The cytotoxicity of 3-Bromopyruvate in breast cancer cells depends on extracellular pH
Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell ...
Source: BJ Cell - February 2, 2015 Category: Biochemistry Authors: J Azevedo-Silva, O Queirós, A Ribeiro, F Baltazar, K H Young, P L Pedersen, A Preto, M Casal Tags: BJ Metabolism Source Type: research
In vitro translation in a hybrid cell free lysate with exogenous cellular ribosomes
Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In this manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensibility, reproducibility and the ability to translate specialized mRNAs in less than one hour. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs...
Source: BJ Cell - January 28, 2015 Category: Biochemistry Authors: B Panthu, D Decimo, L Balvay, T Ohlmann Tags: BJ Gene Source Type: research
Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein
ABCB6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities including confocal and electron microscopy we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide binding (NBD) and transmembran...
Source: BJ Cell - January 28, 2015 Category: Biochemistry Authors: K Kiss, N Kucsma, A Brozik, G E. Tusnady, P Bergam, G Van Niel, G Szakacs Tags: BJ Cell Source Type: research
Role of Cysteine-3602 in the Function and Regulation of the Cardiac Ryanodine Receptor
The cardiac Ca2+ release channel (ryanodine receptor type 2, RyR2) is modulated by sulfhydryl reactive agents, but the molecular basis of RyR2 modulation by sulfhydryl reagents is poorly understood. Cysteine-3635 in the skeletal muscle ryanodine receptor (RyR1) is one of the most hyperreactive sulfhydryls and is important for the redox and calmodulin regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (C3602) in the function and regulation of the RyR2 channel. Here we assessed the impact of mutating C3602 (C3602A) on store overload induced Ca2+...
Source: BJ Cell - January 21, 2015 Category: Biochemistry Authors: T Mi, Z Xiao, W Guo, Y Tang, F Hiess, J Xiao, Y Wang, J Z Zhang, L Zhang, R Wang, P P Jones, S Chen Tags: BJ Biomolecules Source Type: research
Ischemia-induced autophagy leads to degradation of gap junction protein Connexin43 in cardiomyocytes
Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on Connexin (Cx)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischemic heart remain unknown. The results obtained in this study, using both HL-1 cells and organotypic heart cultures, show that simulated ischemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischemia-induced degradation of Cx43 results in GJIC impairment ...
Source: BJ Cell - January 21, 2015 Category: Biochemistry Authors: T Martins-Marques, S Catarino, M Zuzarte, C Marques, P Matafome, P Pereira, H Girão Tags: BJ Cell Source Type: research
The VPS-20 Subunit of the Endosomal Sorting Complex ESCRT-III Exhibits an Open Conformation in the Absence of Upstream Activation
Members of the endosomal sorting complex required for transport (ESCRT) machinery function in membrane remodeling processes during multivesicular endosome biogenesis, cytokinesis, retroviral budding, and plasma membrane repair. During lumenal vesicle formation at endosomes, the ESCRT-II complex and the ESCRT-III subunit VPS-20 play a specific role in regulating assembly of ESCRT-III filaments, which promote vesicle scission. Previous work suggests that Vps20 isoforms, like other ESCRT-III subunits, exhibits an autoinhibited, closed conformation in solution, and its activation depends on an association with ESCRT-II specifi...
Source: BJ Cell - January 15, 2015 Category: Biochemistry Authors: A L Schuh, M Hanna, K Quinney, L Wang, A Sarkeshik, J R Yates III, A Audhya Tags: BJ Cell Source Type: research
Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75)
Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 (connexin43 (Cx43)-interacting protein of 75 kDa), a protein involved in proteasomal degradation, as a novel Cx43 interacting protein. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 a...
Source: BJ Cell - January 12, 2015 Category: Biochemistry Authors: J L Kopanic, B Schlingmann, M Koval, A F. Lau, P L Sorgen, V F Su Tags: BJ Cell Source Type: research
Myc-interacting zinc-finger protein 1 positively regulates Wnt signaling by protecting Dishevelled from Dapper1-mediated degradation
In this study, we report that Myc-interacting zinc-finger protein 1 (MIZ1) interacts with Dpr1, and this interaction attenuates the ability of Dpr1 to induce Dvl2 degradation, thus enhancing Wnt signaling. Mechanistically, MIZ1 is translocated from the nucleus to the cytoplasm upon Wnt3a stimulation or over-expression of Dpr1 and Dvl2, disrupting the interaction between Dpr1 and Dvl2. Furthermore, MIZ1 can promote the proliferation of breast cancer MDA-MB-231 and BT-549 cells through Wnt signaling and reverse the anti-proliferative effect of Dpr1 on colorectal cancer Caco-2. Together, our findings establish a novel layer o...
Source: BJ Cell - January 5, 2015 Category: Biochemistry Authors: Y Huang, P Wang, H Chen, Y Ding, Y Chen Tags: BJ Signal Source Type: research
Differential calcium handling by the cis and trans regions of the Golgi apparatus
High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. Additionally, Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis-/cis to medial Golgi (cGo) or the trans-Golgi (tGo). Ca2+ homeostasis in these compartments and in the ER has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We find that the cGo accumulates Ca2+ to high concentrations (150-300 µM)...
Source: BJ Cell - December 16, 2014 Category: Biochemistry Authors: F J Aulestia, M Teresa Alonso, J Garcia-Sancho Tags: BJ Signal Source Type: research
Unraveling the pivotal role of ALIX in MVB sorting and silencing of activated EGFR
In this study, we demonstrate that despite its non-essential roles in degradation of activated EGFR, ALIX plays a critical role in MVB sorting and silencing of activated EGFR. EGF stimulation of mammalian cell lines induces ALIX interaction with ubiquitinated EGFR through the ALIX V domain and increases ALIX association with membrane-bound CHMP4 through the ALIX Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of ALIX interaction with membrane-bound CHMP4, inhibition of ALIX dimerization through the V domain or ALIX knockdown dramatically inhibits MVB sorting of activated EGFR and p...
Source: BJ Cell - December 16, 2014 Category: Biochemistry Authors: S Sun, X Zhou, W Zhang, G E. Gallick, J Kuang Tags: BJ Cell Source Type: research
The sigma 1 receptors are present in monomeric and oligomeric forms in living cells in the presence and absence of ligands
The sigma-1 receptor (S1R) is a membrane 223-amino acid protein that resides in the endoplasmic reticulum and the plasma membrane of some mammalian cells. The S1R is regulated by a various synthetic molecules including (+)-pentazocine, cocaine and haloperidol, and endogenous molecules such as sphingosine, dimethyltryptamine and dehydroepiandrosterone. Ligand regulated protein chaperone functions linked to oxidative stress and neurodegenerative disorders such as ALS and neuropathic pain have been attributed to the S1R. Several client proteins that interact with S1R have been identified including various types of ion ...
Source: BJ Cell - December 15, 2014 Category: Biochemistry Authors: A K Mishra, T A Mavlyutov, D R Singh, G Biener, J Yang, J A Oliver, A E Ruoho, V Raicu Tags: BJ Biomolecules Source Type: research
TARP {gamma}-8 glycosylation regulates the surface expression of AMPA receptors
Transmembrane AMPA receptor regulatory protein (TARP) γ-8 is an auxiliary subunit of AMPA receptors that is widely distributed in the hippocampus. It has been shown that TARP γ-8 promotes surface expression of AMPA receptors; however, how TARP γ-8 regulates the expression of AMPA receptors remains unclear. In the present study, we examined the effect of TARP glycosylation on AMPA receptor trafficking. We first show that TARP γ-8 is an N-glycosylated protein, which contains two glycosylation sites, Asn-53 and Asn-56, and compare this to the glycosylation of TARP γ-2 and the AMPAR auxiliary...
Source: BJ Cell - December 11, 2014 Category: Biochemistry Authors: C Zheng, K Chang, Y Suh, K W. Roche Tags: BJ Cell Source Type: research
Ubiquitin-C-terminal hydrolases cleave isopeptide and peptide-linked ubiquitin from structured proteins but do not edit ubiquitin homopolymers
Modification of proteins with ubiquitin (Ub) occurs through a variety of topologically distinct Ub linkages, including Ube2W-mediated monoubiquitylation of N-terminal alpha amines to generate peptide-linked linear mono-Ub fusions. Protein ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs), many of which show striking preference for particular Ub linkage types. Here, we have screened for DUBs that preferentially cleave N-terminal Ub from protein substrates but do not act on ubiquitin homopolymers. We show that members of the Ub C-terminal hydrolase (UCH) family of DUBs demonstrate this preferenc...
Source: BJ Cell - December 9, 2014 Category: Biochemistry Authors: J S Bett, M Ritorto, R Ewan, E G Jaffray, S Virdee, J W Chin, A Knebel, T Kurz, M Trost, M H Tatham, R T. Hay Tags: BJ Biomolecules Source Type: research
MiR-98 delays skeletal muscle differentiation by downregulating E2F5
A genome-wide screen had previously shown that knocking down miR-98 and let-7g, two miRNAs of the let-7 family, leads to a dramatic increase of terminal myogenic differentiation. Here we report that a transcriptomic analysis of human myoblasts where miR-98 was knocked down revealed that approximately 240 genes were sensitive to miR-98 depletion. Among these potential targets of miR-98, we identified the transcriptional repressor E2F5, and showed that it is a direct target of miR-98. Knocking down simultaneously E2F5 and miR-98 almost fully restored normal differentiation, indicating that E2F5 is involved in the regulation ...
Source: BJ Cell - November 25, 2014 Category: Biochemistry Authors: J Kropp, C Degerny, N Morozova, J Pontis, A Harel-bellan, A Polesskaya Tags: BJ Cell Source Type: research
Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment
Evasion of cell death is one crucial capability acquired by tumor cells to ward off anti-tumor therapies, and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of Apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signaling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and mass spectrometry, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cance...
Source: BJ Cell - November 21, 2014 Category: Biochemistry Authors: E Varfolomeev, A Izrael-Tomasevic, K Yu, D Bustos, T Goncharov, L D Belmont, A Masselot, C E Bakalarski, D S Kirkpatrick, D Vucic Tags: BJ Signal Source Type: research
