Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes

1. Download : Download high-res image (182KB)
2. Download : Download full-size image

Highlights

• •Insulin Affects the Phosphorylation of G2L1, MARK2, CLIP2, EB1, AGAP3, and CKAP5.
• •Insulin Increases CLASP2 +TIP Density and Decreases CLASP2 +TIP Velocity.
• •Insulin Stimulates CLASP2 and G2L1 Trailing Along Microtubules.
• •Insulin Stimulates α-Tubulin Acetylation at Lysine 40 and Microtubule Stabilization.

     

Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)

1. Download : Download high-res image (128KB)
2. Download : Download full-size image

Highlights

• •Two-step cross-linking coupled with affinity purification to facilitate structural analysis of protein complexes.
• •Integrated QXL-MS workflow for studying condition-dependent structural changes of protein complexes.
• •Mechanistic insights on in vivo H₂O₂-induced conformational dynamics of proteasome complexes.

     

Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 μM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.     

A Conserved residue in the yeast Bem1p SH3 domain maintains the high level of binding specificity required for function

The yeast Bem1p SH3b and Nbp2p SH3 domains are unusual because they bind to peptides containing the same consensus sequence, yet they perform different functions and display low sequence similarity. In this work, by analyzing the interactions of these domains with six biologically relevant peptides containing the consensus sequence, they are shown to possess finely tuned and distinct binding specificities. We also identify a residue in the Bem1p SH3b domain that inhibits binding, yet is highly conserved for the purpose of preventing nonspecific interactions. Substitution of this residue results in a marked reduction of in vivo function that is caused by titration of the domain away from its proper targets through nonspecific interactions with other proteins. This work provides a clear illustration of the importance of intrinsic binding specificity for the function of protein-protein interaction modules, and the key role of "negative" interactions in determining the specificity of a domain.     

Appl1 Is Dispensable for Mouse Development, and Loss of Appl1 Has Growth Factor-selective Effects on Akt Signaling in Murine Embryonic Fibroblasts

The adaptor protein APPL1 (adaptor protein containing pleckstrin homology (PH), phosphotyrosine binding (PTB), and leucine zipper motifs) was first identified as a binding protein of AKT2 by yeast two-hybrid screening. APPL1 was subsequently found to bind to several membrane-bound receptors and was implicated in their signal transduction through AKT and/or MAPK pathways. To determine the unambiguous role of Appl1 in vivo, we generated Appl1 knock-out mice. Here we report that Appl1 knock-out mice are viable and fertile. Appl1-null mice were born at expected Mendelian ratios, without obvious phenotypic abnormalities. Moreover, Akt activity in various fetal tissues was unchanged compared with that observed in wild-type littermates. Studies of isolated Appl1^−/− murine embryonic fibroblasts (MEFs) showed that Akt activation by epidermal growth factor, insulin, or fetal bovine serum was similar to that observed in wild-type MEFs, although Akt activation by HGF was diminished in Appl1^−/− MEFs. To rule out a possible redundant role played by the related Appl2, we used small interfering RNA to knock down Appl2 expression in Appl1^−/− MEFs. Unexpectedly, cell survival was unaffected under normal culture conditions, and activation of Akt was unaltered following epidermal growth factor stimulation, although Akt activity did decrease further after HGF stimulation. Furthermore, we found that Appl proteins are required for HGF-induced cell survival and migration via activation of Akt. Our studies suggest that Appl1 is dispensable for development and only participate in Akt signaling under certain conditions.     

Cross-linking Proteomics Indicates Effects of Simvastatin on the TLR2 Interactome and Reveals ACTR1A as a Novel Regulator of the TLR2 Signal Cascade

1. Download : Download high-res image (107KB)
2. Download : Download full-size image

Highlights

• •An innovative co-IP crosslinking proteomics study was designed for the TLR2 interactome.
• •Proteomic profiling revealed combinatorial effects of simvastatin and Pam3CSK4 on the TLR2 interactome.
• •ACTR1A and MARCKSL1 proteins were identified as potential interactors of TLR2 during the immune response.
• •ACTR1A has important modulatory actions on the TLR2 pro-inflammatory signaling cascade.

     

SETD1A Methyltransferase Is Physically and Functionally Linked to the DNA Damage Repair Protein RAD18

1. Download : Download high-res image (98KB)
2. Download : Download full-size image

Highlights

• •Protein interaction screen of SETD1A/COMPASS complex subunits.
• •Unexpected interaction with DNA damage protein RAD18 was confirmed for SETD1A, but not for other subunits.
• •SETD1A and/or RAD18 influence each other's mRNA and protein expression levels, and disruption of either gene elicits a similar DNA damage sensitivity phenotype.

     

ComplexBrowser: A Tool for Identification and Quantification of Protein Complexes in Large-scale Proteomics Datasets

1. Download : Download high-res image (53KB)
2. Download : Download full-size image

Highlights

• •Automated analysis of protein complexes in proteomic experiments.
• •Quantitative measurement of the coordinated changes in protein complex components.
• •Interactive visualizations for exploratory analysis of proteomic results.

     

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) is an AB₅ toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1.     

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE),

1. Download : Download high-res image (162KB)
2. Download : Download full-size image

Highlights

• •Multiplex epitope mapping/antigenic determinant identification in the gas phase.
• •Intact transition and controlled dissociation of immune complexes by MS.
• •Simultaneous identification and amino acid sequence determination of epitopes.
• •Simplified in-solution sample handling because of ion manipulation and filtering by MS.

     

A Minimum Distance Estimation Approach to Estimate the Recombination Fraction from a Marker Locus in Robust Linkage Analysis for Quantitative Traits

OLSON and WIJSMAN (1993) have proposed a robust linkage analysis between quantitative traits and a marker locus using all relative pairs. We extend their work to estimate the recombination fraction using a two-step procedure. In the first step Generalised Estimating Equations are solved. After robust linkage analysis minimum distance estimation is applied in the second step. Our approach requires a single codominant marker locus only. The relevant parameters of the genetic model can also be estimated by this method in the presence of linkage. We illustrate our approach by simulations.     

A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: Application to gramicidin-S

Summary A global optimization method for intensity-restrained structure refinement, based on variable target function (VTF) analysis, is illustrated using experimental data on a model peptide, gramicidin-S (GS) dissolved in DMSO. The method (referred to as VARTIGO for variable target intensity-restrained global optimization) involves minimization of a target function in which the range of NOE contacts is gradually increased in successive cycles of optimization in dihedral angle space. Several different starting conformations (including all-trans) have been tested to establish the validity of the method. Not all optimizations were successful, but these were readily identifiable from their large NOE R-factors. We also show that it is possible to simultaneously optimize the rotational correlation time along with the dihedral angles. The structural features of GS thus obtained from the successful optimizations are in excellent agreement with the available experimental data. A comparison is made with structures generated from an intensity-restrained single target function (STF) analysis. The results on GS suggest that VARTIGO refinement is capable of yielding better quality structures. Our work also underscores the need for a simultaneous analysis of different NOE R-factors in judging the quality of optimized structures. The NOESY data on GS in DMSO appear to provide evidence for the presence of two orientations for the ornithine side chain, in fast exchange. The NOESY spectra for this case were analyzed using a relaxation rate matrix which is a weighted average of the relaxation rate matrices for the individual conformations.