TMEM16A-TMEM16B chimeras to investigate the structure-function relationship of calcium-activated chloride channels
TMEM16A and TMEM16B proteins are Ca2+ activated Cl- channels (CaCCs) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a ten-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimeric proteins in which different domains of the TMEM16A protein were replaced by equivalent domains of TMEM16B. Replacement of the N-terminus, transmenbrane domains 1 and 2 (TMD1-2), the first intracellular loop, and TMD3-4 did not change channel pr...
Source: BJ Cell - April 10, 2013 Category: Biochemistry Authors: P Scudieri, E Sondo, E Caci, R Ravazzolo, L JV Galietta Tags: BJ Signal Source Type: research
Ube2W conjugates ubiquitin to {alpha}-amino groups of protein N-termini
The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the coordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxyl group of ubiquitin and the ε-amino group of a target lysine. However, ubiquitin is also known to conjugate to the sulphydryl of cysteine side chains and the α-amino group of protein N-termini, although the enzymes responsible for disc...
Source: BJ Cell - April 8, 2013 Category: Biochemistry Authors: M H Tatham, A Plechanovová, E G Jaffray, H Salmen, R T. Hay Tags: BJ Signal Source Type: research
Conformational defects underlie proteosomal degradation of Dent's disease-causing mutants of CLC-5
In this study we studied two misprocessed mutants: C221R located in the membrane domain and R718X, truncating the carboxy terminal domain. Both mutants exhibited enhanced protease susceptibility relative to the normal protein in limited proteolysis studies, providing direct evidence that they are misfolded. Interestingly, the membrane localized mutation: C221R, led to enhanced protease susceptibility of the cytosolic amino terminal region and the carboxy terminal truncation mutation: R718X led to enhanced protease susceptibility of both the cytosolic carboxyl and the membrane domains. Together, these studies support the id...
Source: BJ Cell - April 8, 2013 Category: Biochemistry Authors: C D'Antonio, S Molinski, S Ahmadi, L Huan, L Wellhauser, C E Bear Tags: BJ Disease Source Type: research
ABCA8 stimulates sphingomyelin production in oligodendrocyte
This report represents the first extensive expression and functional study of ABCA8 in the human brain and our data strongly suggest that ABCA8 regulates lipid metabolism in oligodendrocyte and potentially plays a role in myelin formation and maintenance. (Source: BJ Cell)
Source: BJ Cell - April 5, 2013 Category: Biochemistry Authors: W Scott Kim, J T Hsiao, S Bhatia, E N Glaros, A S Don, S Tsuruoka, C Shannon Weickert, G M Halliday Tags: BJ Cell Source Type: research
Zymogen activation of neurotrypsin and neurotrypsin-dependent agrin cleavage on the cell surface are enhanced by glycosaminoglycans
The serine peptidase neurotrypsin is stored in presynaptic nerve endings and secreted in an inactive, zymogenic form by synaptic activity. After activation, which requires activity of postsynaptic NMDA receptors, neurotrypsin cleaves the heparan sulfate proteoglycan agrin at active synapses. The resulting C-terminal 22-kDa fragment of agrin induces dendritic filopodia, which are considered to be precursors of new synapses. Here, we investigated the role of glycosaminoglycans in the activation of neurotrypsin and neurotrypsin-dependent agrin cleavage. We found binding of neurotrypsin to the glycosaminoglycan side chains of ...
Source: BJ Cell - April 5, 2013 Category: Biochemistry Authors: C Gisler, D Lüscher, P Schätzle, S Dürr, A Baici, G Galliciotti, R Reif, M F. Bolliger, B Kunz, P Sonderegger Tags: BJ Cell Source Type: research
An analysis of splicing, actin-binding properties, heterodimerisation and molecular interactions of the non-muscle {alpha}-actinins.
The non-muscle α-actinin isoforms (actinin-1 and -4) are closely related, dimeric actin filament-crosslinking proteins. Despite high sequence similarity, unique properties have been ascribed to actinin-4 in particular. For example, actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney, is overexpressed in several cancers and can translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions we have comprehensively compared these proteins in terms of alternative splicing, actin-binding properties, heterodimer formation and ...
Source: BJ Cell - April 4, 2013 Category: Biochemistry Authors: K Suzanne Foley, P W Young Tags: BJ Cell Source Type: research
Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development
Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest it is essential for schizont development and/or RBC invasion. Here, by tagging endogenous CDPK1 with GFP, we demonstrate that CDPK1 localises to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into the erythrocyte. While a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerat...
Source: BJ Cell - April 3, 2013 Category: Biochemistry Authors: M F Azevedo, P R Sanders, E Krejany, C Q Nie, P Fu, L A Bach, G Wunderlich, B S Crabb, P R Gilson Tags: BJ Cell Source Type: research
The distribution and functional properties of Pelizaeus Merzbacher-like disease-linked Cx47 mutations on Cx47/Cx47 homotypic and Cx47/Cx43 heterotypic gap junctions
Gap junctions (GJs) allow direct intercellular communication, and consist of connexins (Cxs). In the mammalian central nervous system, oligodendrocytes express Cx47, Cx32 and Cx29, whereas astrocytes express Cx43, Cx30 and Cx26. Homotypic Cx47/Cx47 GJs couple oligodendrocytes, and heterotypic Cx47/Cx43 channels are the primary GJs at oligodendrocyte/astrocyte junctions. Interestingly, autosomal recessive mutations in the gene GJC2 encoding Cx47 have been linked to a central hypomyelinating disease termed, Pelizaeus Merzbacher-like disease (PMLD). Our aim is to determine the cellular distribution and functional properties o...
Source: BJ Cell - April 2, 2013 Category: Biochemistry Authors: M Kim, G B Gloor, D Bai Tags: BJ Disease Source Type: research
Specific phosphorylation of the PfRh2b invasion ligand of Plasmodium falciparum
Red blood cell invasion by the malaria parasite Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor-ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. Here we report on the phosphorylation of the cytoplasmic domain (CPD) of P. falciparum reticulocyte homologue protein 2b (Rh2b). First, we identified S3233 as the sole phosphoacceptor site in the CPD for in vitro phosphorylation by parasite extract. We provide several lines of evidence that this phosphorylation is mediated by Plasmodium ca...
Source: BJ Cell - April 2, 2013 Category: Biochemistry Authors: K Engelberg, A S Paul, B Prinz, M Kono, W Ching, D Heincke, T Dobner, T Spielmann, M Duraisingh, T Wolf Gilberger Tags: BJ Disease Source Type: research
Structural Model of a Putrescine-Cadaverine Permease from Trypanosoma cruzi Predicts Residues Vital for Transport and Ligand Binding
The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the amino acid, polyamine, organocation (APC) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key amino acid residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed based upon the atomic coordinates of the E. coli AdiC arginine-agmatine antiporter crystal str...
Source: BJ Cell - March 28, 2013 Category: Biochemistry Authors: R Soysa, H Venselaar, J Poston, B Ullman, M Hasne Tags: BJ Cell Source Type: research
GlgS, previously described as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli
Escherichia coli glycogen metabolism involves regulation of the glgBXCAP operon expression and allosteric control of GlgC-mediated catalysis of ATP and glucose-1-phosphate (G1P) to ADP-glucose linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC negative allosteric regulator AMP. Transcriptomic analyses carried out in this work revealed that, compared with their isogenic BW25113 wild type strain, glgS null (ΔglgS) mutants have increase...
Source: BJ Cell - March 28, 2013 Category: Biochemistry Authors: M Rahimpour, M Montero, G Almagro, A M Viale, A Sevilla, M Cánovas, F José Muñoz, E Baroja-Fernández, A Bahaji, G Eydallin, H Dose, R Takeuchi, H Mori, J Pozueta-Romero Tags: BJ Gene Source Type: research
The Arf GAP AGAP2 Interacts with {beta}Arrestin2 and Regulates {beta}2-Adrenergic Receptor Recycling and Erk Activation
AGAP2 is a multidomain Arf GAP (ADP ribosylation factor-directed GTPase-activating protein) that was shown to promote the fast-recycling of transferrin receptors. Here, we tested the hypothesis that AGAP2 regulates trafficking of β2-adrenergic receptors. We found that AGAP2 formed a complex with βArrestin1 and βArrestin2, proteins that are known to regulate β2-adrenergic receptor signaling and trafficking. AGAP2 colocalized with βArrestin2 on the plasma membrane, and knockdown of AGAP2 expression reduced plasma membrane association of βArrestin2 upon β2-adrenergic receptor a...
Source: BJ Cell - March 26, 2013 Category: Biochemistry Authors: Y Wu, Y Zhao, X Ma, Y Zhu, J Patel, Z Nie Tags: BJ Cell Source Type: research
Evidence of a novel role for Pygopus in ribosomal RNA transcription
Increased protein synthesis during cell proliferation is accompanied by a compensatory increase in efficient ribosome production, but the mechanisms by which cells adapt to this requirement are not fully understood. Here, we present evidence that Pygopus, a protein originally identified as a core component of the Wnt/β-catenin transcription complex is also involved in ribosomal (r)RNA transcription during cancer cell growth. Pygopus was detected in the nucleoli of several transformed cell lines and was associated with Treacle and UBF, proteins essential for ribosome biogenesis in development and cancer. Pygopus was ...
Source: BJ Cell - March 22, 2013 Category: Biochemistry Authors: P G.P. Andrews, Z He, Y R Tzenov, C M Popadiuk, K R Kao Tags: BJ Disease Source Type: research
Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status
We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53 dependent mechanism. To more specifically examine this relationship we ectopically co-expressed mutant p53R273H and HSF1 in the human mammary epithelial cell line, MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, thu...
Source: BJ Cell - March 20, 2013 Category: Biochemistry Authors: C H Nguyen, B J Lang, R CC Chai, J L Vieusseux, M M Kouspou, J T Price Tags: BJ Cell Source Type: research
Mitochondrial respiratory chain complex I is inactivated by NADPH oxidase Nox4
Reactive oxygen species (ROS) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. NADPH oxidase 4 (Nox4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. Here we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrial morphology, stabilized mitochondrial membrane potential, and decreased production of hydrogen peroxide in mitochondria. High-resolution respirometry in permeabilized cells combined w...
Source: BJ Cell - March 20, 2013 Category: Biochemistry Authors: R Koziel, H Pircher, M Kratochwil, B Lener, M Hermann, N A Dencher, P Jansen-Dürr Tags: BJ Cell Source Type: research
