S-palmitoylation regulates biogenesis of core glycosylated wild-type and F508del CFTR in a post-ER compartment
In this study, we used metabolic labeling to confirm that wild-type and F508del CFTR are palmitoylated, and show that blocking palmitoylation with the pharmacologic inhibitor 2-bromopalmitate (2-BP) decreases steady state levels of both wild-type and low temperature-corrected F508del CFTR, disrupts post-ER maturation, and reduces ion channel function at the cell surface. Protein acyl transferases (PATs) comprise a family of 23 gene products that contain a DHHC motif and mediate palmitoylation. Recombinant expression of specific PATs led to increased levels of CFTR protein and enhanced palmitoylation as judged by western bl...
Source: BJ Cell - January 29, 2014 Category: Biochemistry Authors: M L. McClure, H Wen, J Fortenberry, J S. Hong, E J. Sorscher Tags: BJ ChemBio Source Type: research
Differential functions of calpain 1 during epithelial cell death and adipocyte differentiation in mammary gland involution
Calpains become activated in mammary gland early during weaning, cleaving several proteins located mainly in the cell membrane, but also in other organelles such as lysosomes, mitochondria and nuclei. By immunofluorescence and western blot analysis, we demonstrated the nuclear translocation of calpain-1 and calpain-2, together with the cleavage of several cytoplasmic nucleoporins in epithelial cells of the lobulo-alveolar compartment. In vivo and in vitro calpain inhibition prevented this nucleoporin degradation. Besides, calpain-1 was also present in the nucleus of non-epithelial mammary tissue cells, concomitant with adi...
Source: BJ Cell - January 27, 2014 Category: Biochemistry Authors: T Arnandis, I Ferrer-Vicens, L Torres, C García, E R. Garcia-Trevijano, R Zaragoza, J R. Viña Tags: BJ Cell Source Type: research
Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit
Cdc50 is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. Herein, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase miltefosine transporter (LiMT), on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservati...
Source: BJ Cell - January 21, 2014 Category: Biochemistry Authors: S García-Sánchez, M P. Sánchez-Cañete, F Gamarro, S Castanys Tags: BJ Cell Source Type: research
Identification of interferon-{gamma} as a new molecular target of liver X receptor
In this study, we investigated the effects of LXR activation on IFN-γ expression at different levels. At the molecular level, we surprisingly observed that LXR ligand (T0901317) induced macrophage and T cell IFN-γ protein expression which was associated with increased mRNA and secreted protein levels in culture medium. In contrast, selective inhibition of LXRα or/and LXRβ expression by siRNA reduced IFN-γ expression. The promoter analysis defined the multiple LXR responsive elements (LXREs) in the proximal region of the IFN-γ promoter. EMSA and ChIP assays indicated that LXR activa...
Source: BJ Cell - January 20, 2014 Category: Biochemistry Authors: Q Wang, X Ma, Y Chen, L Zhang, M Jiang, X Li, R Xiang, R Miao, D P. Hajjar, Y Duan, J Han Tags: BJ Cell Source Type: research
C-terminal peptides modelling constitutive PrPC processing demonstrate ameliorated toxicity predisposition consequent to {alpha}-cleavage
Misfolding of cellular prion protein (PrPC) to β-strand rich conformations (PrPres) constitutes a key event in prion disease pathogenesis. PrPC can undergo either of two constitutive endoproteolytic events known as α- and β-cleavage, yielding C-terminal fragments known as C1 and C2, respectively. It is unclear whether C-terminal fragments generated through α- and β-cleavage, especially C2, directly influence pathogenesis. Consequently, we compared the biophysical properties and neurotoxicity of recombinant human PrP fragments recapitulating α- and β-cleavage, namely huPrP112...
Source: BJ Cell - January 20, 2014 Category: Biochemistry Authors: V A. Johanssen, T Johanssen, C L. Masters, A F. Hill, K J. Barnham, S J. Collins Tags: BJ Disease Source Type: research
A dual-targeted aminoacyl-tRNA synthetase in Plasmodium falciparum charges cytosolic and apicoplast tRNACys
Plasmodium parasites possess two endosymbiotic organelles: a mitochondrion, and a relict plastid called the apicoplast. To accommodate the translational requirements of these organelles in addition to its cytosolic translation apparatus, the parasite must maintain a supply of charged tRNA molecules in each of these compartments. Here we investigate how the parasite manages these translational requirements for charged tRNACys with only a single gene for cysteinyl-tRNA synthetase (CysRS). We demonstrate that the single PfCysRS transcript is alternatively spliced, and, using a combination of endogenous and heterologous taggin...
Source: BJ Cell - January 16, 2014 Category: Biochemistry Authors: J S. Pham, R Sakaguchi, L M. Yeoh, N S. De Silva, G I. McFadden, Y Hou, S A. Ralph Tags: BJ Cell Source Type: research
Mature VLDL Triggers the Biogenesis of a Distinct Vesicle from the Trans Golgi Network for its Export to the Plasma Membrane
Post-Golgi trafficking of mature VLDL is crucial in maintaining the normal triacylglycerol (TAG) homeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the trans-Golgi network (TGN) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich post-TGN VLDL transport vesicles (PG-VTV) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions whereas protein transport vesicles were present in relatively higher density fractions of...
Source: BJ Cell - January 16, 2014 Category: Biochemistry Authors: T Hossain, A Riad, S Siddiqi, S Parthasarathy, S A. Siddiqi Tags: BJ Cell Source Type: research
Insulin-stimulated leptin secretion requires calcium and PI3K-Akt activation
Numerous studies have focused on the regulation of leptin signaling and leptin functions in energy homeostasis. However, little is known about how leptin secretion is regulated. Here we studied leptin storage and secretion regulation in 3T3-L1 and primary adipocytes. Leptin is stored in membrane-bound vesicles that are localized predominantly in the endoplasmic reticulum (ER) and close to the plasma membrane of both 3T3-L1 and primary adipocytes. Insulin increases leptin secretion as early as 15 min without affecting the leptin mRNA level. Interestingly, protein synthesis inhibitor cycloheximide and ER-Golgi trafficking bl...
Source: BJ Cell - January 10, 2014 Category: Biochemistry Authors: Y Wang, Y Ali, C Lim, W Hong, Z Pang, W Han Tags: BJ Cell Source Type: research
Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/13 identify the PA-domain as a determinant for endosomal localisation
In this report we also identify a functional role for the PA-domain, which is required for endosomal localisation of these proteins. While PA-domain point mutations of RNF13 and RNF167 identified in human tumours are ligase active, the resultant mutant proteins are mislocalised within the cell. Thus, the PA-domain E3-ligases examined here appear to require both E3 ligase activity as well as an intact PA-domain to efficiently target and ubiquitylate their cellular substrates. (Source: BJ Cell)
Source: BJ Cell - January 3, 2014 Category: Biochemistry Authors: J R. van Dijk, Y Yamazaki, R H. Palmer Tags: BJ Cell Source Type: research
Neuregulin-1{beta} induces embryonic stem cell cardiomyogenesis via ErbB3/ErbB2 receptors
Neuregulin-1β (NRG-1β) serves multiple functions during embryonic heart development by signaling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1β induces cardiomyogenesis of mouse embryonic stem cells (mESCs) at the late stage of differentiation through ErbB4 receptor activation. Here we systematically examined NRG-1β induction of cardiac myocytes in mESCs and identified a novel time window, the early first 48 hours, for NRG-1β-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast to the late...
Source: BJ Cell - December 24, 2013 Category: Biochemistry Authors: J Hao, C L. Galindo, T Tran, D B. Sawyer Tags: BJ Signal Source Type: research
Transcriptionally-correlated sub-cellular dynamics of MBNL1 during lens development and their implication for the molecular pathology of Myotonic Dystrophy Type 1.
Myotonic Dystrophy Type 1 (DM1) is caused by elongation of a CTG repeat in the dystrophia myotonica protein kinase (DMPK) gene. mRNA transcripts containing the resulting CUGexp repeats form accumulations, or foci, in the nucleus of the cell. The pathogenesis of Myotonic dystrophy type 1 (DM1) is proposed to result from inappropriate patterns of alternative splicing caused by sequestration of the developmentally regulated alternative splicing factor muscleblind-like 1 (MBNL1), by these foci. Since eye lens cataract is a common feature of DM1 we have examined the distribution and dynamics of MBNL1 in lens epithelial cell lin...
Source: BJ Cell - December 20, 2013 Category: Biochemistry Authors: S M Coleman, A R Prescott, J E Sleeman Tags: BJ Cell Source Type: research
Sequestration of the abrin A chain to the nucleus by BASP1 increases resistance of cells to abrin toxicity
Abrin, a type II ribosome inactivating protein, comprises of A and B subunits wherein the A subunit harbors toxin activity and the B subunit has galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. An earlier study from our laboratory showed that different cell lines exhibited differences of as much as ~ 200-fold in abrin toxicity prompting studies to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain,...
Source: BJ Cell - December 19, 2013 Category: Biochemistry Authors: S Gadadhar, N Bora, V Tiwari, A A. Karande Tags: BJ Cell Source Type: research
The Cdc48-Vms1 complex maintains 26S proteasome architecture
The 26S proteasome is responsible for most regulated protein turnover and for the degradation of aberrant proteins in eukaryotes. The assembly of this ~2.5 MDa multi-catalytic protease requires several dedicated chaperones, and once assembled, substrate selectivity is mediated by ubiquitin conjugation. After modification with ubiquitin, substrates are escorted to the proteasome by myriad factors, including Cdc48. Cdc48 also associates with numerous co-factors, but to date it is unclear whether each cofactor facilitates proteasome delivery. We discovered that yeast lacking a conserved Cdc48 cofactor, Vms1, accumulate protea...
Source: BJ Cell - December 19, 2013 Category: Biochemistry Authors: J R Tran, J L Brodsky Tags: BJ Cell Source Type: research
Heparanase-mediated cleavage of macromolecular heparin accelerates release of granular components of mast cells from extracellular matrices
Heparanase cleaves macromolecular heparin in the secretory granules of connective tissue-type mast cells. We investigated roles of the cleavage under a microenvironment mimicking where the mast cells physiologically reside. A connective tissue-type mast cell line MST and mouse peritoneal cell-derived mast cells stored macromolecular heparin in the secretory granules. The cells expressing heparanase stored fragmented heparin (≈10 kDa) due to heparanase-dependent cleavage of the heparin. We produced an artificial collagen-based extracellular matrix and placed the live cells or glycosaminoglycans purified from the cell...
Source: BJ Cell - December 18, 2013 Category: Biochemistry Authors: N Higashi, M Waki, M Sue, Y Kogane, H Shida, N Tsunekawa, A Hasan, T Sato, A Kitahara, T Kasaoka, Y Hayakawa, M Nakajima, T Irimura Tags: BJ Biomolecules Source Type: research
Effects of alternative splicing on function of Bestrophin-1 calcium-activated chloride channels
The proposed Ca2+-activated Cl- channel protein Bestrophin 1 (Best1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously iden...
Source: BJ Cell - December 17, 2013 Category: Biochemistry Authors: Y Kuo, I F. Abdullaev, M C. Hyzinski-Garcia, A A. Mongin Tags: BJ Cell Source Type: research
