondrial membrane potential and increased reactive oxygen species level that enhances the likelihood of cell death. These findings thus provide guidance for the design of organelle-targeted nanomaterials in tumor therapy.

Variation in community structure and network characteristics of spent mushroom substrate (SMS) compost microbiota driven by time and environmental conditions.

Global mushroom production is growing rapidly, raising concerns about polluting effects of spent mushroom substrate (SMS) and interest in uses in composts. In this study, SMS composting trials and high-throughput sequencing were carried out to investigate to better understand how the structure, co-occurrence patterns, and functioning of bacterial and fungal communities vary through compost time and across environmental conditions. The results suggested that both bacterial and fungal microbiota displayed significant variation in community composition across different composting stages. Enzyme activity levels showed both directional and fluctuating changes during composting, and the activity dynamics of carboxymethyl cellulase, polyphenol oxidase, laccase, and catalase correlated significantly with the succession of microbial community composition. The co-occurrence networks are "small-world" and modularized and the topological properties of each subnetwork were significantly influenced by the environmental factors. Finally, seed germination and seedling experiments were performed to verify the biosafety and effectiveness of the final composting products.