Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.     

Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.     

Elevated concentrations of naturally occurring heavy metals inversely correlate with reproductive output and body mass of the Kagu Rhynochetos jubatus

To assess the effects of naturally occurring heavy metals on wild birds, we compared reproductive success and heavy metal contents in feathers of Kagu Rhynochetos jubatus living on ultramafic (rich in heavy metals) soil with those of Kagu living on non‐ultramafic soil. From 2003 to 2016, we monitored breeding of 19 Kagu families by radiotracking and video‐monitoring, and collected rump down feathers from 69 wild Kagu. The metal concentrations in Kagu feathers correlated with the concentrations in the soil. The mean numbers of eggs laid and fledglings per year of Kagu families on non‐ultramafic soil were about four times higher, and home‐ranges three times smaller, than those of Kagu on ultramafic soil. Mass of eggs and the proportion of eggs that developed to fledglings were similar in the two areas, whereas the mass of adult Kagu on non‐ultramafic soil was nearly 10% higher than that of adult Kagu living on ultramafic soil. The impact of naturally occurring heavy metals on Kagu breeding productivity and body mass appears to act through their effects on food supply rather than being caused directly by metal toxicity. The results imply that conservation of Kagu might be more effective in non‐ultramafic areas, as populations can recover much faster on these soils and Kagu can then recolonize and bolster populations in ultramafic areas.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Akt is a neutral amplifier for Th cell differentiation

Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.     

Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.     

Arabidopsis RPT2a encoding the 26S proteasome subunit is required for various aspects of root meristem maintenance, and regulates gametogenesis redundantly with its homolog, RPT2b

The 26S proteasome plays fundamental roles in the degradation of short-lived regulatory proteins, thereby controlling diverse cellular processes. In Arabidopsis, the essential RPT2 subunit is encoded by two highly homologous genes: RPT2a and RPT2b. Currently, only RPT2a has been reported to regulate various developmental processes, including the maintenance of the root apical meristem (RAM), although the roles of RPT2a in the RAM are still obscure. Here, we analyzed the cell type-specific requirement for RPT2a. When RPT2a was expressed locally in the rpt2a mutant, pleiotropic defects in the RAM, such as cell death and distorted cellular organization, were rescued differently, suggesting that RPT2a regulates various specific activities, which converge to maintain the RAM. On the other hand, the homologous RPT2b was also expressed in meristems, and the expression of RPT2b protein under the control of the RPT2a promoter complemented the rpt2a RAM defects, although the rpt2b mutant showed no obvious defect in all developmental aspects we examined. These results show that RPT2b might work in the RAM, but is dispensable for RAM maintenance in the presence of RPT2a. In contrast, the rpt2a rpt2b double mutant was lethal in male and female gametophytes, suggesting that RPT2a and RPT2b are redundantly required for gametogenesis. Furthermore, we showed that similar meristematic and gametophytic defects were caused by mutations in other subunit genes, RPT5a and RPT5b, suggesting that proper activity of the proteasome, not an RPT2-specific function, is required. Taken together, our results suggest that RPT2a and RPT2b contribute differently to the proteasome activity required for each developmental context.     

Development of an ELISA system for tick-borne encephalitis virus infection in rodents

Tick-borne encephalitis (TBE) virus causes severe encephalitis with serious sequelae in humans. An epizootiological survey of wild rodents is effective to detect TBE virus-endemic areas; however, limited serological diagnostic methods are available to detect anti-TBE virus antibodies in wild rodents. In this study, ELISAs for the detection of rodent antibodies against the TBE virus were developed using two recombinant proteins, domain III of the E protein (EdIII) and subviral particles (SPs), as the antigens. As compared with the neutralization test, the ELISA using EdIII had 77.1% sensitivity and 80.0% specificity, and the ELISA using SPs had 91.4% sensitivity and 100% specificity. Furthermore, when the ELISAs were applied to the epizootiological survey in the TBE virus-endemic area, both of the ELISAs was able to detect wild rodents with TBE virus-specific antibodies. This is the first study to show that ELISAs using recombinant antigens can be safe and useful in the detection of TBE virus-infected wild rodents in epizootiological research.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.     

The Impact of Model Building on the Transmission Dynamics under Vaccination: Observable (Symptom-Based) versus Unobservable (Contagiousness-Dependent) Approaches

Background

The way we formulate a mathematical model of an infectious disease to capture symptomatic and asymptomatic transmission can greatly influence the likely effectiveness of vaccination in the presence of vaccine effect for preventing clinical illness. The present study aims to assess the impact of model building strategy on the epidemic threshold under vaccination.

Methodology/Principal Findings

We consider two different types of mathematical models, one based on observable variables including symptom onset and recovery from clinical illness (hereafter, the “observable model”) and the other based on unobservable information of infection event and infectiousness (the “unobservable model”). By imposing a number of modifying assumptions to the observable model, we let it mimic the unobservable model, identifying that the two models are fully consistent only when the incubation period is identical to the latent period and when there is no pre-symptomatic transmission. We also computed the reproduction numbers with and without vaccination, demonstrating that the data generating process of vaccine-induced reduction in symptomatic illness is consistent with the observable model only and examining how the effective reproduction number is differently calculated by two models.

Conclusions

To explicitly incorporate the vaccine effect in reducing the risk of symptomatic illness into the model, it is fruitful to employ a model that directly accounts for disease progression. More modeling studies based on observable epidemiological information are called for.