Oral Limonite Supplement Ameliorates Glucose Intolerance in Diabetic and Obese Mice.

INTRODUCTION: Diabetes mellitus is a serious threat to public health worldwide. It causes a substantial economic burden, mental and physical disabilities, poor quality of life, and high mortality. Limonite is formed when iron-rich materials from the underground emerge and oxidized on the ground surface. It is currently used to purify contaminated water, absorption of irritant gases, and improve livestock breeding. Limonite can change the composition of environmental microbial communities. In the present study, we evaluated whether limonite can ameliorate glucose metabolism abnormalities by remodeling the gut microbiome. METHODS: The investigation was performed using mouse models of streptozotocin-induced diabetes mellitus and high-calorie diet-induced metabolic syndrome. RESULTS: Oral limonite supplement was associated with significant body weight recovery, reduced glycemia with improved insulin secretion, increased number of regulatory T cells, and abundant beneficial gut microbial populations in mice with diabetes mellitus compared to control. Similarly, mice with obesity fed with limonite supplements had significantly reduced body weight, insulin resistance, steatohepatitis, and systemic inflammatory response with significant gut microbiome remodeling. CONCLUSION: This study demonstrates that limonite supplement ameliorates abnormal glucose metabolism in diabetes mellitus and obesity. Gut microbiome remodeling, inhibition of inflammatory cytokines, and the host immune response regulation may explain the limonite's beneficial activity under pathological conditions in vivo.

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe.

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.

Processing and maturation of carboxypeptidase Y and alkaline phosphatase in Schizosaccharomyces pombe.

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.

A-mating-type gene expression can drive clamp formation in the bipolar mushroom Pholiota microspora (Pholiota nameko).

In the bipolar basidiomycete Pholiota microspora, a pair of homeodomain protein genes located at the A-mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. microspora to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3-hox1 or A3-hox2) from the A3 monokaryon strain was transformed into the A4 monokaryon strain, the transformants produced many pseudoclamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamplike cells in the transformants was significantly increased to ca. 50%. We therefore concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12-163 x NGW19-6). The results of real-time reverse transcription-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. microspora. Thus, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A-mating-type genes alone is sufficient to drive true clamp formation.

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomyces pombe.

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids.

Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

Although the fission yeast Schizosaccharomyces pombe has been used for high-level heterologous protein production, the productivity of secreted human serum transferrin (hTF) has been low, presumably, because the protein harbors twenty disulfide bonds and two N-glycosylation sites. In the present study, we found that overexpression of endogenous putative protein disulfide isomerase (PDI) improved productivity. Whole genome sequence analysis of S. pombe revealed five putative PDI genes and overexpression of two of them, SPAC17H9.14c and SPBC3D6.13c (SpPdi2p or SpPdi3p, respectively), significantly improved the productivity of secreted hTF. GFP-fused SpPdi2p and SpPdi3p were found to localize to the endoplasmic reticulum. Co-overexpression of SpPdi2p or SpPdi3p with hTF coupled with modifications to the growth medium reported in our previous study were able to increase the level of secreted hTF approximately 30-fold relative to conventional conditions.

Autophagy in the fission yeast Schizosaccharomyces pombe.

Autophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast.

Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation.

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome-vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1Delta and atg12Delta mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.

Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system.

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well-characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high-level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein-expressing host systems are still required for the production of heterologous proteins involved in post-translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission-yeast strains.

Genomic structure of the A mating-type locus in a bipolar basidiomycete, Pholiota nameko.

In the bipolar basidiomycete, Pholiota nameko, the homeodomain protein, A4-hox1, located at the A mating-type locus, is known to regulate mating compatibility. In the present study, we investigated the genomic structure of the P. nameko A mating-type locus and its flanking region. A second homeodomain gene (A4-hox2) was discovered upstream of A4-hox1; this together with the conserved gene order around the A mating-type locus and their similar transcription direction were found in P. nameko, another bipolar mushroom, Coprinellus disseminatus, and two tetrapolar mushrooms, Coprinopsis cinerea and Laccaria bicolor. Analysis of the deduced protein sequences of the homeodomain protein genes from two strains of P. nameko show that the putative functional domains differ from those of the homeodomain proteins of the tetrapolar mushrooms, C. cinerea and L. bicolor.

A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate.

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure.

Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae. We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole. Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system. We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation.

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain.

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.

Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica.

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.

Paz2 and 13 other PAZ gene products regulate vacuolar engulfment of peroxisomes during micropexophagy.

BACKGROUND: In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded through direct engulfment by the vacuole in a process known as micropexophagy, but the mechanism of micropexophagy is not known. RESULTS: To gain molecular insights into micropexophagy, we used fluorescence time-lapse microscopy, coupled with gene-tagging mutagenesis to isolate P. pastoris mutants defective in micropexophagy. The relevant genes have been designated PAZ genes. Morphological and genetic analyses enabled us to postulate a schematic model for micropexophagy. This new model invokes the generation of new vacuolar compartments as an intermediate structure during micropexophagy. Different classes of paz mutants arrest micropexophagy at distinct stages of the process. Most of APG-related paz mutants ceased micropexophagy at Stage 1c and that GCN-family paz mutants ceased micropexophagy at Stage 2. The paz2Delta strain shows a unique phenotype. Paz2 is the homologue of Saccharomyces cerevisiae Apg8, which is necessary for macroautophagy in that yeast. Our analysis revealed that in P. pastoris, Paz2 plays a key role in repressing the engulfment of peroxisomes by the vacuole before the onset of micropexophagy. Paz2 is proteolytically processed by another autophagy-related Paz protein Paz8, but this processing is not required for the ability of Paz2 to suppress aberrant micropexophagy. CONCLUSION: Micropexophagy has been dissected into a multistep reaction that involves 14 identified Paz gene products. Our studies indicate that Paz2 controls the engulfment of peroxisomes by the vacuole, pointing to a novel early function of this protein.