Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10.

Antiviral signaling, immune response and cell metabolism are dysregulated by SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome-Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.

Preclinical Characterization of Pharmacologic NAD+ Boosting as a Promising Therapeutic Approach in Rheumatoid Arthritis.

OBJECTIVE: We analyzed NAD+ metabolism in patients with rheumatoid arthritis (RA), its association with disease activity and clinical outcomes of RA, and the therapeutic potential of pharmacologic NAD+ boosting. METHODS: Our study included 253 participants. In the first cohort, comprising 153 RA patients and 56 healthy donors, we assessed NAD+ levels and NAD+ -related gene pathways. We analyzed 92 inflammatory molecules by proximity extension assay. In the second cohort, comprising 44 RA patients starting anti-tumor necrosis factor (anti-TNF) drugs, we evaluated changes in NAD+ levels and their association with clinical response after 3 months. Mechanistic studies were performed ex vivo on peripheral blood mononuclear cells (PBMCs) from patients with RA to test the beneficial effects of NAD+ boosters, such as nicotinamide and nicotinamide riboside. RESULTS: Reduced NAD+ levels were found in RA samples, in line with altered activity and expression of genes involved in NAD+ consumption (sirtuins, poly[ADP-ribose] polymerase, CD38), transport (connexin 43), and biosynthesis (NAMPT, NMNATs). Unsupervised clustering analysis identified a group of RA patients with the highest inflammatory profile, the lowest NAD+ levels, and the highest disease activity (as shown by the Disease Activity Score in 28 joints). NAD+ levels were modulated by anti-TNF therapy in parallel with the clinical response. In vitro studies using PBMCs from RA patients showed that nicotinamide riboside and nicotinamide increased NAD+ levels via NAMPT and NMNAT and reduced their prooxidative, proapoptotic, and proinflammatory status. CONCLUSION: RA patients display altered NAD+ metabolism, directly linked to their inflammatory and disease activity status, which was reverted by anti-TNF therapy. The preclinical beneficial effects of NAD+ boosters, as shown in leukocytes from RA patients, along with their proven clinical safety, might pave the way for the development of clinical trials using these compounds.

Metabolic shift underlies tumor progression and immune evasion in S-nitrosoglutathione reductase-deficient cancer.

S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR-low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8+ T cells. Notably, GSNOR-low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR-Cas9-mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR-deficiency conferred higher tumorigenic and tumor-initiating capacities. Moreover, GSNOR-KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR-KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2-deoxyglucose (2DG), and a fragmented mitochondrial network. Real-time metabolic analysis revealed that GSNOR-KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient-derived xenografts and organoids from clinical GSNOR-low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulation of hepatic coenzyme Q biosynthesis by dietary omega-3 polyunsaturated fatty acids.

Dietary fats are important for human health, yet it is not fully understood how different fats affect various health problems. Although polyunsaturated fatty acids (PUFAs) are generally considered as highly oxidizable, those of the n-3 series can ameliorate the risk of many age-related disorders. Coenzyme Q (CoQ) is both an essential component of the mitochondrial electron transport chain and the only lipid-soluble antioxidant that animal cells can synthesize. Previous work has documented the protective antioxidant properties of CoQ against the autoxidation products of PUFAs. Here, we have explored in vitro and in vivo models to better understand the regulation of CoQ biosynthesis by dietary fats. In mouse liver, PUFAs increased CoQ content, and PUFAs of the n-3 series increased preferentially CoQ10. This response was recapitulated in hepatic cells cultured in the presence of lipid emulsions, where we additionally demonstrated a role for n-3 PUFAs as regulators of CoQ biosynthesis via the upregulation of several COQ proteins and farnesyl pyrophosphate levels. In both models, n-3 PUFAs altered the mitochondrial network without changing the overall mitochondrial mass. Furthermore, in cellular systems, n-3 PUFAs favored the synthesis of CoQ10 over CoQ9, thus altering the ratio between CoQ isoforms through a mechanism that involved downregulation of farnesyl diphosphate synthase activity. This effect was recapitulated by both siRNA silencing and by pharmacological inhibition of farnesyl diphosphate synthase with zoledronic acid. We highlight here the ability of n-3 PUFAs to regulate CoQ biosynthesis, CoQ content, and the ratio between its isoforms, which might be relevant to better understand the health benefits associated with this type of fat. Additionally, we identify for the first time zoledronic acid as a drug that inhibits CoQ biosynthesis, which must be also considered with respect to its biological effects on patients.

Cdkn1a transcript variant 2 is a marker of aging and cellular senescence.

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.

Therapeutic Potential and Immunomodulatory Role of Coenzyme Q10 and Its Analogues in Systemic Autoimmune Diseases.

Coenzyme Q10 (CoQ10) is a mitochondrial electron carrier and a powerful lipophilic antioxidant located in membranes and plasma lipoproteins. CoQ10 is endogenously synthesized and obtained from the diet, which has raised interest in its therapeutic potential against pathologies related to mitochondrial dysfunction and enhanced oxidative stress. Novel formulations of solubilized CoQ10 and the stabilization of reduced CoQ10 (ubiquinol) have improved its bioavailability and efficacy. Synthetic analogues with increased solubility, such as idebenone, or accumulated selectively in mitochondria, such as MitoQ, have also demonstrated promising properties. CoQ10 has shown beneficial effects in autoimmune diseases. Leukocytes from antiphospholipid syndrome (APS) patients exhibit an oxidative perturbation closely related to the prothrombotic status. In vivo ubiquinol supplementation in APS modulated the overexpression of inflammatory and thrombotic risk-markers. Mitochondrial abnormalities also contribute to immune dysregulation and organ damage in systemic lupus erythematosus (SLE). Idebenone and MitoQ improved clinical and immunological features of lupus-like disease in mice. Clinical trials and experimental models have further demonstrated a therapeutic role for CoQ10 in Rheumatoid Arthritis, multiple sclerosis and type 1 diabetes. This review summarizes the effects of CoQ10 and its analogs in modulating processes involved in autoimmune disorders, highlighting the potential of these therapeutic approaches for patients with immune-mediated diseases.

Regulation of coenzyme Q biosynthesis pathway in eukaryotes.

Coenzyme Q (CoQ, ubiquinone/ubiquinol) is a ubiquitous and unique molecule that drives electrons in mitochondrial respiratory chain and an obligatory step for multiple metabolic pathways in aerobic metabolism. Alteration of CoQ biosynthesis or its redox stage are causing mitochondrial dysfunctions as hallmark of heterogeneous disorders as mitochondrial/metabolic, cardiovascular, and age-associated diseases. Regulation of CoQ biosynthesis pathway is demonstrated to affect all steps of proteins production of this pathway, posttranslational modifications and protein-protein-lipid interactions inside mitochondria. There is a bi-directional relationship between CoQ and the epigenome in which not only the CoQ status determines the epigenetic regulation of many genes, but CoQ biosynthesis is also a target for epigenetic regulation, which adds another layer of complexity to the many pathways by which CoQ levels are regulated by environmental and developmental signals to fulfill its functions in eukaryotic aerobic metabolism.

NQO1 protects obese mice through improvements in glucose and lipid metabolism.

Chronic nutrient excess leads to metabolic disorders and insulin resistance. Activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. Here, inducible activation of Nrf2 in mice and transgenesis of the Nrf2 target, NQO1, conferred protection from diet-induced metabolic defects through preservation of glucose homeostasis, insulin sensitivity, and lipid handling with improved physiological outcomes. NQO1-RNA interaction mediated the association with and inhibition of the translational machinery in skeletal muscle of NQO1 transgenic mice. NQO1-Tg mice on high-fat diet had lower adipose tissue macrophages and enhanced expression of lipogenic enzymes coincident with reduction in circulating and hepatic lipids. Metabolomics data revealed a systemic metabolic signature of improved glucose handling, cellular redox, and NAD+ metabolism while label-free quantitative mass spectrometry in skeletal muscle uncovered a distinct diet- and genotype-dependent acetylation pattern of SIRT3 targets across the core of intermediary metabolism. Thus, under nutritional excess, NQO1 transgenesis preserves healthful benefits.

Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-α therapy effectiveness.

BACKGROUND: Radiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory form of arthritis in which tumor necrosis factor (TNF)-α, a potent inducer of inflammatory response and a key regulator of innate immunity and of Th1 immune responses, plays a central role. NETosis is a mechanism of innate immune defense that is involved in diverse rheumatology diseases. Nevertheless, spontaneous NETosis generation in r-axSpA, its association to disease pathogenesis, and the NETosis involvement on anti-TNF-α therapy's effects has never been explored. METHODS: Thirty r-axSpA patients and 32 healthy donors (HDs) were evaluated. Neutrophil extracellular trap (NET) formation, mediators of signal-transduction cascade required for NETosis induction and cell-free NETosis-derived products were quantified. An additional cohort of 15 r-axSpA patients treated with infliximab (IFX) for six months were further analyzed. In vitro studies were designed to assess the effects of IFX in NETosis generation and the inflammatory profile triggered. RESULTS: Compared to HDs, neutrophils from r-axSpA patients displayed augmented spontaneous NET formation, elevated expression of NET-associated signaling components, nuclear peptidylarginine deiminase 4 translocation and increased citrullinated histone H3. Furthermore, patients exhibited altered circulating levels of cell-free NETosis-derived products (DNA, nucleosomes and elastase). Additional studies revealed that cell-free NETosis-derived products could be suitable biomarkers for distinguish r-axSpA patients from HDs. Correlation studies showed association between cell-free NETosis-derived products and clinical inflammatory parameters. Besides, nucleosomes displayed potential as a biomarker for discriminate patients according to disease activity. IFX therapy promoted a reduction in both NETosis generation and disease activity in r-axSpA patients. Mechanistic in vitro studies further unveiled the relevance of IFX in reducing NET release and normalizing the augmented inflammatory activities promoted by NETs in mononuclear cells. CONCLUSIONS: This study reveals that NETosis is enhanced in r-axSpA patients and identifies the NETosis-derived products as potential disease activity biomarkers. In addition, the data suggests the potential role of NET generation analysis for assessment of therapeutic effectiveness in r-axSpA.

Protective Role of Nrf2 in Renal Disease.

Chronic kidney disease (CKD) is one of the fastest-growing causes of death and is predicted to become by 2040 the fifth global cause of death. CKD is characterized by increased oxidative stress and chronic inflammation. However, therapies to slow or prevent CKD progression remain an unmet need. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that plays a key role in protection against oxidative stress and regulation of the inflammatory response. Consequently, the use of compounds targeting Nrf2 has generated growing interest for nephrologists. Pre-clinical and clinical studies have demonstrated that Nrf2-inducing strategies prevent CKD progression and protect from acute kidney injury (AKI). In this article, we review current knowledge on the protective mechanisms mediated by Nrf2 against kidney injury, novel therapeutic strategies to induce Nrf2 activation, and the status of ongoing clinical trials targeting Nrf2 in renal diseases.