The BAG proteins: a ubiquitous family of chaperone regulators

The BAG (Bcl-2 associated athanogene) family is a multifunctional group of proteins that perform diverse functions ranging from apoptosis to tumorigenesis. An evolutionarily conserved group, these proteins are distinguished by a common conserved region known as the BAG domain. BAG genes have been found in yeasts, plants, and animals, and are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in carcinogenesis, HIV infection, and Parkinson’s disease. These proteins are therefore potential therapeutic targets, and their expression in cells may serve as a predictive tool for such diseases. In plants, the Arabidopsis thaliana genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. Three members contain a calmodulin-binding domain possibly reflecting differences between plant and animal programmed cell death. This review summarizes current understanding of BAG proteins in both animals and plants. Received 21 November 2007; received after revision 17 December 2007; accepted 2 January 2008     

A complex of Shc and Ran-GTPase localises to the cell nucleus

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 20 October 2008; received after revision 04 December 2008; accepted 15 December 2008     

Cell migration to the chemokine CXCL8: Paxillin is activated and regulates adhesion and cell motility

The chemokine CXCL8 is a powerful inducer of directional cell motility, primarily during inflammation. In this study, we found that CXCL8 stimulation led to paxillin phosphorylation in normal neutrophils, and that both CXCL8 receptors (CXCR1 and CXCR2) mediated CXCL8-induced paxillin phosphorylation. In CXCR2-transfected cells, the process depended on G_αi and G_αs coupling to CXCR2. Dominant negative (DN) paxillin increased CXCL8-induced adhesion and migration, indicating that endogenous paxillin keeps migration at submaximal levels. Furthermore, using activating antibodies to β1 integrins, analyses with focal adhesion kinase (FAK) DN variant (FRNK) and co-immunoprecipitations of FAK and paxillin, we found that β1 integrin ligation cooperates with CXCL8-induced stimulation, leading to FAK activation and thereafter to FAK-mediated paxillin phosphorylation. Our findings indicate that paxillin keeps directional motility at a restrained magnitude, and suggest that perturbations in its activation may lead to chemotactic imbalance and to pathological conditions associated with excessive or reduced leukocyte migration. R. Mintz, T. Meshel: These authors contributed equally to this work. Received 31 July 2008; received after revision 14 December 2008; accepted 16 December 2008     

The perspectives of studying multi-domain protein folding

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding. Received 3 December 2008; accepted 23 December 2008     

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction

The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 11 August 2008; received after revision 18 December 2008; accepted 22 December 2008     

Breaking biological symmetry in membrane proteins: The asymmetrical orientation of PsaC on the pseudo-C2 symmetric Photosystem I core

The elucidation of assembly pathways of multi-subunit membrane proteins is of growing interest in structural biology. In this study, we provide an analysis of the assembly of the asymmetrically oriented PsaC subunit on the pseudo C₂-symmetric Photosystem I core. Based on a comparison of the differences in the NMR solution structure of unbound PsaC with that of the X-ray crystal structure of bound PsaC, and on a detailed analysis of the PsaC binding site surrounding the F_X iron-sulfur cluster, two models can be envisioned for what are likely the last steps in the assembly of Photosystem I. Here, we dissect both models and attempt to address heretofore unrecognized issues by proposing a mechanism that includes a thermodynamic perspective. Experimental strategies to verify the models are proposed. In closing, the evolutionary aspects of the assembly process will be considered, with special reference to the structural arrangement of the PsaC binding surface. Received 22 October 2008; received after revision 17 November 2008; accepted 05 December 2008     

Functions and pathologies of BiP and its interaction partners

The endoplasmic reticulum (ER) is involved in a variety of essential and interconnected processes in human cells, including protein biogenesis, signal transduction, and calcium homeostasis. The central player in all these processes is the ER-lumenal polypeptide chain binding protein BiP that acts as a molecular chaperone. BiP belongs to the heat shock protein 70 (Hsp70) family and crucially depends on a number of interaction partners, including co-chaperones, nucleotide exchange factors, and signaling molecules. In the course of the last five years, several diseases have been linked to BiP and its interaction partners, such as a group of infectious diseases that are caused by Shigella toxin producing E. coli. Furthermore, the inherited diseases Marinesco-Sj?gren syndrome, autosomal dominant polycystic liver disease, Wolcott-Rallison syndrome, and several cancer types can be considered BiP-related diseases. This review summarizes the physiological and pathophysiological characteristics of BiP and its interaction partners. Received 20 November 2008; received after revision 09 December 2008; accepted 12 December 2008     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

hShroom1 links a membrane bound protein to the actin cytoskeleton

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with β-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton. D. E. Dye, S. Karlen: These authors contributed equally to this work. Received 09 October 2008; received after revision 23 November 2008; accepted 09 December 2008     

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Activation of δ-opioid receptors (DOR) attenuates anoxic K⁺ leakage and protects cortical neurons from anoxic insults by inhibiting Na⁺ influx. It is unknown, however, which pathway(s) that mediates the Na⁺ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na⁺ influxes mediated by voltage-gated Na⁺ channels; (2) DOR activation inhibited Na⁺ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K⁺ derangement; and (3) DOR activation had little effect on Na⁺/Ca²⁺ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K⁺ derangement by restricting Na⁺ influx mediated by Na⁺ channels and NMDA receptors, and that non-NMDA receptors and Na⁺/Ca²⁺ exchangers, although involved in anoxic K⁺ derangement in certain degrees, are less likely the targets of DOR signal. Received 26 November 2008; received after revision 26 December 2008; accepted 13 January 2009     

Functions and transport of silicon in plants

Silicon exerts beneficial effects on plant growth and production by alleviating both biotic and abiotic stresses including diseases, pests, lodging, drought, and nutrient imbalance. Recently, two genes (Lsi1 and Lsi2) encoding Si transporters have been identified from rice. Lsi1 (low silicon 1) belongs to a Nod26-like major intrinsic protein subfamily in aquaporin, while Lsi2 encodes a putative anion transporter. Lsi1 is localized on the distal side of both exodermis and endodermis in rice roots, while Lsi2 is localized on the proximal side of the same cells. Lsi1 shows influx transport activity for Si, while Lsi2 shows efflux transport activity. Therefore, Lsi1 is responsible for transport of Si from the external solution to the root cells, whereas Lsi2 is an efflux transporter responsible for the transport of Si from the root cells to the apoplast. Coupling of Lsi1 with Lsi2 is required for efficient uptake of Si in rice. Received 21 December 2007; received after revision 29 April 2008; accepted 15 May 2008     

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 September 2008; received after revision 17 December 2008; accepted 06 January 2009     

Loss of FrmA leads to increased cell-cell adhesion and impaired multi-cellular development of Dictyostelium cells

Cell-cell adhesion is a critical property of all multi-cellular organisms and its correct regulation is critical during development, differentiation, tissue building and maintenance, and many immune responses. The multi-talin-like FERM domain containing protein, FrmA, is required during starvation-induced multi-cellular development of Dictyostelium cells. Loss of FrmA leads to increased cell-cell adhesion and results in impaired multi-cellular development, slug migration and fruiting bodies. Further, mixing experiments show that FrmA null cells are excluded from the apex of wild-type mounds, to which cells that normally form the organising centre known as the tip sort. These data suggest a critical role for FrmA in regulating cell-cell adhesion, multi-cellular development and, in particular, the formation of the organising centre known as the tip. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 28 August 2008; received after revision 10 October 2008; accepted 21 October 2008     

Dissection of a novel molecular determinant mediating Golgi to trans-Golgi network transition

Two major functions of the Golgi apparatus (GA) are formation of complex glycans and sorting of proteins destined for various subcellular compartments or secretion. To fulfill these tasks proper localization of the accessory proteins within the different sub-compartments of the GA is crucial. Here we investigate structural determinants mediating transition of the two glycosyltransferases β-1,4- galactosyltransferase 1 (gal-T1) and the α-1,3-fucosyltransferase 6 (fuc-T6) from the trans-Golgi cisterna to the trans-Golgi network (TGN). Upon treatment with the ionophore monensin both glycosyltransferases are found in TGN-derived swollen vesicles, as determined by confocal fluorescence microscopy and density gradient fractionation. Both enzymes carry a signal consisting of the amino acids E₅P₆ in gal-T1 and D₂P₃ in fuc-T6 necessary for the transition of these glycosyltransferases from the trans-Golgi cisterna to the TGN, but not for their steady state localization in the trans-Golgi cisterna. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 30 July 2008; received after revision 17 September 2008; accepted 29 September 2008     

Mitochondrial association of alpha-synuclein causes oxidative stress

α-Synuclein is a neuron-specific protein that contributes to the pathology of Parkinson’s disease via mitochondria-related mechanisms. The present study investigated possible interaction of α-synuclein with mitochondria and consequences of such interaction. Using SHSY cells overexpressing α-synuclein A53T mutant or wild-type, as well as isolated rat brain mitochondria, the present study shows that α-synuclein localizes at the mitochondrial membrane. In both SHSY cells and isolated mitochondria, interaction of α-synuclein with mitochondria causes release of cytochrome c, increase of mitochondrial calcium and nitric oxide, and oxidative modification of mitochondrial components. These findings suggest a pivotal role for mitochondria in oxidative stress and apoptosis induced by α-synuclein. Received 27 December 2007; received after revision 7 February 2008; accepted 8 February 2008     

Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart

Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from type 1 diabetic rats. PKCβ₂ co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from diabetic hearts. Augmented phosphorylation of mitochondrial aconitase in diabetic hearts was found to be associated with an increase in its reverse activity (isocitrate to aconitate), while the rate of the forward activity was unchanged. Similar results were obtained on phosphorylation of mitochondrial aconitase by PKCβ₂ in vitro. These results demonstrate the regulation of mitochondrial aconitase activity by PKC-dependent phosphorylation. This may influence the activity of the tricarboxylic acid cycle, and contribute to impaired mitochondrial function and energy metabolism in diabetic hearts. Received 31 October 2008; received after revision 17 December 2008; accepted 2 January 2009     

CD20-related signaling pathway is differently activated in normal and dystrophic circulating CD133+ stem cells

Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133⁺ cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133⁺ stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca²⁺]_i) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca²⁺]_i is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133⁺ stem cells, supporting the assumption of a “CD20-related calcium impairment-affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133⁺ stem cells. Received 15 October 2008; received after revision 27 November 2008; accepted 05 December 2008