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Cancer Research Communications Jul 2024Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by...
Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases. Myristoylation is emerging as a promising cancer therapeutic target, however the molecular determinants of sensitivity to N-myristoyltransferase inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that N-myristoyltransferases are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter Translocase of Inner Mitochondrial Membrane 17 homologue A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis N-myristoyltransferase-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.
PubMed: 38949950
DOI: 10.1158/2767-9764.CRC-23-0428 -
International Maritime Health 2024Saturation diving is a standard method of intervention for commercial diving during offshore operations. Current saturation procedures achieve a high level of safety...
BACKGROUND
Saturation diving is a standard method of intervention for commercial diving during offshore operations. Current saturation procedures achieve a high level of safety with regards to decompression sickness but still put the divers under multiple stressors: 1) Environmental stress (long confinement, heat/cold, dense gases, high oxygen levels), 2) Work stress (muscular fatigue, psychological pressure, breathing equipment, etc.), 3) venous gas emboli associated with decompression, 4) Inflammation related to oxidative stress and microparticles. We present the results of a saturation divers monitoring campaign performed in the North Sea Danish sector, on the Tyra field, during 2022. The study was supported by TotalEnergies, the field operator, and performed by Boskalis Subsea Services, the diving contractor, onboard the diving support vessel Boka Atlantis. The objective was twofold: document the level of diving stress during saturation operations in the Danish sector, and compare the performances of two saturation procedures, the Boskalis and the NORSOK procedures.
MATERIALS AND METHODS
Fourteen divers volunteered for the study. The monitoring package include weight and temperature measurements, psychomotor tests (objective evaluation) and questionnaires (subjective evaluation), Doppler bubble detection and bioimpedance. The results were presented in a radar diagram that provides a general view of the situation.
RESULTS
The data were analysed along 3 dimensions: work and environmental, desaturation bubbles, oxidative stress and inflammation. The results showed little or no variations from the reference values. No bubbles were detected after excursion dives and the final decompression, except for two divers with a grade 1 after arriving at surface. No statistical difference could be found between the Boskalis and the NORSOK saturation procedures.
CONCLUSIONS
At a depth of 40-50 msw corresponding to the Danish sector, the two saturation procedures monitored induce no or little stress to the divers. The divers know how to manage their diet, equilibrate their hydration and pace their effort. Data available on divers' post saturation period show a recovery over the 24-48 hours following the end of the decompression. Further research should focus on diving deeper than 100 msw where a greater stress can be anticipated.
Topics: Humans; Diving; Decompression Sickness; North Sea; Adult; Male; Oxygen Saturation; Middle Aged; Stress, Physiological; Denmark; Monitoring, Physiologic
PubMed: 38949219
DOI: 10.5603/imh.99606 -
JPMA. the Journal of the Pakistan... Jun 2024To compare the efficacy of tocotrienol and tocopherol in the management of patients with atherosclerotic cardiovascular diseases. (Comparative Study)
Comparative Study
OBJECTIVE
To compare the efficacy of tocotrienol and tocopherol in the management of patients with atherosclerotic cardiovascular diseases.
METHODS
The systematic review was conducted in line with Preferred Reporting Items for Systematic Reviews and Meta- Analyses guidelines 2020, and comprised literature search from 2002 till January 5, 2023, on PubMed, Google Scholar, Cochrane Library, Google, Wiley-Inter Science Library, Medline, SpringerLink, Taylor and Francis databases. The search was conducted using key words, such as: "tocopherol", "tocotrienol", "vitamin E", "dyslipidaemia", "cardiovascular diseases" "cardioprotective", "hypercholesterolemia" and "atherosclerosis" along with Boolean operators. Human clinical studies regarding the use of tocotrienol or tocopherol or comparison of its efficacy in patients having atherosclerosis, dyslipidaemia leading to cardiovascular diseases, and studies including details of efficacy of any of the four alpha, beta, gamma, delta isomers of tocopherol or tocotrienol were included. Pertinent data from the eligible studies was retrieved and reviewed.
RESULTS
Of the 516 articles identified, 26 (5%) articles met eligibility criteria. Of them 5(19%) were subjected to detailed analysis. Tocotrienol showed significant anti-oxidant efficacy at (250 mg/d) by decreasing cholesterol and serum inflammatory biomarkers i.e C-reactive protein (40%), malondialdehyde (34%), gamma-glutamyl transferase (22%) (p<0.001). Total anti-oxidant status (TAS) levels raised 22% (p<0.001) and Inflammatory cytokines i.e resistin, interleukin (IL)-1, IL-12, Interferon-gamma were decreased 15-17% (p<0.05-0.01) respectively by tocotrienol. Several microRNA (miRNA-133a, miRNA-223, miRNA-214, miRNA-155) were modulated by δ-tocotrienol. Whereas, tocopherol showed heterogeneity of results by either decreasing or increasing the risk of mortality in atherosclerotic cardiovascular diseases.
CONCLUSION
Compared to tocopherol, tocotrienol was found to be safe and potential candidate for improving cardiovascular health in the management of atherosclerotic cardiovascular diseases.
Topics: Humans; Tocotrienols; Atherosclerosis; Tocopherols; Antioxidants; Cardiovascular Diseases; Dyslipidemias; Cholesterol
PubMed: 38948984
DOI: 10.47391/JPMA.9227 -
BioRxiv : the Preprint Server For... Jun 2024Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative...
Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
PubMed: 38948861
DOI: 10.1101/2024.06.20.598218 -
BioRxiv : the Preprint Server For... Jun 2024Oxidative stress is implicated in the pathogenesis and progression of abdominal aortic aneurysm (AAA). Antioxidant delivery as a therapeutic for AAA is of substantial...
BACKGROUND
Oxidative stress is implicated in the pathogenesis and progression of abdominal aortic aneurysm (AAA). Antioxidant delivery as a therapeutic for AAA is of substantial interest although clinical translation of antioxidant therapy has met with significant challenges due to limitations in achieving sufficient antioxidant levels at the site of AAA. We posit that nanoparticle-based approaches hold promise to overcome challenges associated with systemic administration of antioxidants.
METHODS
We employed a peptide-based nanoplatform to overexpress a key modulator of oxidative stress, superoxide dismutase 2 (SOD2). The efficacy of systemic delivery of SOD2 mRNA as a nanotherapeutic agent was studied in two different murine AAA models. Unbiased mass spectrometry-enabled proteomics and high-dimensional bioinformatics were used to examine pathways modulated by SOD2 overexpression.
RESULTS
The murine SOD2 mRNA sequence was mixed with p5RHH, an amphipathic peptide capable of delivering nucleic acids to form self-assembled nanoparticles of ∼55 nm in diameter. We further demonstrated that the nanoparticle was stable and functional up to four weeks following self-assembly when coated with hyaluronic acid. Delivery of SOD2 mRNA mitigated the expansion of small AAA and largely prevented rupture. Mitigation of AAA was accompanied by enhanced SOD2 protein expression in aortic wall tissue. Concomitant suppression of nitric oxide, inducible nitric oxide synthase expression, and cell death was observed. Proteomic profiling of AAA tissues suggests that SOD2 overexpression augments levels of microRNAs that regulate vascular inflammation and cell apoptosis, inhibits platelet activation/aggregation, and downregulates mitogen-activated protein kinase signaling. Gene set enrichment analysis shows that SOD2 mRNA delivery is associated with activation of oxidative phosphorylation, lipid metabolism, respiratory electron transportation, and tricarboxylic acid cycle pathways.
CONCLUSIONS
These results confirm that SOD2 is key modulator of oxidative stress in AAA. This nanotherapeutic mRNA delivery approach may find translational application in the medical management of small AAA and the prevention of AAA rupture.
PubMed: 38948794
DOI: 10.1101/2024.06.17.599454 -
BioRxiv : the Preprint Server For... Apr 2024The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether...
BACKGROUND
The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects.
METHODS
The effect of Ang-(1-5) (1µM) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT -receptor (AT R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT R-knockout mice. Binding of Ang-(1-5) to the AT R was confirmed and the preferred conformations determined by docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics.
RESULTS
Key findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at eNOS and eNOS and thereby (2) increased NO release from HAEC and AT R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT R-KO or in PD123319-treated mice and which were more potent than effects of the established AT R-agonist C21. (5) According to modelling, Ang-(1-5) binds to the AT R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease.
CONCLUSIONS
Ang-(1-5) is a potent, endogenous AT R-agonist.
PubMed: 38948791
DOI: 10.1101/2024.04.05.588367 -
BioRxiv : the Preprint Server For... Jun 2024Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T)...
UNLABELLED
Sickle cell disease is caused by a mutation in the beta subunit of hemoglobin (HbSS) that drives Hb fiber formation when the protein is in the deoxygenated (tense, T) state. The drug voxelotor was recently approved to treat sickle cell disease by preventing HbSS fiber formation. Voxelotor acts as an allosteric inhibitor of polymerization by maintaining the HbSS protein in the relaxed (R) conformation, limiting polymerization of T-state fibers. Normal blood cells contain small amounts of natural Hb fibers and a few percent of the Fe ferric form, metHb, incapable of binding oxygen. Although the drug Voxelotor is now in use, the effect of the drug on the oxidized metHb state has not been reported. Here we assessed the influence of voxelotor on normal human metHb. We compared the aggregation of metHb at two pH values (5.5 and 7.1). MetHb is known to form organized fiber structures at or below pH 5.5. We find that voxelotor significantly enhances fiber formation of metHb R-state at pH 5.5, consistent with the mode of action for this drug in maintaining the Hb R conformation. The opposite effect is observed at physiological pH values. Voxelotor significantly decreases the rate of metHb aggregate formation at pH 7.1 but did not affect protein stability. Notably, drug binding drives metHb into novel spherical particles with a morphology never seen before for Hb. The formation of these particles should be considered in patients being treated for sickle cell disease with voxelotor.
WHY IT MATTERS
Voxelotor is an FDA-approved drug for sickle cell anemia, known to prevent hemoglobin fiber formation. Here, we investigate its effect on methemoglobin, the form of hemoglobin in which iron takes on the ferric Fe state. Our study examines voxelotor's impact on methemoglobin aggregation and stability. At pH 7.1, we found voxelotor to have an effect on methemoglobin solubility as evidenced by the formation of novel methemoglobin spherical structures. We observe that voxelotor significantly increases methemoglobin fiber formation at pH 5.5 but, notably, reduces methemoglobin aggregation at physiological pH levels. Minimal impact on methemoglobin thermodynamic stability is noted. These findings suggest voxelotor's potential therapeutic efficacy for various hemoglobinopathies, including conditions characterized by Heinz body formation.
PubMed: 38948767
DOI: 10.1101/2024.06.16.599216 -
BioRxiv : the Preprint Server For... Jun 2024Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit...
Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit tissue overgrowth, as well as an increased risk of childhood neoplasms in the liver and kidney. To understand the impact of these 11p15 changes, specifically in the liver, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to generate paired, cell-type-specific transcriptional and chromatin accessibility profiles of both BWS-liver and nonBWS-liver nontumorous tissue. Our integrated RNA+ATACseq multiomic approach uncovered hepatocyte-specific enrichment and activation of the peroxisome proliferator-activated receptor α (PPARA) - a liver metabolic regulator. To confirm our findings, we utilized a BWS-induced pluripotent stem cell (iPSC) model, where cells were differentiated into hepatocytes. Our data demonstrates the dysregulation of lipid metabolism in BWS-liver, which coincided with observed upregulation of PPARA during hepatocyte differentiation. BWS liver cells exhibited decreased neutral lipids and increased fatty acid β-oxidation, relative to controls. We also observed increased reactive oxygen species (ROS) byproducts in the form of peroxidated lipids in BWS hepatocytes, which coincided with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism.
PubMed: 38948745
DOI: 10.1101/2024.06.14.599077 -
Several common methods of making vesicles (except an emulsion method) capture intended lipid ratios.BioRxiv : the Preprint Server For... Jun 2024Researchers choose different methods of making giant unilamellar vesicles in order to satisfy different constraints of their experimental designs. A challenge of using a...
UNLABELLED
Researchers choose different methods of making giant unilamellar vesicles in order to satisfy different constraints of their experimental designs. A challenge of using a variety of methods is that each may produce vesicles of different lipid compositions, even if all vesicles are made from a common stock mixture. Here, we use mass spectrometry to investigate ratios of lipids in vesicles made by five common methods: electroformation on indium tin oxide slides, electroformation on platinum wires, gentle hydration, emulsion transfer, and extrusion. We made vesicles from either 5-component or binary mixtures of lipids chosen to span a wide range of physical properties: di(18:1)PC, di(16:0)PC, di(18:1)PG, di(12:0)PE, and cholesterol. For a mixture of all five of these lipids, ITO electroformation, Pt electroformation, gentle hydration, and extrusion methods result in only minor shifts (≤ 5 mol%) in lipid ratios of vesicles relative to a common stock solution. In contrast, emulsion transfer results in ∼80% less cholesterol than expected from the stock solution, which is counterbalanced by a surprising overabundance of saturated PC-lipid relative to all other phospholipids. Experiments using binary mixtures of some of the lipids largely support results from the 5-component mixture. Exact values of lipid ratios variations likely depend on the details of each method, so a broader conclusion is that experiments that increment lipid ratios in small steps will be highly sensitive to the method of lipid formation and to sample-to-sample variations, which are low (roughly ±2 mol% in the 5-component mixture and either scale proportionally with increasing mole fraction or remain low). Experiments that increment lipid ratios in larger steps or that seek to explain general trends or new phenomena will be less sensitive to the method used.
SIGNIFICANCE STATEMENT
Small changes to the amounts and types of lipids in membranes can drastically affect the membrane's behavior. Unfortunately, it is unknown whether (or to what extent) different methods of making vesicles alter the ratios of lipids in membranes, even when identical stock solutions are used. This presents challenges for researchers when comparing data with colleagues who use different methods. Here, we measure ratios of lipid types in vesicle membranes produced by five methods. We assess each method's reproducibility and compare resulting vesicle compositions across methods. In doing so, we provide a quantitative basis that the scientific community can use to estimate whether differences between their results can be simply attributed to differences between methods or to sample-to-sample variations.
PubMed: 38948736
DOI: 10.1101/2024.02.21.581444 -
BioRxiv : the Preprint Server For... Jun 2024Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species...
Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2- based and functional reporter screens in to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting ERGU-1 causes excessive H2O2 and transcriptional gene up- regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, E ndoplasmic R eticulum Gu ardian A egis of R edox D efense. Human and homologs of ERGU-1 can rescue mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.
PubMed: 38948723
DOI: 10.1101/2024.06.19.599784