Defining the apoptotic trigger: the interaction of cytochrome c and cardiolipin [Membrane Biology]

October 20th, 2015 by O'Brien, E. S., Nucci, N. V., Fuglestad, B., Tommos, C., Wand, A. J.

The interaction between cytochrome c and the anionic lipid cardiolipin has been proposed as a primary event in the apoptotic signaling cascade. Numerous studies that have examined the interaction of cytochrome c with cardiolipin embedded in a variety of model phospholipid membranes have suggested that partial unfolding of the protein is a precursor to the apoptotic response. However, these studies lacked site resolution and used model systems with negligible or a positive membrane curvature, which is distinct from the large negative curvature of the invaginations of the inner mitochondrial membrane where cytochrome c resides. We have used reverse micelle encapsulation to mimic the potential effects of confinement on the interaction of cytochrome c with cardiolipin. Encapsulation of oxidized horse cytochrome c in 1-decanoyl-rac-glycerol/lauryldimethylamine-N-oxide/hexanol reverse micelles prepared in pentane yields NMR spectra essentially identical to the protein in free aqueous solution. The structure of encapsulated ferricytochrome c was determined to high precision (bb ~ 0.23 A) using NMR-based methods and is closely similar to the cryogenic crystal structure (bb ~ 1.2 A). Incorporation of cardiolipin into the reverse micelle surfactant shell causes localized chemical shift perturbations of the encapsulated protein, providing the first view of the cardiolipin/cytochrome c interaction interface at atomic resolution. Three distinct sites of interaction are detected: the so-called A- and L-sites, plus a previously undocumented interaction centered on residues Phe36, Gly37, Thr58, Trp59, and Lys60. Importantly, in distinct contrast to earlier studies of this interaction, the protein is not significantly disturbed by the binding of cardiolipin in the context of the reverse micelle.

Twist1 is Essential for Tooth Morphogenesis and Odontoblast Differentiation [Molecular Bases of Disease]

October 20th, 2015 by

Twist1 is a basic helix-loop-helix (bHLH)- containing transcription factor that is expressed in the dental mesenchyme during the early stages of tooth development. To better delineate its roles in tooth development, we generated Twist1 conditional knockout embryos (Twist2Cre/+;Twist1fl/fl) by breeding Twist1 floxed mice (Twist1fl/fl) with Twist2-Cre knock-in mice (Twist2Cre/+). The Twist2Cre/+;Twist1fl/fl embryos formed smaller tooth germs and abnormal cusps during early tooth morphogenesis. Molecular and histological analyses showed that the developing molars of the Twist2Cre/+;Twist1fl/fl embryos had reduced cell proliferation and expression of fibroblast growth factors (Fgfs) 3, 4, 9 and 10, and FGF receptors (Fgfrs) 1 and 2 in the dental epithelium and mesenchyme. In addition, 3-week-old renal capsular transplants of the E18.5 Twist2Cre/+;Twist1fl/fl molars showed malformed crowns and cusps with defective crown dentin and enamel. Immunohistochemical analyses revealed that the implanted mutant molars had defects in odontoblast differentiation and delayed ameloblast differentiation. Furthermore, in vitro chromatin immuno-precipitation (ChIP) assays demonstrated that Twist1 was able to bind to a specific region of the Fgf10 promoter. In conclusion, our findings suggest that Twist1 plays crucial roles in regulating tooth development and that it may exert its functions through the FGF signaling pathway.

Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3 [Protein Structure and Folding]

October 20th, 2015 by

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells, and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation and epitope mapping of 8H9 based on structure determination, modeling and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5 Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (ScFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescent cell sorting. Six mutations (three in the CDR and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nM KD) compared to parental hu8H9 scFv (144 nM KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to sub-nanomolar KD) had 2- to 9-fold enhancements in affinity compared to their parental forms, potent in vitro antibody dependent cell-mediated cytotoxicity (0.1-0.3 μg/mL EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the precise molecular epitope of 8H9 was determined to be the FG loop of B7-H3, a region critical to its function in immunologic blockade, unique among anti-B7-H3 antibodies published to date.
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N-helix and cysteines inter-regulate human mitochondrial VDAC-2 function and biochemistry [Bioenergetics]

October 20th, 2015 by Maurya, S. R., Mahalakshmi, R.

Human voltage-dependent anion channel-2 (hVDAC-2) functions primarily as the crucial anti-apoptotic protein in the outer mitochondrial membrane, and additionally as a gated bidirectional metabolite transporter. The N-terminal helix (NTH), involved in voltage sensing, bears an additional 11-residue extension (NTE) only in hVDAC-2. In this study, we assign a unique role for the NTE as influencing the chaperone-independent refolding kinetics and overall thermodynamic stability of hVDAC-2. Our electrophysiology data shows that N-helix is crucial for channel activity while NTE sensitizes this isoform to voltage gating. Additionally, hVDAC-2 possesses the highest cysteine content, possibly for regulating reactive oxygen species content. We identify interdependent contributions of the N-helix and cysteines to channel function, and the measured stability in micellar environments with differing physico-chemical properties. The evolutionary demand for the NTE in the presence of cysteines clearly emerges from our biochemical and functional studies, providing insight into factors that functionally demarcate hVDAC-2 from the other VDACs.

The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase is Palmitoylated and Contains Tightly Bound Zinc [Membrane Biology]

October 20th, 2015 by Gottlieb, C. D., Zhang, S., Linder, M. E.

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 moles of zinc per mole DHHC3 protein. Taken together, our data demonstrate coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.

Genetic screen reveals link between maternal-effect sterile gene mes-1 and P. aeruginosa-Induced neurodegeneration in C. elegans [Neurobiology]

October 16th, 2015 by Wu, Q., Cao, X., Yan, D., Wang, D., Aballay, A.

Increasing evidence indicates that immune responses to microbial infections may contribute to neurodegenerative diseases. Here, we show that Pseudomonas aeruginosa infection of Caenorhabditis elegans causes a number of neural changes that are hallmarks of neurodegeneration. Using an unbiased genetic screen to identify genes involved in the control of P. aeruginosa-induced neurodegeneration, we identified mes-1, which encodes a receptor tyrosine kinase-like protein that is required for unequal cell divisions in the early embryonic germline. We showed that sterile but not fertile mes-1 animals were resistant to neurodegeneration induced by P. aeruginosa infection. Similar results were observed using animals carrying a mutation in the maternal-effect gene pgl-1, which is required for postembryonic germline development, and the germline-deficient strains glp-1 and glp-4. Additional studies indicated that the FOXO transcription factor DAF-16 is required for resistance to P. aeruginosa-induced neurodegeneration in germline-deficient strains. Thus, our results demonstrate that P. aeruginosa infection results in neurodegeneration phenotypes in C. elegans that are controlled by the germline in a cell-nonautonomous manner.
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Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the RAGE/Raf/MEK/ERK Pathway. [Molecular Bases of Disease]

October 15th, 2015 by Meng, H.-Z., Zhang, W.-L., Liu, F., Yang, M.-W.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells: cell proliferation was stimulated after exposure to low-dose AGE-BSA, but cell apoptosis was stimulated after exposure to high-dose AGE-BSA. The low-dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf / MEK / ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short-term effects of low-dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy and regulated the proliferation and function of hFOB1.19 cells.
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Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-Homology Domains [Protein Structure and Folding]

October 15th, 2015 by

Many multi-component protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

BMP-2 Activates NFATc1 via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling [Gene Regulation]

October 15th, 2015 by

Bone remodeling is controlled by dual actions of osteoclasts (OC) and osteoblasts (OB). The calcium sensitive NFATc1 transcription factor, as an OC signature gene, regulates differentiation of OC downstream of BMP-2-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1 we analyzed bones from the OB-specific BMP-2 knock out (BMP-2 cKO) mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OB. We showed that BMP-2, via receptor-specific Smad pathway, regulates expression of NFATc1 in OB. PI 3 kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by calcium-dependent serine threonine phosphatase, calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. BAPTA-AM, an inhibitor of intracellular calcium abundance blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity resulting in NFATc1 nuclear translocation. Cyclosporine A (CSA), which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription confirming its autoregulation. Together these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk between Smad, PI 3 kinase/Akt and Ca2+ signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis [Enzymology]

October 15th, 2015 by

ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome (SMC) proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.
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