The Inhibitory Mechanism of the {zeta} Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related {alpha}-Proteobacteria. [Enzymology]

November 6th, 2015 by

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria, different to bacterial ϵ and mitochondrial IF1 inhibitors. The N-terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Rios, E., Mendoza-Hernandez, G., Ramirez-Silva, L., Perez-Hernandez, G., and Garcia-Trejo, J.J. (2014) FASEB J. 24, 599-608) by a hitherto unknown quaternary structure that was modeled here by structural homology and protein docking. The F1 and F1-ζ models of Paracoccus denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1 at the open catalytic αE-βE interface, and two partial γ rotations lock the N-terminus of ζ in an inhibition-general core region, blocking further γ rotation, whilst the ζ globular domain anchors it to the closed αDP-βDP interface. Heterologous inhibition of the F1-ATPase of Paracoccus denitrificans by the mitochondrial IF1 supported both, the modelled ζ binding site at the αDP-βDP-γ interface, as well as the endosymbiotic α-proteobacterial origin of mitochondria. In sum, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor-stator interface where the mitochondrial IF1 or the bacterial ϵ bind, with a proposed pawl mechanism coupled to the rotation of the central γ subunit working as a ratchet, but with structural differences that makes it a unique control mechanism of the nanomotor to favour the ATP synthase activity over the ATPase turnover in the α-proteobacteria.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The Inhibitory Mechanism of the {zeta} Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related {alpha}-Proteobacteria. [Enzymology]

Specificity of collybistin-phosphoinositide interactions: Impact of the individual protein domains [Neurobiology]

November 6th, 2015 by

The regulatory protein collybistin (CB)2 recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full length CB2 (CB2SH3+) harboring the N-terminal SH3-domain adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A-E262A mutation, which conformationally 'opens' CB2SH3+ and allows the PH domain to properly bind lipids depending on the phosphoinositide species with a preference for PI(3)P and PI(4)P. This type of membrane tethering under the control of the release of the SH3-domain of CB is essential for regulating gephyrin clustering.

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-Activated Calcium Modulator 1 (Orai1) [Membrane Biology]

November 6th, 2015 by Palty, R., Isacoff, E. Y.

Calcium flux through store operated calcium entry (SOCE) is a central regulator of intracellular calcium signaling. The two key components of the store operated calcium release activated calcium (CRAC) channel are the Ca2+ sensing protein stromal interaction molecule 1 (STIM1) and the channel pore forming protein Orai1. During SOCE activation calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1 activating domain (CAD). In order to gate Orai1 channels the exposed STIM1 activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study we show that the N and C terminal domains of Orai1 synergistically contribute to interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-Activated Calcium Modulator 1 (Orai1) [Membrane Biology]

In silico prediction of human sulfotransferase 1E1 activity guided by pharmacophores from molecular dynamics simulations [Computational Biology]

November 5th, 2015 by

Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28 % of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on In silico prediction of human sulfotransferase 1E1 activity guided by pharmacophores from molecular dynamics simulations [Computational Biology]

MicroRNA-7 Compromises p53-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1 [Signal Transduction]

November 5th, 2015 by Hong, C.-F., Lin, S.-Y., Chou, Y.-T., Wu, C.-W.

We previously demonstrated that EGFR upregulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3'UTR of SMARCD1, and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3'UTR of SMARCD1 and downregulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 downregulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that while SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was downregulated in non-small cell lung cancer (NSCLC) patients and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated miRNA pathway in the SWI/SNF chromatin remodeling complex suggest that EGFR-mediated miR-7 suppressed the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on MicroRNA-7 Compromises p53-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1 [Signal Transduction]

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium Independent Membrane Binding [Molecular Biophysics]

November 5th, 2015 by

Perforin is an essential component in the cytotoxic lymphocyte mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process, and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. While many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium Independent Membrane Binding [Molecular Biophysics]

The voltage-dependent anion channel 1 mediates amyloid beta toxicity and represents a potential target for Alzheimer’s disease therapy [Cell Biology]

November 5th, 2015 by

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is over-expressed in post-mortem brains of Alzheimer's disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid beta (Aβ). In this study, we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance and microscale thermophoresis. Pre-incubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance about two-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol, as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization and cytochrome release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial- and plasma membrane-VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The voltage-dependent anion channel 1 mediates amyloid beta toxicity and represents a potential target for Alzheimer’s disease therapy [Cell Biology]

Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex [Cell Biology]

November 5th, 2015 by Mattera, R., Guardia, C. M., Sidhu, S., Bonifacino, J. S.

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, however, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. [Protein Synthesis and Degradation]

November 5th, 2015 by

Runx2, a master regulator of osteoblast differentiation is tightly regulated both at transcriptional and post-translational levels. Post translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, over-expression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function, however; both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized animals (induction of surgical menopause) that show reduced Runx2 and enhanced Fbw7α while this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation. [Protein Synthesis and Degradation]

Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-Chain Fatty Acyl-CoAs and a Novel CoA Induced Fit Mechanism [Enzymology]

November 4th, 2015 by

PaaI thioesterases are members of the TE13 thioesterase family which catalyse the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8 Å resolution, to reveal two double hotdog domains arranged in a back-to-back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle X-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn37, Asp52, and Thr68 are important for catalysis, and size exclusion chromatography analysis and X-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N-terminus, and a loop region involving Tyr38Tyr39. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-Chain Fatty Acyl-CoAs and a Novel CoA Induced Fit Mechanism [Enzymology]