The cytosolic chaperone Hsc70 promotes traffic to the cell surface of intracellular retained melanocortin-4 receptor mutants

Inherited modifications in protein structure frequently cause a loss-of-function by interfering with protein synthesis, transport, or stability. For the obesity-linked melanocortin-4 receptor (MC4R) and other G protein-coupled receptors, many mutants are intracellular retained. The biogenesis and trafficking of G protein-coupled receptors are regulated by multiple factors, including molecular chaperone networks. Here, we have investigated the ability of the cytosolic cognate 70-kDa heat-shock protein (Hsc70) chaperone system to modulate cell surface expression of MC4R. Clinically occurring MC4R mutants S58C, P78L, and D90N were demonstrated to have reduced trafficking to the plasma membrane and to be retained at the endoplasmic reticulum (ER). Analyses by fluorescence recovery after photobleaching revealed that the mobility of MC4R mutant protein at the ER was reduced, implying protein misfolding. In cells expressing MC4R, overexpression of Hsc70 resulted in increased levels of wild-type and mutant receptors at the cell surface. MC4R and Hsc70 coimmunoprecipitated, and fluorescence recovery after photobleaching analyses showed that increasing cellular levels of Hsc70 promoted the mobility of ER retained MC4R. Moreover, expression of HSJ1b, a cochaperone that enhances degradation of Hsc70 clients, reduced cellular levels of MC4R. Hsp70 and Hsp90 chaperone systems collaborate in the cellular processing of clients. For MC4R, inhibition of endogenous Hsp90 by geldanamycin reduced receptor levels. By contrast, expression of the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase) increased cellular levels of MC4R. Finally, we demonstrate that signaling of intracellular retained MC4R mutants is increased in cells overexpressing Hsc70. These data indicate that cytosolic chaperone systems can facilitate rescue of intracellular retained MC4R by improving folding. They also support proteostasis networks as a potential target for MC4R-linked obesity.     

Accuracies of genomic breeding values in American Angus beef cattle using K-means clustering for cross-validation

Background

Genomic selection is a recently developed technology that is beginning to revolutionize animal breeding. The objective of this study was to estimate marker effects to derive prediction equations for direct genomic values for 16 routinely recorded traits of American Angus beef cattle and quantify corresponding accuracies of prediction.

Methods

Deregressed estimated breeding values were used as observations in a weighted analysis to derive direct genomic values for 3570 sires genotyped using the Illumina BovineSNP50 BeadChip. These bulls were clustered into five groups using K-means clustering on pedigree estimates of additive genetic relationships between animals, with the aim of increasing within-group and decreasing between-group relationships. All five combinations of four groups were used for model training, with cross-validation performed in the group not used in training. Bivariate animal models were used for each trait to estimate the genetic correlation between deregressed estimated breeding values and direct genomic values.

Results

Accuracies of direct genomic values ranged from 0.22 to 0.69 for the studied traits, with an average of 0.44. Predictions were more accurate when animals within the validation group were more closely related to animals in the training set. When training and validation sets were formed by random allocation, the accuracies of direct genomic values ranged from 0.38 to 0.85, with an average of 0.65, reflecting the greater relationship between animals in training and validation. The accuracies of direct genomic values obtained from training on older animals and validating in younger animals were intermediate to the accuracies obtained from K-means clustering and random clustering for most traits. The genetic correlation between deregressed estimated breeding values and direct genomic values ranged from 0.15 to 0.80 for the traits studied.

Conclusions

These results suggest that genomic estimates of genetic merit can be produced in beef cattle at a young age but the recurrent inclusion of genotyped sires in retraining analyses will be necessary to routinely produce for the industry the direct genomic values with the highest accuracy.     

The hyper-fluorescent trichome phenotype of the brt1 mutant of Arabidopsis is the result of a defect in a sinapic acid: UDPG glucosyltransferase

Sinapoylmalate is a major phenylpropanoid that is accumulated in Arabidopsis. Its presence causes the adaxial surface of leaves to fluoresce blue under UV light, and mutations that lead to lower levels of sinapoylmalate decrease UV-induced leaf fluorescence. The Arabidopsis bright trichomes 1 (brt1) mutant was first identified in a screen for mutants that exhibit a reduced epidermal fluorescence phenotype; however, subsequent examination of the mutant revealed that its trichomes are hyper-fluorescent. The results from genetic mapping and complementation analyses showed that BRT1 (At3g21560) encodes UGT84A2, a glucosyltransferase previously shown to be capable of using sinapic acid as a substrate. Residual levels of sinapoylmalate and sinapic acid:UDP-glucose glucosyltransferase activity in brt1 leaves suggest that BRT1 is one member of a family of partially redundant glycosyltransferases that function in Arabidopsis sinapate ester biosynthesis. RT-PCR analysis showed that BRT1 is expressed through all stages of plant life cycle, a result consistent with the impact of the brt1 mutation on both leaf sinapoylmalate levels and seed sinapoylcholine content. Finally, the compound accumulated in brt1 trichomes was identified as a sinapic acid-derived polyketide, indicating that when sinapic acid glycosylation is reduced, a portion of it is instead activated to its CoA thioester, which then serves as a substrate for chalcone synthase.     

Chemically Induced Conditional Rescue of the Reduced Epidermal Fluorescence8 Mutant of Arabidopsis Reveals Rapid Restoration of Growth and Selective Turnover of Secondary Metabolite Pools

The phenylpropanoid pathway is responsible for the biosynthesis of diverse and important secondary metabolites including lignin and flavonoids. The reduced epidermal fluorescence8 (ref8) mutant of Arabidopsis (Arabidopsis thaliana), which is defective in a lignin biosynthetic enzyme p-coumaroyl shikimate 3′-hydroxylase (C3′H), exhibits severe dwarfism and sterility. To better understand the impact of perturbation of phenylpropanoid metabolism on plant growth, we generated a chemically inducible C3′H expression construct and transformed it into the ref8 mutant. Application of dexamethasone to these plants greatly alleviates the dwarfism and sterility and substantially reverses the biochemical phenotypes of ref8 plants, including the reduction of lignin content and hyperaccumulation of flavonoids and p-coumarate esters. Induction of C3′H expression at different developmental stages has distinct impacts on plant growth. Although early induction effectively restored the elongation of primary inflorescence stem, application to 7-week-old plants enabled them to produce new rosette inflorescence stems. Examination of hypocotyls of these plants revealed normal vasculature in the newly formed secondary xylem, presumably restoring water transport in the mutant. The ref8 mutant accumulates higher levels of salicylic acid than the wild type, but depletion of this compound in ref8 did not relieve the mutant’s growth defects, suggesting that the hyperaccumulation of salicylic acid is unlikely to be responsible for dwarfism in this mutant.Phenylpropanoids including flavonoids, hydroxycinnamate esters, and lignin have been shown to play important roles in many aspects of plant growth and development. Flavonoids are important for flower pigmentation and pollen viability in some species (Coe et al., 1981; Mo et al., 1992; Taylor and Jorgensen, 1992; Mol et al., 1998), and sinapate esters, a class of hydroxycinnamate esters found in Arabidopsis (Arabidopsis thaliana) and related members of the Brassicaceae, are important UV protectants (Landry et al., 1995). Lignin is a major component of the plant cell wall, where it confers mechanical strength to plants, and is important for the vascular system to conduct long-distance water transport. Reducing lignin content or manipulating its composition is of great interest in an applied context because of the polymer’s negative impact on the utilization of cellulosic biomass for feed, paper manufacture, and biofuel production (Li et al., 2008).The lignin biosynthetic pathway has been largely elucidated during the last two decades (for review, see Bonawitz and Chapple, 2010; Vanholme et al., 2013). In Arabidopsis and other species, down-regulation or mutation of genes and enzymes early in the pathway leads to drastic lignin reduction and a concomitant inhibition of plant growth. For example, knocking out four Phe ammonia-lyase genes (PAL) in Arabidopsis decreases lignin content by 75% and results in stunted and sterile plants (Rohde et al., 2004; Huang et al., 2010). Arabidopsis reduced epidermal fluorescence3 (ref3) and ref8 mutants, which are defective in cinnamate 4-hydroxylase (C4H) and p-coumaroyl shikimate 3′-hydroxylase (C3′H), respectively, as well as RNA interference (RNAi) plants in which hydroxycinnamoyl-CoA shikimate:hydroxycinnamoyl transferase (HCT) was suppressed, also display severe growth defects and sterility (Franke et al., 2002b; Hoffmann et al., 2004; Abdulrazzak et al., 2006; Besseau et al., 2007; Schilmiller et al., 2009; Li et al., 2010). The association between lignin modification and plant growth reduction has also been reported in several other species, including poplar (Populus spp.), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa; Piquemal et al., 1998; Pinçon et al., 2001; O’Connell et al., 2002; Reddy et al., 2005; Leplé et al., 2007; Shadle et al., 2007). Despite its wide occurrence, it is not yet clear how the perturbation of phenylpropanoid metabolism influences plant growth and development (Bonawitz and Chapple, 2013). Considering the biological roles of lignin in providing mechanical strength and hydrophobicity in the vascular system, lignin deficiency may directly impact plant growth. Alternatively, various nonlignin phenylpropanoids are produced through the phenylpropanoid pathway, and deficiency or accumulation of those compounds may also contribute to the alteration of plant growth. For example, decreasing PAL activity by either suppressing PAL expression or applying PAL inhibitors resulted in reduced levels of salicylic acid (SA) and reduced systemic-acquired resistance to pathogens in tobacco and Arabidopsis (Mauch-Mani and Slusarenko, 1996; Pallas et al., 1996; Huang et al., 2010). Several Arabidopsis nonphenylpropanoid mutants containing increased SA content also display dwarfism (Bowling et al., 1994; Petersen et al., 2000; Li et al., 2001; Lee et al., 2007). These observations suggest a possible link between SA homeostasis and plant growth. A recent study showed that Arabidopsis plants with reduced HCT expression have elevated levels of SA and reducing the SA accumulation in these plants alleviated their dwarfism (Gallego-Giraldo et al., 2011). Some soluble phenylpropanoids such as dehydrodiconiferyl alcohol glycosides had been shown to have a cell division-promoting effect and therefore might also contribute to the growth defects of the plants in which the phenylpropanoid metabolism is perturbed (Binns et al., 1987; Lynn et al., 1987; Teutonico et al., 1991; Orr and Lynn, 1992).To better understand how phenylpropanoid metabolism impacts plant growth and to probe secondary metabolite synthesis and turnover, we investigated temporal changes in lignification, plant growth, and phenylpropanoid levels in the Arabidopsis ref8 mutant using a chemically inducible system. Here, we report that the ability of C3′H to restore growth of the ref8 mutant depends on when it is activated during the development of the plants. Our data also revealed selective turnover of different phenylpropanoid metabolite pools upon C3′H induction. Finally, unlike a recent report of the importance of SA in HCT-RNAi-induced dwarfing, our results suggest that the accumulation of SA is unlikely to be the cause for growth inhibition in ref8 plants.     

Impact of tail loss on the behaviour and locomotor performance of two sympatric Lampropholis skink species

Caudal autotomy is an anti-predator behaviour that is used by many lizard species. Although there is an immediate survival benefit, the subsequent absence of the tail may inhibit locomotor performance, alter activity and habitat use, and increase the individuals' susceptibility to future predation attempts. We used laboratory experiments to examine the impact of tail autotomy on locomotor performance, activity and basking site selection in two lizard species, the delicate skink (Lampropholis delicata) and garden skink (L. guichenoti), that occur sympatrically throughout southeastern Australia and are exposed to an identical suite of potential predators. Post-autotomy tail movement did not differ between the two Lampropholis species, although a positive relationship between the shed tail length and distance moved, but not the duration of movement, was observed. Tail autotomy resulted in a substantial decrease in sprint speed in both species (28-39%), although this impact was limited to the optimal performance temperature (30°C). Although L. delicata was more active than L. guichenoti, tail autotomy resulted in decreased activity in both species. Sheltered basking sites were preferred over open sites by both Lampropholis species, although this preference was stronger in L. delicata. Caudal autotomy did not alter the basking site preferences of either species. Thus, both Lampropholis species had similar behavioural responses to autotomy. Our study also indicates that the impact of tail loss on locomotor performance may be temperature-dependent and highlights that future studies should be conducted over a broad thermal range.     

Phylogenetic diversity of stress signalling pathways in fungi

Background  

Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts.     

An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

Background  

Tunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea.