Growth differentiation factor 15 (GDF15; also known as MIC-1) is a divergent member of the TGF-β superfamily and is associated with body-weight regulation in humans and rodents. However, the cognate ...receptor of GDF15 is unknown. Here we show that GDF15 binds specifically to GDNF family receptor α-like (GFRAL) with high affinity, and that GFRAL requires association with the coreceptor RET to elicit intracellular signaling in response to GDF15 stimulation. We also found that GDF15-mediated reductions in food intake and body weight of mice with obesity were abolished in GFRAL-knockout mice. We further found that GFRAL expression was limited to hindbrain neurons and not present in peripheral tissues, which suggests that GDF15-GFRAL-mediated regulation of food intake is by a central mechanism. Lastly, given that GDF15 did not increase energy expenditure in treated mice with obesity, the anti-obesity actions of the cytokine are likely driven primarily by a reduction in food intake.
With the solar power panels as integrated surface elements in new building constructions, solar power will be economic attractive in urban areas, close to the power consumption. In the new dense ...urban areas, the rooftop areas will be attractive for other purposes, like terraces and green roofs. The solar power panels may instead be placed at the facades of the buildings – this is especially relevant at high geographic latitudes, where the altitude of the sun is low. It may even make sense to mount panels at all facades, better distributing the generation over the day.
The energy produced by bifacial photovoltaic (PV) arrays can be augmented via albedo enhancements. However, the value of the additional energy must outweigh the costs for such modifications to be ...economically viable. In this work, the electrical performance and economic value of six 13 kWp crystalline-silicon (c-Si) PV arrays with distinct configurations are evaluated. The system designs include horizontal single axis trackers (HSAT) and 25° fixed-tilt structures, monofacial and bifacial PV panels, and low and high ground albedo. The value of the system designs is assessed using onsite electrical measurements and spot prices from the Nord Pool electricity market. We find that HSAT systems increase the annual value factor (VF) by 4% and decrease levelized cost of energy (LCOE) by 3.5 EUR/MWh relative to fixed-tilt systems. The use of bifacial panels can increase the VF by 1% and decrease LCOE by 4.0 EUR/MWh. However, a negligible VF increase and modest LCOE decrease was found in systems with bifacial panels and ground albedo enhancements. Although our results show that albedo enhancements result in lower LCOE than designs without, the uncertainty in upfront and ongoing costs of altering the ground in utility-scale PV parks makes the solution presently unadvisable.
Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of ...disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid, DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4′-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 μg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.
The use of auxotrophic
Saccharomyces cerevisiae
strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the ...metabolic pathways for amino acid (
LEU2
) and pyrimidine (
URA3
) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers
LEU2
d and
URA3
d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the
LEU2
and
URA3
promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the
URA3
promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the
LEU2
promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the
URA3
d-based expression systems showed a high potential for industrial protein production and for further academic studies.
We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T ...conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel β-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel β-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.
Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during ...the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.
PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, ...and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.
Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during ...the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.
Battery energy storage systems (BESS) have started to be part of the photovoltaic (PV) system design to allow the further penetration of PV into the grid. This study deals with the sizing (power and ...energy capacity) of a BESS for residential households which are represented by a typical load consumption profile, they have electrical air conditioning and electrical water heating (water boiler) resembling a common residential house in Cyprus. The BESS sizing is done considering the local energy consumption profile and the energy efficiency class of the house and the water boiler. The BESS is sized in order to achieve zero PV grid export and to increase self-consumption.
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