Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme
The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase family of enzymes. These enzymes catalyse the introduction, reduction and isomerisation of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. W...
Source: BJ Cell - May 19, 2015 Category: Biochemistry Authors: O B. V. Oka, H Ying Yeoh, N John Bulleid Tags: BJ Biomolecules Source Type: research
Deletion of the amino acid transporter Slc6a14 suppresses tumor growth in spontaneous mouse models of breast cancer
SLC6A14 mediates Na + /Cl --coupled concentrative uptake of a broad-spectrum of amino acids. It is expressed at low levels in many tissues but upregulated in certain cancers. Pharmacologic blockade of SLC6A14 causes amino acid starvation in ER+ breast cancer cells and suppresses their proliferation in vitro and in vivo. Here we interrogated the role of this transporter in breast cancer by deleting Slc6a14 in mice and monitoring the consequences of this deletion in models of spontaneous breast cancer (PyMT oncogene-Tg mouse and MMTV/Neu-Tg mouse). Slc6a14-knockout mice are viable, fertile, and phenotypically n...
Source: BJ Cell - May 13, 2015 Category: Biochemistry Authors: E Babu, Y D Bhutia, S Ramachandran, J P Gnana-Prakasam, P D Prasad, M Thangaraju, V Ganapathy Tags: BJ Disease Source Type: research
The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4-Not nuclease module
In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key event in regulated mRNA degradation. A major enzyme involved in deadenylation is the Ccr4-Not deadenylase complex, which can be recruited to its target mRNA by RNA-binding proteins or the miRNA repression complex. In addition to six non-catalytic components, the complex contains two enzymatic subunits with ribonuclease activity: Ccr4 and Caf1 (Pop2). In vertebrates, each deadenylase subunit is encoded by two paralogues: Caf1, which can interact with the anti-proliferative protein BTG2, is encoded by CNOT7 and CN...
Source: BJ Cell - May 6, 2015 Category: Biochemistry Authors: M Maryati, B Airhihen, G Sebastiaan Winkler Tags: BJ Gene Source Type: research
Acyl-CoA Binding Domain Containing 3 Modulates NAD{+} Metabolism through Activating Poly(ADP-ribose) Polymerase 1
Nicotinadmide adenine dinucleotide (NAD+) plays essential roles in cellular energy homeostasis and redox state functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD+-consuming enzymes, this molecule may be also involved in many other cellular and biological outcomes such as chromatin remodeling, gene transcription, genomic integrity, cell division, calcium signaling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme, and dysfunctional PARP1 has been linked with the onset and development of vario...
Source: BJ Cell - May 5, 2015 Category: Biochemistry Authors: Y Chen, S Bang, S Park, H Shi, S F Kim Tags: BJ Signal Source Type: research
Critical role of evolutionarily conserved glycosylation at Asn211 in the intracellular trafficking and activity of sialyltransferase ST3Gal-II
ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn92 and Asn211). While Asn92 is only conserved in mammalian species, Asn211 is highly conserved in mammals, birds and fish. The current study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mu...
Source: BJ Cell - April 28, 2015 Category: Biochemistry Authors: F M. Ruggiero, A A. Vilcaes, R Iglesias-Bartolomé, J L. Daniotti Tags: BJ Cell Source Type: research
Lopimune -Induced Mitochondrial Toxicity is attenuated by increased uncoupling protein - 2 level in treated mouse hepatocyes
The objective of this study is to evaluate mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2) in mouse hepatocytes following lopimune administration. Mitochondria were extracted from mouse liver using differential centrifugation and hepatocytes were isolated by collagenase perfusion procedure. Mitochondrial respiration was measured using Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometry using 2′,7′-Dichlorofluorescin diacetate probe and UCP2 protein expression was detected by western blot. We found th...
Source: BJ Cell - April 13, 2015 Category: Biochemistry Authors: S El Hoss, G M. Bahr, K S. Echtay Tags: BJ Cell Source Type: research
Secreted Frizzled-related protein 3 (sFRP3)-mediated suppression of Interleukin-6 receptor release by A disintegrin and Metalloprotease 17 (ADAM17) is abrogated in the osteoarthritis-associated rare double variant of sFRP3
This study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R is linked to inflammation, cartilage degeneration, and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also sugges...
Source: BJ Cell - April 7, 2015 Category: Biochemistry Authors: M Oldefest, S Düsterhöft, C Desel, S Thysen, C Fink, B Rabe, R Lories, J Grötzinger, I Lorenzen Tags: BJ Cell Source Type: research
Probing the cis-arrangement of prototype tight junction proteins claudin-1 and claudin-3
Claudins form a large family of tight junction (TJ) proteins featuring four transmembrane segments (TM1 to 4), two extra- and one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the...
Source: BJ Cell - April 7, 2015 Category: Biochemistry Authors: S Milatz, J Piontek, J Schulzke, I E. Blasig, M Fromm, D Günzel Tags: BJ Biomolecules Source Type: research
Depletion of the Polyamines Spermidine and Spermine by Overexpression of Spermidine/spermine N1-Acetyltransferase1 (SAT1) Leads to Mitochondria-Mediated Apoptosis in Mammalian Cells
The polyamines putrescine, spermidineand spermine are intimately involved in the regulation of cellular growth and viability. Transduction of HEK293T cells with an adenovirus (AdSAT1) encoding a key polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase1 (SAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/ propidium iodide Fluorescent Activated Cell Sorter (FACS) analyses, Terminal Uridine Nucleotide End- Labeling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1 transduced cells (at 24-72 h), but not in ce...
Source: BJ Cell - April 7, 2015 Category: Biochemistry Authors: S Mandal, A Mandal, M Park Tags: BJ Metabolism Source Type: research
RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin
RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologs, have remained poorly understood. We show that RECQ1 associates with FEN-1 in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5’-flap DNA substrates containing nontelomeric or...
Source: BJ Cell - March 16, 2015 Category: Biochemistry Authors: F Sami, X Lu, S Parvathaneni, R Roy, R K. Gary, S Sharma Tags: BJ Biomolecules Source Type: research
Akt-mediated regulation of antidepressant-sensitive serotonin transporter function, cell surface expression and phosphorylation
The serotonin (5-HT) transporter (SERT) controls serotonergic neurotransmission in the brain by rapid clearance of 5-HT from the synaptic cleft into presynaptic neurons. SERTs are primary target for antidepressants for therapeutic intervention of mood disorders. Our previous studies have identified the involvement of several signaling pathways and protein kinases in regulating SERT function, trafficking and phosphorylation. However, whether the protein kinase B/Akt regulates SERT function is not known. Here, we made novel observation that inhibition of Akt resulted in the down regulation of SERT function through the regula...
Source: BJ Cell - March 11, 2015 Category: Biochemistry Authors: J Rajamanickam, B Annamalai, T Rahbek-Clemmensen, S Sundaramurthy, U Gether, L D Jayanthi, S Ramamoorthy Tags: BJ Signal Source Type: research
Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion
Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 Diabetes, but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 cells and compared it to insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells, respectively, secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. While glycolytic metab...
Source: BJ Cell - March 3, 2015 Category: Biochemistry Authors: J A Stamenkovic, L E Andersson, A E Adriaenssens, A Bagge, V V Sharoyko, F Gribble, F Reimann, C B Wollheim, H Mulder, P Spégel Tags: BJ Metabolism Source Type: research
Conversion of Non-adipogenic Fibroblasts into Adipocytes by a Defined Hormone Mixture
We report here for the first time that NIH-3T3 fibroblasts, which are generally considered to be non-adipogenic, can be converted into mature adipocytes by treatment with a defined hormone mixture comprising epidermal growth factor (EGF), hepatocyte growth factor (HGF), dexamethasone (Dex), and insulin. Furthermore, NIH-3T3 cells transplanted into obese immunodeficient NOD-SCID mice formed adipocytes in vivo. Interestingly, the mixture elicited conversion of NIH-3T3 cells directly into adipocytes without a preceding pre-adipocyte stage. Functional analysis revealed that each component of the mixture was necessary for the i...
Source: BJ Cell - March 2, 2015 Category: Biochemistry Authors: T Nie, X Hui, X Gao, B Nie, L Mao, X Tang, R Yuan, K Li, P Li, A Xu, P Liu, S Ding, W Han, G Cooper, D Wu Tags: BJ Cell Source Type: research
Distinct lipid effects on tBid and Bim activation of membrane permeabilization by pro-apoptotic Bax
After exposure to stressful stimuli, apoptotic signals can be relayed to mitochondria by pro-apoptotic activator proteins, tBid and Bim, which activate Bax and or Bak to induce mitochondrial outer membrane permeabilization (MOMP). These protein-protein and protein-membrane interactions are critical for apoptosis regulation since MOMP irreversibly leads to cell death. While the distinct roles of tBid and Bim as sensors of different types of stress are well recognized, it is not known whether the molecular mechanisms whereby they initiate MOMP are the same. Here we compare membrane permeabilization by Bax activated by either...
Source: BJ Cell - February 25, 2015 Category: Biochemistry Authors: A Shamas-Din, S Bindner, X Chi, B Leber, D W Andrews, C Fradin Tags: BJ Cell Source Type: research
The Leishmania donovani histidine acid ecto-phosphatase LdMAcP: insight into its structure and function
Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. Here we report data contributing to the molecular/structural and functional characterization of the Leishmania donovani LdMAcP, member of the histidine acid phosphatase family. LdMAcP, is membrane anchored and shares high sequence identity with the major secreted Leishmania donovani acid phosphatases (LdSAcPs).Sequence comparison of the LdMAcP orthologs in Leishmania spp revealed strain polymorphism and species specificity for the Leishmania donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus ...
Source: BJ Cell - February 19, 2015 Category: Biochemistry Authors: A Papadaki, A S. Politou, D Smirlis, M P. Kotini, K Kourou, T Papamarcaki, H Boleti Tags: BJ Cell Source Type: research
