Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.Reactive oxygen species (ROS) are the chemical species formed by the incomplete reduction of oxygen and includes superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), singlet oxygen and hydroxyl radicals (·OH).^{1, 2} ROS is generated primarily as a by-product of cellular metabolism through leakage of electrons by electron transport chain (ETC) in mitochondria and from other sources such as plasma membrane, peroxisomes and endoplasmic reticulum.^{3, 4} ROS acts as signaling molecule when present at an appropriate level through the covalent modification of specific cysteine residues of redox-sensitive target proteins.⁵ The optimum level of ROS is maintained by equilibrium between its production and scavenging through the involvement of antioxidant system. An alteration in this equilibrium gives rise to oxidative stress, leading to cellular damage that finally precipitates into neurodegenerative disorders, cancer and metabolic disorders such as diabetes.^{6, 7, 8, 9, 10, 11}Therefore, for the maintenance of redox equilibrium, surveillance on generation of ROS is as critical as ROS scavenging. Mitochondria being the primary source of ROS generation by ETC assume the important center for maintenance of ROS levels. The factors that control ROS production by ETC complexes are not well defined. Mitochondria harbors a number of ROS scavenging enzymes such as intermembrane space-associated Cu–Zn superoxide dismutase (Cu-Zn SOD), matrix-localized MnSOD, isoforms of peroxiredoxins and glutaredoxins.¹² Together, these proteins help in maintaining the appropriate cellular ROS level. Maintenance of optimum level of ROS is important for developing tissues and stem cells and in metabolically active tissues where the high-energy demand makes them more vulnerable to oxidative stress. In addition to this, cancer cells having aberrant metabolism tend to have enhanced ROS that helps in tumor progression; however, abnormally high ROS may lead to apoptosis.^{13, 14, 15, 16, 17} Thus, in cancer cells, for their prolonged survival, there exists an evolved mechanism to maintain the balance of redox state through upregulation of many antioxidant enzymes^{9, 10, 11} and a number of signaling molecules modulating the expression level of these enzymes.^{18, 19}In earlier reports, overexpression of ‘Magmas'' was observed in patient samples of prostate cancer, pituitary adenoma, in various developmental stages and in metabolically active human tissues.²⁰ Originally, Magmas was identified as a protein involved in granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling and was found to localize in mitochondria.^{21, 22} Magmas belongs to type IV class of J-proteins and acts as co-chaperone of mitochondrial heat shock protein 70 (mtHsp70). It is an inner membrane-associated protein and an essential component of mitochondrial protein translocation machinery. Magmas inhibits the activity of its J-protein counterpart DnaJC19 in stimulating ATPase activity of mtHsp70 at the transport channel and regulates the import of nuclear-encoded mitochondrial proteins into the matrix.²²Although overexpression of Magmas in energy-demanding tissues and cancer cells is well known, the physiological advantage provided by the enhanced expression of the protein is still enigmatic. In the present study, we have provided compelling evidence favoring an additional function of Magmas as a ‘ROS regulator''. The overexpression of Magmas led to reduction in ROS and increased cellular tolerance to oxidative stress, whereas its downregulation elevated the cellular ROS level and made the cells more susceptible to ROS-mediated apoptosis. To maintain the redox equilibrium and provide cytoprotection against oxidative stress, Magmas controls ROS production by enhancing the ETC complex activity. On the other hand, it increases the activity of two major antioxidant enzymes such as MnSOD and glutathione peroxidase (GPx) to promote ROS scavenging. In summary, we propose a novel essential role of Magmas as a ‘ROS regulatory protein'' in the maintenance of cellular redox homeostasis and imparting cytoprotection under oxidative stress.     

Comparative Study of Functional Response of Common Hemipteran Bugs of East Calcutta Wetlands,India

The common and abundant hemipteran water bugs Anisops bouvieri, Diplonychus rusticus, D. annulatus, of the wetlands of East Kolkata are known predators of a wide range of aquatic insects including the mosquito larvae. In the laboratory their predation were assessed in respect to short term and long term periods using the larvae of Culex quinquefasciatus to reveal their possible role in regulating the dipteran population in nature. The attack rate (a) and handling time (T_h ) of these predators varied with respect to the prey size. For the backswimmers A. bouvieri the values for a and T_h for the small prey were 5.47 L and 18.72 min respectively, while in case of the belostomatid bugs, the values for the same were 5.37 L and 8.64 min (for D. rusticus), 5.81 L and 20.16 min (for D. annulatus). The predation rate varied with prey and predator densities for both the prey sizes. It was revealed that on an average A. bouvieri can kill and consume 10–82 and 6–44, D. rusticus 10–118 and 10–84 and D. annulatus 10–70 and 10–138 small and large sized prey per day, respectively. However the mutual interference (m) values of the three predators varied with the prey size and ranged between 0.053–0.326 for A. bouvieri, 0.0381–0.066 for D. rusticus and 0.0556–0.115 for D. annulatus, respectively. In the long term experiments A. bouvieri killed between 6–119 small preys and 3–31 large preys, D. rusticus killed 50–94 small preys and 50–96 large preys and D. annulatum were found to kill between 14–74 small prey and 50–131 large prey per day, respectively. The clearance rates were found to be proportional to the predator density as well to the prey size and density, and differed between the predator species significantly. These data are supportive of qualifying the water bugs, A. bouvieri, D. rusticus, and D. annulatus as potential biological resources in regulating the population of mosquito larvae in the wet‐lands. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Benchmarking of a Bayesian single cell RNAseq differential gene expression test for dose–response study designs

The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose–response study designs used in safety assessments. To benchmark DGEA methods for dose–response scRNAseq experiments, we proposed a multiplicity corrected Bayesian testing approach and compare it against 8 other methods including two frequentist fit-for-purpose tests using simulated and experimental data. Our Bayesian test method outperformed all other tests for a broad range of accuracy metrics including control of false positive error rates. Most notable, the fit-for-purpose and standard multiple group DGEA methods were superior to the two group scRNAseq methods for dose–response study designs. Collectively, our benchmarking of DGEA methods demonstrates the importance in considering study design when determining the most appropriate test methods.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386–563 and 386–510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.     

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.     

Radiation-induced inactivation of dihydroorotate dehydrogenase in dilute aqueous solution.

The inactivation of dihydroorotate dehydrogenase by gamma irradiation in dilute aqueous solution has been investigated. The activity of the enzyme decreased exponentially as a function of the absorbed dose under aerated and nitrous oxide-saturated conditions. The contributions of the individual radical species derived from water radiolysis were estimated from the inactivation results observed under aerated, argon-saturated, and nitrous oxide-saturated conditions. The hydrogen atom and hydroxyl radical were found to be important in enzyme inactivation. The effect of selected inorganic radical anions such as Br.2-, I.2-, and (SCN).2- on the enzyme activity was also studied, and the results implicate the possible involvement of cysteine and tyrosine residues in the catalytic activity of dihydroorotate dehydrogenase. Changes in the kinetic parameters (Michaelis-Menten constant, Km, and maximal velocity, Vmax) due to irradiation under the conditions investigated suggest that radiation-induced inactivation is due to modification of the substrate binding sites and that of the active site residues in the enzyme. Evidence for the reduction of iron-sulfur centers in the enzyme during the inactivation process has been put forward from the difference spectrum of the irradiated dihydroorotate dehydrogenase. It has also been shown by electrophoretic studies that radiation-induced inactivation was not due to any fragmentation of the protein structure or the formation of any intermolecular crosslinking.     

Quantifying the effects of switchgrass (Panicum virgatum) on deep organic C stocks using natural abundance 14C in three marginal soils

Perennial bioenergy crops have been shown to increase soil organic carbon (SOC) stocks, potentially offsetting anthropogenic C emissions. The effects of perennial bioenergy crops on SOC are typically assessed at shallow depths (<30 cm), but the deep root systems of these crops may also have substantial effects on SOC stocks at greater depths. We hypothesized that deep (>30 cm) SOC stocks would be greater under bioenergy crops relative to stocks under shallow‐rooted conventional crop cover. To test this, we sampled soils to between 1‐ and 3‐m depth at three sites in Oklahoma with 10‐ to 20‐year‐old switchgrass (Panicum virgatum) stands, and collected paired samples from nearby fields cultivated with shallow rooted annual crops. We measured root biomass, total organic C, ¹⁴C, ¹³C, and other soil properties in three replicate soil cores in each field and used a mixing model to estimate the proportion of recently fixed C under switchgrass based on ¹⁴C. The subsoil C stock under switchgrass (defined over 500–1500 kg/m² equivalent soil mass, approximately 30–100 cm depth) exceeded the subsoil stock in neighboring fields by 1.5 kg C/m² at a sandy loam site, 0.6 kg C/m² at a site with loam soils, and showed no significant difference at a third site with clay soils. Using the mixing model, we estimated that additional SOC introduced after switchgrass cultivation comprised 31% of the subsoil C stock at the sandy loam site, 22% at the loam site, and 0% at the clay site. These results suggest that switchgrass can contribute significantly to subsoil organic C—but also indicated that this effect varies across sites. Our analysis shows that agricultural strategies that emphasize deep‐rooted grass cultivars can increase soil C relative to conventional crops while expanding energy biomass production on marginal lands.     

Substrate competition and specificity at the active site of amylopullulanase from Clostridium thermohydrosulfuricum

A highly thermostable pullulanase purified from Clostridium thermohydrosulfuricum strain 39E displayed dual activity with respect to glycosidic bond cleavage. The enzyme cleaved alpha-1,6 bonds in pullulan, while it showed alpha-1,4 activity against malto-oligosaccharides. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as the two competing substrates were used to distinguish the dual specificity of the enzyme from the single substrate specificity known for pullulanases and alpha-amylases.