ELL Associated Factor 2 (EAF2) Inhibits Transforming Growth Factor {beta} Signaling through a Direct Interaction with Smad3 [Gene Regulation]

September 14th, 2015 by Liu, X., Chen, Z., Ouyang, G., Song, T., Liang, H., Liu, W., Xiao, W.

A series of in vitro and in vivo studies have shown that EAF2 can affect multiple signaling pathways involved in cellular processes. However, the molecular mechanisms underlying its effects have remained elusive. Here we report the discovery of a new functional link between EAF2 and TGF-β signaling. Promoter reporter assays indicated that EAF2 suppresses Smad3 transcriptional activity, resulting in inhibition of TGF-β signaling. Coimmunoprecipitation assays showed that EAF2 specifically interacts with Smad3 in vitro and in vivo, but not with other Smad proteins. In addition, we observed that EAF2 binding does not alter Smad3 phosphorylation, but causes Smad3 cytoplasmic retention, competes with Smad4 for binding to Smad3, and prevents p300/Smad3 complex formation. Furthermore, we demonstrated that EAF2 suppresses both TGF-β-induced G1 cell cycle arrest and TGF-β-induced cell migration. This study identifies and characterizes a novel repressor of TGF-β signaling.
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Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG) [Protein Structure and Folding]

September 14th, 2015 by

Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP vs. cAMP is critical. However, the mechanism of cGMP-vs.-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic-nucleotide-binding domain of PKG (CNB-B) binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-vs.-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP- and cAMP-bound forms of PKG CNB-B. We show that although cGMP-activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.

Structural and Functional Insights into the Cryoprotection of Membranes by the Intrinsically Disordered Dehydrins [Protein Structure and Folding]

September 14th, 2015 by

Dehydration can be caused by desiccation due to a lack of environmental water, or by freezing due to a lack of liquid water. Plants have evolved a large family of proteins called late embryogenesis abundant (LEA) proteins, which include the intrinsically disordered dehydration protein (dehydrin) family, to combat these abiotic stresses. While transcription and translation studies have shown a correlation between dehydration stress and the presence of dehydrins, the biochemical mechanisms have remained somewhat elusive. We examine here the effect and structure of a small model dehydrin (Vitis riparia K2) on the protection of membranes from freeze-thaw stress. This protein is able to bind to liposomes containing phosphatidic acid, and protect the liposomes from fusing after freeze-thaw treatment. The presence of K2 did not measurably affect liposome surface accessibility or lipid mobility, but did lower its membrane transition temperature by 3°C. Using sodium dodecyl sulfate as a membrane model, we examined the NMR structure of K2 in the presence and absence of the micelle. Biochemical, NMR, and in silico docking experiments show that the conserved, lysine-rich segments are involved in the binding of the dehydrin to a membrane while the poorly conserved φ-segments play no role in binding or protection.
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Structural basis for a unique ATP synthase core complex from Nanoarcheaum Equitans [Protein Structure and Folding]

September 14th, 2015 by

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structure and biophysical studies of the regulatory subunit, NeqB, the apo NeqAB, and NeqAB in complex with nucleotides, ADP and AMP-PNP. NeqB is approximately 20 amino acids shorter at its C-terminus than its homologs but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

A Kissing-Loop is Important for BtuB Riboswitch Ligand Sensing and Regulatory Control [Gene Regulation]

September 14th, 2015 by Lussier, A., Bastet, L., Chauvier, A., Lafontaine, D. A.

RNA-based genetic regulation is exemplified by metabolite-binding riboswitches that modulate gene expression through conformational changes. Crystal structures show that the Escherichia coli btuB riboswitch contains a kissing-loop interaction that is in close proximity to the bound ligand. To analyze the role of the kissing-loop interaction in the riboswitch regulatory mechanism, we used RNase H cleavage assays to probe the structure of nascent riboswitch transcripts produced by the E. coli RNA polymerase. By monitoring the folding of the aptamer, kissing-loop and riboswitch expression platform, we established the conformation of each structural component in the absence or presence of bound adenosylcobalamin. We found that the kissing-loop interaction is not essential for ligand binding. However, we showed that kissing-loop formation improves ligand binding efficiency and is required to couple ligand binding to the riboswitch conformational changes involved in regulating gene expression. These results support a mechanism by which the btuB riboswitch modulates the formation of a tertiary structure to perform metabolite sensing and regulate gene expression.

AMP-activated Protein Kinase Stimulates Warburg-Like Glycolysis and Activation of Satellite Cells during Muscle Regeneration [Metabolism]

September 14th, 2015 by Fu, X., Zhu, M.-J., Dodson, M. V., Du, M.

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent, but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a non-canonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating non-canonical Shh promote proliferation of satellite cells, which is abolished due to satellite cell-specific AMPKα1 knockout. Taken together, AMPKα1 is a critical mediator linking non-canonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.

Robust glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI family member protein is critical for oxidative stress resistance in Saccharomyces cerevisiae [Protein Synthesis and Degradation]

September 14th, 2015 by

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. Accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, and leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, but their functions are largely dependent on glutathione cofactor whose availability is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein; a member of ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae plays an indispensable role in regulating redox homeostasis. Our results highlight that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared to another paralog, Hsp34. On the other hand, glyoxalase defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. In addition to that, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress. Besides, Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1 which is implicated in the familial form of Parkinson disease complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying importance of these proteins in the maintenance of ROS homeostasis across phylogeny.
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Effect of Cholesterol Reduction on Receptor Signaling in Neurons [Neurobiology]

September 14th, 2015 by Fukui, K., Ferris, H. A., Kahn, C. R.

Diabetes mellitus is associated with a variety of complications, including alterations in the central nervous system (CNS). We have recently shown that diabetes results in a reduction of cholesterol synthesis in the brain due to decreased insulin stimulation of SREBP2 mediated cholesterol synthesis in neuronal and glial cells. In the present study, we have explored the effects of the decrease in cholesterol on neuronal cell function using GT1-7 hypothalamic cells subjected to cholesterol depletion in vitro using three independent methods: 1) exposure to methyl-beta-cyclodextrin (MBCD), 2) treatment with the HMG-CoA reductase inhibitor simvastatin and 3) shRNA-mediated knockdown of SREBP2. All three methods produced 20-31% reductions in cellular cholesterol content, similar to the decrease in cholesterol synthesis observed in diabetes. All cholesterol-depleted neuron-derived cells, independent of the method of reduction, exhibited decreased phosphorylation/ activation of IRS-1 and Akt following stimulation by insulin, IGF-1 or the neurotrophins (NGF and BDNF). ERK phosphorylation/activation was also decreased after MBCD and statin treatment, but increased in cells following SREBP2 knockdown. In addition, apoptosis in the presence of amyloid-β; was increased. Reduction in cellular cholesterol also resulted in increased basal autophagy and impairment of induction of autophagy by glucose deprivation. Together, these data indicate that a reduction in neuron-derived cholesterol content, similar to that observed in diabetic brain, creates a state of insulin and growth factor resistance that could contribute to CNS-related complications of diabetes, including increased risk of neurodegenerative diseases such as Alzheimer′s disease.

Activation of Toll-like Receptor (TLR) 4 Attenuates Adaptive Thermogenesis via Endoplasmic Reticulum Stress [Metabolism]

September 14th, 2015 by Okla, M., Wang, W., Kang, I., Pashaj, A., Carr, T., Chung, S.

Adaptive thermogenesis is the cellular process transforming chemical energy into heat in response to cold. A decrease in adaptive thermogenesis is a contributing factor to obesity. However, the molecular mechanisms responsible for the compromised adaptive thermogenesis in obese subjects have not yet been elucidated. In this study, we hypothesized that TLR4 activation and subsequent inflammatory responses are key regulators to suppress adaptive thermogenesis. To test this hypothesis, C57BL/6 mice were either fed a palmitate-enriched high fat (HF) diet or administered with chronic low-dose LPS before cold acclimation. TLR4 stimulation by HF diet or LPS were both associated with reduced core body temperature and heat release. Impairment of thermogenic activation was correlated with diminished expression of brown-specific markers and mitochondrial dysfunction in subcutaneous white adipose tissue (sWAT). Defective sWAT browning was concomitant with elevated levels of ER stress and autophagy. Consistently, TLR4 activation by LPS abolished cAMP-induced upregulation of uncoupling protein 1 (UCP1) in primary human adipocytes, which was reversed by silencing of C/EBP homologous protein (CHOP). Moreover, the inactivation of ER stress by genetic deletion of CHOP or chemical chaperone, conferred a resistance to the LPS-induced suppression of adaptive thermogenesis. Collectively, our data implicate the existence of a novel signaling network that links TLR4 activation, ER stress, and mitochondrial dysfunction, thereby antagonizing thermogenic activation of sWAT. Our results also suggest that TLR4/ER stress axis activation may be a responsible mechanism for obesity-mediated defective BAT activation.

Self-assembly is prerequisite for catalysis of Fe(II) oxidation by catalytically active subunits of ferritin [Protein Structure and Folding]

September 14th, 2015 by Honarmand Ebrahimi, K., Hagedoorn, P.-L., Hagen, W. R.

Fe(III)-storage by Ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical-shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a diiron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal-ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site were obliterated. However, metal-ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions towards the ferroxidase center directly through the protein shell, and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.