Increased production of isobutanol from xylose through metabolic engineering of Saccharomyces cerevisiae overexpressing transcription factor Znf1 and exogenous genes.

Only trace amount of isobutanol is produced by the native Saccharomyces cerevisiae via degradation of amino acids. Despite several attempts using engineered yeast strains expressing exogenous genes, catabolite repression of glucose must be maintained together with high activity of downstream enzymes, involving iron-sulfur assimilation and isobutanol production. Here, we examined novel roles of nonfermentable carbon transcription factor Znf1 in isobutanol production during xylose utilization. RNA-seq analysis showed that Znf1 activates genes in valine biosynthesis, Ehrlich pathway and iron-sulfur assimilation while coupled deletion or downregulated expression of BUD21 further increased isobutanol biosynthesis from xylose. Overexpression of ZNF1 and xylose-reductase/dehydrogenase (XR-XDH) variants, a xylose-specific sugar transporter, xylulokinase, and enzymes of isobutanol pathway in the engineered S. cerevisiae pho13gre3Δ strain resulted in the superb ZNXISO strain, capable of producing high levels of isobutanol from xylose. The isobutanol titer of 14.809 ± 0.400 g/L was achieved, following addition of 0.05 g/L FeSO4.7H2O in 5 L bioreactor. It corresponded to 155.88 mg/g xylose consumed and + 264.75% improvement in isobutanol yield. This work highlights a new regulatory control of alternative carbon sources by Znf1 on various metabolic pathways. Importantly, we provide a foundational step toward more sustainable production of advanced biofuels from the second most abundant carbon source xylose.

A structure-based virtual high-throughput screening, molecular docking, molecular dynamics and MM/PBSA study identified novel putative drug-like dual inhibitors of trypanosomal cruzain and rhodesain cysteine proteases.

Virtual screening a collection of ~ 25,000 ChemBridge molecule collection identified two nitrogenous heterocyclic molecules, 12 and 15, with potential dual inhibitory properties against trypanosomal cruzain and rhodesain cysteine proteases. Similarity search in DrugBank found the two virtual hits with novel chemical structures with unreported anti-trypanosomal activities. Investigations into the binding mechanism by molecular dynamics simulations for 100 ns revealed the molecules were able to occupy the binding sites and stabilise the protease complexes. Binding affinities calculated using the MM/PBSA method for the last 20 ns showed that the virtual hits have comparable binding affinities to other known inhibitors from literature suggesting both molecules as promising scaffolds with dual cruzain and rhodesain inhibition properties, i.e. 12 has predicted ΔGbind values of - 38.1 and - 38.2 kcal/mol to cruzain and rhodesain, respectively, and 15 has predicted ΔGbind values of - 34.4 and - 25.8 kcal/mol to rhodesain. Per residue binding free energy decomposition studies and visual inspection at 100 ns snapshots revealed hydrogen bonding and non-polar attractions with important amino acid residues that contributed to the ΔGbind values. The interactions are similar to those previously reported in the literature. The overall ADMET predictions for the two molecules were favourable for drug development with acceptable pharmacokinetic profiles and adequate oral bioavailability.

Activation of cryptic xylose metabolism by a transcriptional activator Znf1 boosts up xylitol production in the engineered Saccharomyces cerevisiae lacking xylose suppressor BUD21 gene.

BACKGROUND: Xylitol is a valuable pentose sugar alcohol, used in the food and pharmaceutical industries. Biotechnological xylitol production is currently attractive due to possible conversion from abundant and low-cost industrial wastes or agricultural lignocellulosic biomass. In this study, the transcription factor Znf1 was characterised as being responsible for the activation of cryptic xylose metabolism in a poor xylose-assimilating S. cerevisiae for xylitol production. RESULTS: The results suggest that the expression of several xylose-utilising enzyme genes, encoding xylose reductases for the reduction of xylose to xylitol was derepressed by xylose. Their expression and those of a pentose phosphate shunt and related pathways required for xylose utilisation were strongly activated by the transcription factor Znf1. Using an engineered S. cerevisiae strain overexpressing ZNF1 in the absence of the xylose suppressor bud21Δ, xylitol production was maximally by approximately 1200% to 12.14 g/L of xylitol, corresponding to 0.23 g/g xylose consumed, during 10% (w/v) xylose fermentation. Proteomic analysis supported the role of Znf1 and Bud21 in modulating levels of proteins associated with carbon metabolism, xylose utilisation, ribosomal protein synthesis, and others. Increased tolerance to lignocellulosic inhibitors and improved cell dry weight were also observed in this engineered bud21∆ + pLJ529-ZNF1 strain. A similar xylitol yield was achieved using fungus-pretreated rice straw hydrolysate as an eco-friendly and low-cost substrate. CONCLUSIONS: Thus, we identified the key modulators of pentose sugar metabolism, namely the transcription factor Znf1 and the suppressor Bud21, for enhanced xylose utilisation, providing a potential application of a generally recognised as safe yeast in supporting the sugar industry and the sustainable lignocellulose-based bioeconomy.

Identification of new 3-phenyl-1H-indole-2-carbohydrazide derivatives and their structure-activity relationships as potent tubulin inhibitors and anticancer agents: A combined in silico, in vitro and synthetic study.

Virtual screening of commercially available molecular entities by using CDRUG, structure-based virtual screening, and similarity identified eight new derivatives of 3-phenyl-1H-indole-2-carbohydrazide with anti-proliferative activities. The molecules were tested experimentally for inhibition of tubulin polymerisation, which revealed furan-3-ylmethylene-3-phenyl-1H-indole-2-carbohydrazide (27a) as the most potent candidate. Molecule 27a was able to induce G2/M phase arrest in A549 cell line, similar to other tubulin inhibitors. Synthetic modifications of 27a were focussed on small substitutions on the furan ring, halogenation at R1 position and alteration of furyl connectivity. Derivatives 27b, 27d and 27i exhibited the strongest tubulin inhibition activities and were comparable to 27a. Bromine substitution at R1 position showed most prominent anticancer activities; derivatives 27b-27d displayed the strongest activities against HuCCA-1 cell line and were more potent than doxorubicin and the parent molecule 27a with IC50 values <0.5 µM. Notably, 27b with a 5-methoxy substitution on furan displayed the strongest activity against HepG2 cell line (IC50 = 0.34 µM), while 27d displayed stronger activity against A549 cell line (IC50 = 0.43 µM) compared to doxorubicin and 27a. Fluorine substitutions at the R1 position tended to show more modest anti-tubulin and anticancer activities, and change of 2-furyl to 3-furyl was tolerable. The new derivatives, thiophenyl 26, displayed the strongest activity against A549 cell line (IC50 = 0.19 µM), while 1-phenylethylidene 21b and 21c exhibited more modest anticancer activities with unclear mechanisms of action; 26 and 21c demonstrated G2/M phase arrest, but showed weak tubulin inhibitory properties. Molecular docking suggests the series inhibit tubulin at the colchicine site, in agreement with the experimental findings. The calculated molecular descriptors indicated that the molecules obey Lipinski's rule which suggests the molecules are drug-like structures.

The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A NOVEL MECHANISM CAUSING FAMILIAL HYPERCHOLESTEROLEMIA.

Familial hypercholesterolemia (FH) is characterized by severely elevated low density lipoprotein (LDL) cholesterol. Herein, we identified an FH patient presenting novel compound heterozygote mutations R410S and G592E of the LDL receptor (LDLR). The patient responded modestly to maximum rosuvastatin plus ezetimibe therapy, even in combination with a PCSK9 monoclonal antibody injection. Using cell biology and molecular dynamics simulations, we aimed to define the underlying mechanism(s) by which these LDLR mutations affect LDL metabolism and lead to hypercholesterolemia. Our data showed that the LDLR-G592E is a class 2b mutant, because it mostly failed to exit the endoplasmic reticulum and was degraded. Even though LDLR-R410S and LDLR-WT were similar in levels of cell surface and total receptor and bound equally well to LDL or extracellular PCSK9, the LDLR-R410S was resistant to exogenous PCSK9-mediated degradation in endosomes/lysosomes and showed reduced LDL internalization and degradation relative to LDLR-WT. Evidence is provided for a tighter association of LDL with LDLR-R410S at acidic pH, a reduced LDL delivery to late endosomes/lysosomes, and an increased release in the medium of the bound/internalized LDL, as compared with LDLR-WT. These data suggested that LDLR-R410S recycles loaded with its LDL-cargo. Our findings demonstrate that LDLR-R410S represents an LDLR loss-of-function through a novel class 8 FH-causing mechanism, thereby rationalizing the observed phenotype.

Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other.

Amyloid precursor-like protein 2 (APLP2) and sortilin were reported to individually bind the proprotein convertase subtilisin/kexin type 9 (PCSK9) and regulate its activity on the low-density lipoprotein receptor (LDLR). The data presented herein demonstrate that mRNA knockdowns of APLP2, sortilin, or both in the human hepatocyte cell lines HepG2 and Huh7 do not affect the ability of extracellular PCSK9 to enhance the degradation of the LDLR. Furthermore, mice deficient in APLP2 or sortilin do not exhibit significant changes in liver LDLR or plasma total cholesterol levels. Moreover, cellular overexpression of one or both proteins does not alter PCSK9 secretion, or its activity on the LDLR. We conclude that PCSK9 enhances the degradation of the LDLR independently of either APLP2 or sortilin both ex vivo and in mice. Interestingly, when co-expressed with PCSK9, both APLP2 and sortilin were targeted for lysosomal degradation. Using chemiluminescence proximity and co-immunoprecipitation assays, as well as biosynthetic analysis, we discovered that sortilin binds and stabilizes APLP2, and hence could regulate its intracellular functions on other targets.

A regulator of secretory vesicle size, Kelch-like protein 12, facilitates the secretion of apolipoprotein B100 and very-low-density lipoproteins--brief report.

OBJECTIVE: One of the major risk factors for atherosclerosis is the plasma level of low-density lipoprotein (LDL), which is a product of very-low-density lipoprotein (VLDL). Hepatic apolipoprotein B100 (apoB100) is the essential component that provides structural stability to VLDL particles. Newly translated apoB100 is partially lipidated in the endoplasmic reticulum (ER), forming nascent apoB100-VLDL particles. These particles are further modified to form fully mature VLDLs in the Golgi apparatus. Therefore, the transport of nascent VLDL from the ER to the Golgi represents a critical step during VLDL maturation and secretion and in regulating serum LDL cholesterol levels. Our previous studies showed that apoB100 exits the ER in coat complex II vesicles (COPII), but the cohort of related factors that control trafficking is poorly defined. APPROACH AND RESULTS: Expression levels of Kelch-like protein 12 (KLHL12), an adaptor protein known to assist COPII-dependent transport of procollagen, were manipulated by using a KLHL12-specific small interfering RNA and a KLHL12 expression plasmid in the rat hepatoma cell line, McArdle RH7777. KLHL12 knockdown decreased the secreted and intracellular pools of apoB100, an effect that was attenuated in the presence of an autophagy inhibitor. KLHL12 knockdown also significantly reduced secretion of the most lipidated apoB100-VLDL species and led to the accumulation of apoB100 in the ER. Consistent with these data, KLHL12 overexpression increased apoB100 recovery and apoB100-VLDL secretion. Images obtained from confocal microscopy revealed colocalization of apoB100 and KLHL12, further supporting a direct link between KLHL12 function and VLDL trafficking from the ER. CONCLUSIONS: KLHL12 plays a critical role in facilitating the ER exit and secretion of apoB100-VLDL particles, suggesting that KLHL12 modulation would influence plasma lipid levels.

Comparative Proteomic Analysis of Ridge Gourd Seed (Luffa acutangula (L.) Roxb.) during Artificial Aging.

Seed aging is a complicated process influenced by environmental conditions, impacting biochemical processes in seeds and causing deterioration that results in reduced viability and vigor. In this study, we investigated the seed aging process of ridge gourd, which is one of the most exported commercial seeds in Thailand using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 855 proteins were identified among the two groups (0 d/15 d and 0 d/30 d). The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed proteins revealed that in ridge gourd seeds, the aging process altered the abundance of proteins related to the oxidative stress response, nutrient reservoir, and metabolism pathway. The most identified DEPs were mitochondrial proteins, ubiquitin-proteasome system proteins, ribosomal proteins, carbohydrate metabolism-related proteins, and stress response-related proteins. This study also presented the involvement of aconitase and glutathione pathway-associated enzymes in seed aging, with aconitase and total glutathione being determined as possible suggestive biomarkers for aged ridge gourd seeds. This acquired knowledge has the potential to considerably improve growing methods and seed preservation techniques, enhancing seed storage and maintenance.

Protective effect of Anoectochilus burmannicus extracts and its active compound, kinsenoside on adipocyte differentiation induced by benzyl butyl phthalate and bisphenol A.

Benzyl butyl phthalate (BBP) and bisphenol-A (BPA) are obesogens that have been reported to be associated with obesity. Inhibition of their adipogenic activity could decrease the risk of obesity-related metabolic disorders. This study hypothesized that Anoectochilus burmannicus ethanolic extract (ABE) which has been previously reported its anti-inflammation property and its known active compound, kinsenoside (Kin) abrogate BBP- and BPA-induced adipogenesis. ABE and Kin markedly suppress both BBP- and BPA-stimulated adipogenesis with different modulations on adipogenic-gene expression including C/EBPα, PPARγ, adiponectin, and leptin in 3T3-L1. BBP induced C/EBPα, adiponectin, and leptin mRNA expressions and slightly increased PPARγ mRNA level, whereas BPA markedly induced PPARγ and adiponectin mRNA levels. ABE significantly decreased the expression of C/EBPα and leptin, but not PPARγ and adiponectin in the BBP-treated cells. In the BPA-treated cells, ABE significantly decreased the mRNA expression of C/EBPα and PPARγ, but not adiponectin and leptin. Interestingly, Kin significantly overcame BBP- and BPA-induced C/EBPα, PPARγ, adiponectin, and leptin expressions. This study first provides evidence to support the health benefits of this plant, especially for people exposed to obesogens. Besides, this finding would encourage the conservation and culture of this orchid for development as an economic plant and healthy food.

The Toxicological Assessment of Anoectochilus burmannicus Ethanolic-Extract-Synthesized Selenium Nanoparticles Using Cell Culture, Bacteria, and Drosophila melanogaster as Suitable Models.

Selenium nanoparticles (SeNPs) are worthy of attention and development for nutritional supplementation due to their health benefits in both animals and humans with low toxicity, improved bioavailability, and controlled release, being greater than the Se inorganic and organic forms. Our previous study reported that Anoectochilus burmannicus extract (ABE)-synthesized SeNPs (ABE-SeNPs) exerted antioxidant and anti-inflammatory activities. Furthermore, ABE could stabilize and preserve the biological activities of SeNPs. To promote the ABE-SeNPs as supplementary and functional foods, it was necessary to carry out a safety assessment. Cytotoxicity testing showed that SeNPs and ABE-SeNPs were harmless with no killing effect on Caco2 (intestinal epithelial cells), MRC-5 (lung fibroblasts), HEK293 (kidney cells), LX-2 (hepatic stellate cells), and 3T3-L1 (adipocytes), and were not toxic to isolated human PBMCs and RBCs. Genotoxicity assessments found that SeNPs and ABE-SeNPs did not induce mutations in Salmonella typhimurium TA98 and TA100 (Ames test) as well as in Drosophila melanogaster (somatic mutation and recombination test). Noticeably, ABE-SeNPs inhibited mutation in TA98 and TA100 induced by AF-2, and in Drosophila induced by urethane, ethyl methanesulfonate, and mitomycin c, suggesting their anti-mutagenicity ability. This study provides data that support the safety and anti-genotoxicity properties of ABE-SeNPs for the further development of SeNPs-based food supplements.

Comparative study on structural, biological and functional activities of hydrolysates from Adzuki bean (Vigna angularis) and mung bean (Vigna radiata) protein concentrates using Alcalase and Flavourzyme.

The physicochemical features of mung bean protein (MBP) and adzuki bean protein (ABP) hydrolysates derived from Alcalase (MBPHA, ABPHA) and Flavourzyme (MBPHF, ABPHF) were assessed using FTIR, hydrophobicity, emulsion activity, zeta potential, and health-promoting activities. The results proved that the choice of peptidase and substrate both have a significant effect on the hydrolysates in different physicochemical, structural and functional properties. Size exclusion-HPLC was used to fractionate the MBP and ABP hydrolysates. The results demonstrated that Alcalase hydrolysates included smaller peptides than Flavourzyme hydrolysates, and the chromatogram patterns of the two peptidases were similar. The peptides with the most potent antioxidant and ACE-inhibitory properties were identified using MALDI-TOF-MS. The fraction (F4) of MBPHA exhibited the highest levels of metal chelating activity. The Flavourzyme hydrolysates fraction (F2) and the ABPHA fraction (F2) showed the highest ABTS radical scavenging activity and ACE-inhibitory activity, respectively. Pro-Pro was identified in peptide sequences with ABTS radical scavenging activity as an active component while Pro-Gln was identified in peptide sequences with ACE-inhibitory activity. As a result, Pro-Pro and Pro-Gln, respectively, are likely-one of the characteristics of antioxidant and ACE-inhibitory peptides from MBP and ABP. Compared to mung bean and adzuki bean protein as substrate, Alcalase and Flavourzyme as peptidases significant impacted the development of distinct functionalities and biological activities.

O-linked beta-N-acetylglucosamine (O-GlcNAc): Extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress.

BACKGROUND: Since its discovery in the early 1980s, O-linked-beta-N-acetylglucosamine (O-GlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc, and beta-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of O-GlcNAc's functions has grown rapidly. SCOPE OF REVIEW: In this review, the functions of GlcNAcylation in regulating cellular processes, its extensive crosstalk with protein phosphorylation, and regulation of OGT and O-GlcNAcase will be explored. MAJOR CONCLUSIONS: GlcNAcylation rivals phosphorylation in terms of its abundance, protein distribution and its cycling on and off of proteins. GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling, transcription and the cytoskeleton in response to nutrients and stress. GENERAL SIGNIFICANCE: Abnormal crosstalk between GlcNAcylation and phosphorylation underlies dysregulation in diabetes, including glucose toxicity, and defective GlcNAcylation is involved in neurodegenerative disease and cancer and most recently in AIDS.

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis.

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.