Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential

Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3′ UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3′ UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t_1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t_1/2 (<1 h). MTCH2 read-through–deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Influence of Aloe vera on water absorption and enzymatic in vitro degradation of alginate hydrogel films

This study investigates the influence of Aloe vera on water absorption and the in vitro degradation rate of Aloe vera-Ca-alginate hydrogel films, for wound healing and drug delivery applications. The influence of A. vera content (5%, 15% and 25%, v/v) on water absorption was evaluated by the incubation of the films into a 0.1 M HCl solution (pH 1.0), acetate buffer (pH 5.5) and simulated body fluid solution (pH 7.4) during 24 h. Results show that the water absorption is significantly higher for films containing high A. vera contents (15% and 25%), while no significant differences are observed between the alginate neat film and the film with 5% of A. vera. The in vitro enzymatic degradation tests indicate that an increase in the A. vera content significantly enhances the degradation rate of the films. Control films, incubated in a simulated body fluid solution without enzymes, are resistant to the hydrolytic degradation, exhibiting reduced weight loss and maintaining its structural integrity. Results also show that the water absorption and the in vitro degradation rate of the films can be tailored by changing the A. vera content.     

Organic Carbon Degradation in Anoxic Organic-Rich Shelf Sediments: Biogeochemical Rates and Microbial Abundance

Identifying and explaining bottlenecks in organic carbon mineralization and the persistence of organic matter in marine sediments remain challenging. This study aims to illuminate the process of carbon flow between microorganisms involved in the sedimentary microbial food chain in anoxic, organic-rich sediments of the central Namibian upwelling system, using biogeochemical rate measurements and abundances of Bacteroidetes, Gammaproteobacteria, and sulfate-reducing bacteria at two sampling stations. Sulfate reduction rates decreased by three orders of magnitude in the top 20 cm at one sampling station (280 nmol cm^?3 d^?1 – 0.1 nmol cm^?3 d^?1) and by a factor of 7 at the second station (65 nmol cm^?3 d^?1 – 9.6 nmol cm^?3 d^?1). However, rates of enzymatic hydrolysis decreased by less than a factor of three at both sampling stations for the polysaccharides laminarin (23 nmol cm^?3 d^?1– 8 nmol cm^?3 d^?1 and 22 nmol cm^?3 d^?1– 10 nmol cm^?3 d^?1) and pullulan (11 nmol cm^?3 d^?1– 4 nmol cm^?3 d^?1 and 8 nmol cm^?3 d^?1– 6 nmol cm^?3 d^?1). Increasing imbalance between carbon turnover by hydrolysis and terminal oxidation with depth, the steep decrease in cell specific activity of sulfate reducing bacteria with depth, low concentrations of volatile fatty acids (less than 15 μM), and persistence of dissolved organic carbon, suggest decreasing bioavailability and substrate limitation with depth.     

Isolation of Coal Degrading Fungus from Drilled Core Coal Sample and Effect of Prior Fungal Pretreatment on Chemical Attributes of Extracted Humic Acid

Fungal degradation of low rank coal has appeared as an alternative technique for exploitation of non-fuel options. A fungal isolate, MW1, was isolated and coal sample was subjected to fungal pretreatment. The residual coal was processed for extraction of humic acid for determining the effect of such pretreatment. Extracted humic acid was analyzed on the basis of elemental composition and spectroscopy. Fungal pretreatment caused improvement in oxygen content, E₄/E₆ ratio, and absorption bands related to humic materials. Conclusively, pretreatment resulted in improving chemical attributes of humic acid molecule, thus, warranting supplementary high-tech investigations for the optimization of process upscale.     

Redox Buffer Capacity in Water-Rock-Microbe Interaction Systems in Subsurface Environments

An incubation experiment was conducted to estimate redox buffer capacity of “water-rock-microbe” interaction systems in sedimentary rocks. The water chemistry, microbial growth and community structure were analyzed during the incubations. The dissolved oxygen (DO) concentrations and oxidation-reduction potential (ORP) values decreased notably in the presence of active microorganisms, whereas abiotic reactions did not lead to reducing conditions during incubation. The change in microbial community structure suggests that nitrate-reducing and sulfate-reducing bacteria played an important role in reduction of water by using lignite-derived organic matter. These results show that the microbial role is extremely important for the redox buffering capacity in sedimentary rock environments.     

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.