Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.
Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor β-Klotho
MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor β-Klotho (βKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates βKL by binding to the 3′ UTR of βKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic βKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, βKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti–miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a βKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting βKL. The miR-34a/βKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.
Positive Selection on the Osteoarthritis-Risk and Decreased-Height Associated Variants at the GDF5 Gene in East Asians
GDF5 is a member of the bone morphogenetic protein (BMP) gene family, and plays an important role in the development of the skeletal system. Variants of the gene are associated with osteoarthritis and height in some human populations. Here, we resequenced the gene in individuals from four geographically separated human populations, and found that the evolution of the promoter region deviated from neutral expectations, with the sequence evolution driven by positive selection in the East Asian population, especially the haplotypes carrying the derived alleles of 5′ UTR SNPs rs143384 and rs143383. The derived alleles of rs143384 and rs143383, which are associated with a risk of osteoarthritis and decreased height, have high frequencies in non-Africans and show strong extended haplotype homozygosity and high population differentiation in East Asian. It is concluded that positive selection has driven the rapid evolution of the two osteoarthritis osteoarthritis-risk and decreased height associated variants of the human GDF5 gene, and supports the suggestion that the reduction in body size during the terminal Pleistocene and Holocene period might have been an adaptive process influenced by genetic factors.
A genome-wide association study of post-traumatic stress disorder identifies the retinoid-related orphan receptor alpha (RORA) gene as a significant risk locus.
We describe the results of the first genome-wide association study (GWAS) of post-traumatic stress disorder (PTSD) performed using trauma-exposed white non-Hispanic participants from a cohort of veterans and their intimate partners (295 cases and 196 controls). Several single-nucleotide polymorphisms (SNPs) yielded evidence of association. One SNP (rs8042149), located in the retinoid-related orphan receptor alpha gene (RORA), reached genome-wide significance. Nominally significant associations were observed for other RORA SNPs in two African-American replication samples—one from the veteran cohort (43 cases and 41 controls) and another independent cohort (100 cases and 421 controls). However, only the associated SNP from the veteran African-American replication sample survived gene-level multiple-testing correction. RORA has been implicated in prior GWAS studies of psychiatric disorders and is known to have an important role in neuroprotection and other behaviorally relevant processes. This study represents an important step toward identifying the genetic underpinnings of PTSD.
Dwarf And Low-Tillering Acts as a Direct Downstream Target of a GSK3/SHAGGY-Like Kinase to Mediate Brassinosteroid Responses in Rice
In Arabidopsis thaliana, the GSK3/SHAGGY-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) plays a critical role in the brassinosteroid (BR) signaling pathway by negatively regulating the activities of bri1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 family transcription factors that regulate the expression of downstream BR-responsive genes. In this study, we analyzed the function of a rice (Oryza sativa) GSK3/SHAGGY-like kinase (GSK2), which is one of the orthologs of BIN2. Overexpression of GSK2 (Go) led to plants with typical BR loss-of-function phenotypes, and suppression of GSK2 resulted in enhanced BR signaling phenotypes. DWARF AND LOW-TILLERING (DLT) is a positive regulator that mediates several BR responses in rice. Suppression of DLT can enhance the phenotypes of BR receptor mutant d61-1, and overexpression of DLT obviously suppressed the BR loss-of-function phenotypes of both d61-1 and Go, suggesting that DLT functions downstream of GSK2 to modulate BR responses. Indeed, GSK2 can interact with DLT and phosphorylate DLT. Moreover, brassinolide treatment can induce the dephosphorylation of DLT, leading to the accumulation of dephosphorylated DLT protein. In GSK2 transgenic plants, the DLT phosphorylation level is dictated by the GSK2 level. These results demonstrate that DLT is a GSK2 substrate, further reinforcing that the BIN2/GSK2 kinase has multiple substrates that carry out various BR responses.
Through the incorporation of the metal-chelating amino acid pyTyr into green fluorescent protein (GFP) from jellyfish, photoinduced electron transfer (ET) from the GFP chromophore to a bound Cu(II) ion was shown to occur within one nanosecond in a distance dependent manner. The crystal structure of GFP with pyTyr at a specific position shows the structural basis for the nanomolar binding affinity of pyTyr to Cu(II) ions.
The electron transfer (ET) involves many important biochemical processes in vivo, including photosynthesis. How to transform biological components to achieve efficient and controllable light-induced charge separation is an important problem and a major bottleneck means of synthetic biology to produce renewable energy. Meanwhile, the current experimental measurement of the electron transfer protein are usually rely connection probes to conduct a study on the protein itself contains a residue of histidine or cysteine, so that the method can only be used to study a small soluble protein, limits its application. Light-induced electron transfer (PET) The lead fluorescence quenching is a powerful tool used to explore the dynamic conformational changes of biological macromolecules. However, due to the limitations of current technology, only as an electron donor with a tyrosine or tryptophan.
The study will have a means of the metal chelating ability of non-natural amino acid via codon extended sentinel inserted into the green fluorescent protein (GFP), for the first time to achieve a rapid light-induced electron transfer between the fluorescent protein luminescent center to the copper ion, and measuring electron transfer occurs in a 1 nanosecond (near the center of the light velocity). The crystal structure reveals 3 - pyrazolyl tyrosine on the binding capacity of the copper ions have a high strength. These new methods of The protein dynamic conformational change research provides new means to provide new ideas for the use of synthetic biology means the production of renewable energy, provides a new tool for the design of metalloproteins. The paper also proposed jellyfish green fluorescent protein may be a new point of view of the jellyfish photoreceptors.
Princeton University, USA famous biophysical chemist Prof. Haw Yang council, said: "I believe that the non-natural amino acid coding technology researchers biophysics - the field of protein research will provide a very valuable tool in this article, is to promote one of the development in the field of research, because the use of copper as a quencher, can greatly expand the toolkit based on distance measurements. "
American University of Massachusetts famous bio-inorganic chemist Professor GUO Maolin, council said: "Understanding the biological electron transfer mechanism has been proved to be a challenging and complex scientific problems. Institute of Biophysics, Chinese Academy of Sciences, Dr. Wang and his colleagues developed a new strategy by the metal-chelating non-natural amino acids and encoded into proteins to study this complex issue. their success chelated Cu (II) groups in the green fluorescent protein (GFP) surface coding, 405nm light-induced light-induced electron transfer (PET) from protein chromophore of Cu (II) occurs rapidly, reducing the copper (I) and manufactured in a nanosecond. gene encoding strategy wonderful is that real-time monitoring to provide an opportunity for electron transfer proteins in living cells, which will be even more exciting! "
The Peking University famous biophysical chemist Professor Gao Yiqin council said: "light-induced electron transfer study of protein dynamics is a very useful tool, but its application is generally limited to a relatively simple system this work fine metal ion chelation together amino acids into proteins, which provide a new strategy for protein dynamics studies, this method is likely to significantly improve the applicability of PET in the study of protein dynamics. "
Advanced bone formation in mice with a dominant-negative mutation in the thyroid hormone receptor β gene due to activation of Wnt/β-catenin signaling
Thyroid hormone (T3) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance but the signaling pathways mediating these effects are poorly understood. ThrbPV/PV mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T3-receptor (TRβPV) that cannot bind T3 and interferes with the actions of wild-type TR.ThrbPV/PV mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and ThrbPV/PV mice. Activation of the canonical Wnt signaling in ThrbPV/PV mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during post-natal growth.By contrast, T3 treatment inhibited Wnt signaling in osteoblastic cells, suggesting T3 inhibits the Wnt pathway by facilitating proteasomal degradation of β-catenin and preventing its accumulation in the nucleus.Activation of the Wnt pathway in ThrbPV/PV mice, however, results from a gain-of-function for TRβPV that stabilizes β-catenin despite the presence of increased thyroid hormone levels.These studies demonstrate novel interactions between T3 and Wnt signaling pathways in the regulation of skeletal development and bone formation.
MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes
Rationale: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ.
Objective: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo.
Methods and Results: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin.
Conclusions: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.