WNT10B Enhances Proliferation Through {beta}-catenin and RAC1 GTPase in Human Corneal Endothelial Cells [Signal Transduction]

September 14th, 2015 by Lee, J. G., Heur, M.

The cornea is the anterior, transparent tissue of the human eye that serves as its main refractive element. The corneal endothelial cells are arranged as a monolayer on the posterior surface of the cornea and function as a pump to counteract the leakiness of its basement membrane. Maintaining the cornea in a slightly dehydrated state is critical for maintenance of corneal transparency. Adult human corneal endothelial cells are G1 arrested, even in response to injury, leading to an age-dependent decline in the endothelial cell density. Corneal edema and subsequent vision loss ensues when the endothelial cell density decreases below a critical threshold. Vision loss secondary to corneal endothelial dysfunction is a common indication for transplantation in developed nations. An impending increase in demand for and a current global shortage of donor corneas will necessitate development of treatments for vision loss due to endothelial dysfunction that does not rely on donor corneas. Wnt ligands regulate many critical cellular functions such as proliferation, making them attractive candidates for modulation in corneal endothelial dysfunction. We show WNT10B causes nuclear transport and binding of RAC1 and β-catenin in human corneal endothelial cells, leading to activation of Cyclin D1 expression and proliferation. Our findings indicate WNT10B promotes proliferation in human corneal endothelial cells by simultaneously utilizing both β-catenin dependent and independent pathways, and suggest that its modulation could be used to treat vision loss secondary to corneal endothelial dysfunction.

Posttranslational Regulation of Human DNA Polymerase {iota} [Enzymology]

September 14th, 2015 by

Human DNA polymerases (pols) η and ι are Y- family DNA polymerase paralogs that facilitate translesion synthesis (TLS) past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines, may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the PCNA-interacting region, the Rev1-interacting region, as well as its Ubiquitin Binding Motifs, UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA damaging agents such as UV- light (generating UV-photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand crosslinks), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via K11- and K48- linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle, as no polyubiquitination was observed after treatment with rotenone, or antimycin A, which inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase, KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at K11- and K48- rather than oxidative damage per se.

Pore Hydration States of KcsA Potassium Channels in Membranes [Lipids]

September 14th, 2015 by

Water-filled hydrophobic cavities in channel proteins serve as gateways for transfer of ions across membranes but their properties are largely unknown. We determined water distributions along the conduction pores in two tetrameric channels embedded in lipid bilayers using neutron diffraction: potassium channel KcsA and the trans-membrane domain of M2 protein of Influenza A virus. For the KcsA channel in the closed state, the distribution of water is peaked in the middle of the membrane, showing water in the central cavity adjacent to the selectivity filter. This water is displaced by the channel blocker tetrabutyl-ammonium. The amount of water associated with the channel was quantified, using neutron diffraction and solid-state NMR. In contrast, the M2 proton channel shows a "V"- shaped water profile across the membrane, with a narrow constriction at the center, like the hour-glass shape of its internal surface. These two types of water distribution are therefore very different in their connectivity to the bulk water. The water and protein profiles determined here provide important evidence concerning conformation and hydration of channels in membranes and the potential role of pore hydration in channel gating.

Two Hydrophobic Residues Can Determine The Specificity Of MAP Kinase Docking Interactions [Cell Biology]

September 14th, 2015 by Bardwell, A. J., Bardwell, L.

Mitogen-activated protein kinases (MAPKs) bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK and p38) can bind selectively to D-sites in their authentic substrates and regulators, while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just one or two key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or visa versa. These specificity-determining differences are also found in the D-sites of the ETS-family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK vs. JNK as predicted. These results provide new insights into docking specificity, and suggest that this specificity can evolve rapidly by changes to just one or two amino acids.

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.

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.

Resistance of Dynamin-related protein 1 Oligomers to Disassembly Impairs Mitophagy Resulting in Myocardial Inflammation and Heart failure [Metabolism]

September 14th, 2015 by

We previously reported that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy (DCM). The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python DCM model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast (MEF) model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F MEFs were depolarized and had reduced calcium uptake, with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age, and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis and activation of sterile myocardial inflammation resulting in heart failure.
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