Spartin activates atrophin-1-interacting protein 4 (AIP4) E3 ubiquitin ligase and promotes ubiquitination of adipophilin on lipid droplets

<p>Abstract</p> <p>Background</p> <p>Spartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.</p> <p>Results</p> <p>In this study, we show that spartin is not a substrate for AIP4 activity and that spartin's binding to AIP4 significantly increases self-ubiquitination of this E3 ligase, indicating that spartin disrupts the AIP4 autoinhibitory intramolecular interaction. Correspondingly, spartin has a seven times higher binding affinity to the WW region of AIP4 than the binding of the WW region has to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme-linked immunosorbent assay. We also show that spartin recruits AIP4 to lipid droplets and promotes ubiquitination of lipid droplet-associated protein, adipophilin, which regulates turnover of lipid droplets.</p> <p>Conclusions</p> <p>Our findings demonstrate that spartin acts as an adaptor protein that activates and recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of adipophilin and potentially other lipid-associated proteins. We propose that this is one of the mechanisms by which spartin regulates lipid droplet turnover and might contribute to the pathology of SPG20.</p

Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling

In-Cell Biochemistry Using NMR Spectroscopy

Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry

Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background: We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (Kd) and gp120-to-CD4 inhibition constants (Ki). The PA analogs were selected to satisfy Lipinski’s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity. Principal Findings: PA analog 2-bromopalmitate (2-BP) was most efficacious with Kd,74 nM and Ki,122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher Kd,140 nM and Ki,354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with Kd,364 nM and inhibiting gp120-to-CD4 binding with Ki,1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry. Significance: Considering observed differences between K i and K d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry

Differential branching fraction and angular analysis of the decay B0→K∗0μ+μ−

The angular distribution and differential branching fraction of the decay B 0→ K ∗0 μ + μ − are studied using a data sample, collected by the LHCb experiment in pp collisions at s√=7 TeV, corresponding to an integrated luminosity of 1.0 fb−1. Several angular observables are measured in bins of the dimuon invariant mass squared, q 2. A first measurement of the zero-crossing point of the forward-backward asymmetry of the dimuon system is also presented. The zero-crossing point is measured to be q20=4.9±0.9GeV2/c4 , where the uncertainty is the sum of statistical and systematic uncertainties. The results are consistent with the Standard Model predictions

Opposite-side flavour tagging of B mesons at the LHCb experiment

The calibration and performance of the oppositeside flavour tagging algorithms used for the measurements of time-dependent asymmetries at the LHCb experiment are described. The algorithms have been developed using simulated events and optimized and calibrated with B + →J/ψK +, B0 →J/ψK ∗0 and B0 →D ∗− μ + νμ decay modes with 0.37 fb−1 of data collected in pp collisions at √ s = 7 TeV during the 2011 physics run. The oppositeside tagging power is determined in the B + → J/ψK + channel to be (2.10 ± 0.08 ± 0.24) %, where the first uncertainty is statistical and the second is systematic

Search for CP violation in D+→ϕπ+ and D+s→K0Sπ+ decays

A search for CP violation in D + → ϕπ + decays is performed using data collected in 2011 by the LHCb experiment corresponding to an integrated luminosity of 1.0 fb−1 at a centre of mass energy of 7 TeV. The CP -violating asymmetry is measured to be (−0.04 ± 0.14 ± 0.14)% for candidates with K − K + mass within 20 MeV/c 2 of the ϕ meson mass. A search for a CP -violating asymmetry that varies across the ϕ mass region of the D + → K − K + π + Dalitz plot is also performed, and no evidence for CP violation is found. In addition, the CP asymmetry in the D+s→K0Sπ+ decay is measured to be (0.61 ± 0.83 ± 0.14)%

Measurement of the Bs0→J/ψKS0B_s^0\to J/\psi K_S^0 branching fraction

The $B_s^0\to J/\psi K_S^0$ branching fraction is measured in a data sample corresponding to 0.41$fb^{-1}$ of integrated luminosity collected with the LHCb detector at the LHC. This channel is sensitive to the penguin contributions affecting the sin2$\beta$ measurement from $B^0\to J/\psi K_S^0$ The time-integrated branching fraction is measured to be $BF(B_s^0\to J/\psi K_S^0)=(1.83\pm0.28)\times10^{-5}$. This is the most precise measurement to date

Model-independent search for CP violation in D0→K−K+π−π+ and D0→π−π+π+π− decays

A search for CP violation in the phase-space structures of D0 and View the MathML source decays to the final states K−K+π−π+ and π−π+π+π− is presented. The search is carried out with a data set corresponding to an integrated luminosity of 1.0 fb−1 collected in 2011 by the LHCb experiment in pp collisions at a centre-of-mass energy of 7 TeV. For the K−K+π−π+ final state, the four-body phase space is divided into 32 bins, each bin with approximately 1800 decays. The p-value under the hypothesis of no CP violation is 9.1%, and in no bin is a CP asymmetry greater than 6.5% observed. The phase space of the π−π+π+π− final state is partitioned into 128 bins, each bin with approximately 2500 decays. The p-value under the hypothesis of no CP violation is 41%, and in no bin is a CP asymmetry greater than 5.5% observed. All results are consistent with the hypothesis of no CP violation at the current sensitivity