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Biochemical Journal

Ana Perea, José Ignacio Manzano, Yasuhisa Kimura, Kazumitsu Ueda, Santiago Castanys, Francisco Gamarro
The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, it has been described the involvement of LABCG2 in virulence, autophagy and oxidative stress. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. In order to explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system...
November 21, 2017: Biochemical Journal
Nazma Malik, Thomas Macartney, Annika Hornberger, Karen Anderson, Hannah Tovell, Alan R Prescott, Dario R Alessi
Derailment of the PI3K-AGC protein kinase signalling network contributes to many human diseases including cancer. Recent work has revealed that the poorly studied AGC kinase family member, SGK3 promotes resistance to cancer therapies that target the Class 1 PI3K pathway, by substituting for loss of Akt kinase activity. SGK3 is recruited and activated at endosomes, by virtue of its PX domain binding to PtdIns(3)P. Here we demonstrate that endogenous SGK3 is rapidly activated by growth factors such as IGF1, through pathways involving both Class 1 and Class 3 PI3Ks...
November 17, 2017: Biochemical Journal
Wenxia Chen, Yongbin Yan, Chundong Song, Ying Ding, Tao Du
Studies have demonstrated that microvesicles (MVs) derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSCs) could ameliorate renal ischemia/reperfusion injury (IRI), however, the underlying mechanisms were not clear yet. Here, MVs were isolated and injected intravenously into rats immediately after ischemia of the left kidney, and Erk1/2 activator HGF or inhibitor U0126 was administrated, respectively. Tubular cell proliferation and apoptosis were identified by Ki67 or TUNEL immunostaining. Masson's tri-chrome straining and alpha-smooth muscle actin (α -SMA) staining were used for assessing renal fibrosis...
November 17, 2017: Biochemical Journal
Venkatasubramanian Vidhyasagar, Yujiong He, Manhong Guo, Tanu Talwar, Ravi Shankar Singh, Manisha Yadav, George Katselis, Franco J Vizeacoumar, Erique Lukong, Yuliang Wu
Human Nucleic Acid Binding Protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single stranded (ss) DNA and RNA binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In this study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution, however C9ORF80 exhibits anomalous behavior on SDS-PAGE and gel filtration because of 48% random coil present in the protein...
November 17, 2017: Biochemical Journal
Rahamthulla S Shaik, Min Woo Sung, Stanislav Vitha, Andreas Holzenburg
Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by assembly of cytoskeletal FtsZ proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts except that MinC was lost and replaced by the eukaryotic ARC3. Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy (TEM) in conjunction with single particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division: FtsZ2...
November 14, 2017: Biochemical Journal
Alessia Leonetti, Laura Cervoni, Fabio Polticelli, Yuta Kanamori, Zuleyha Nihan Yurtsever, Enzo Agostinelli, Paolo Mariottini, Pasquale Stano, Manuela Cervelli
Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidise spermine and, after treatment with DTT, revert to SMOX monomer...
November 14, 2017: Biochemical Journal
Konstantin Tomanov, Lilian Nehlin, Ionida Ziba, Andreas Bachmair
The SUMO conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. In order to better define biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO conjugating enzyme (SCE) were tested for in vitro activity. Lys to Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly...
November 13, 2017: Biochemical Journal
Pawel Lis, Sophie Burel, Martin Steger, Matthias Mann, Fiona Brown, Federico Diez, Francesca Tonelli, Janice L Holton, Philip Winglok Ho, Shu-Leong Ho, Meng-Yun Chou, Nicole K Polinski, Terina N Martinez, Paul Davies, Dario R Alessi
Mutations that activate the LRRK2 protein kinase, predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector binding Switch-II motif. In this study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against 9 different LRRK2 phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43)...
November 10, 2017: Biochemical Journal
Ying Fan, Andrew Jm Howden, Adil R Sarhan, Pawel Lis, Genta Ito, Terina N Martinez, Kathrin Brockmann, Thomas Gasser, Dario R Alessi, Esther M Sammler
There is compelling evidence for the role of LRRK2 and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2 mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2...
November 10, 2017: Biochemical Journal
Anna M Barron, Bin Ji, Seiji Kito, Tetsuya Suhara, Makoto Higuchi
The translocator protein (TSPO) has been proposed to act as a key component in a complex important for mitochondrial cholesterol importation, which is the rate-limiting step in steroid hormone synthesis. However, TSPO function in steroidogenesis has recently been challenged by the development of TSPO knockout (TSPO-KO) mice, as they exhibit normal baseline gonadal testosterone and adrenal corticosteroid production. Here we demonstrate that despite normal androgen levels in young male TSPO-KO mice, TSPO deficiency alters steroidogenic flux and results in reduced total steroidogenic output...
November 10, 2017: Biochemical Journal
E De Meulenaere, J B Bailey, F A Tezcan, D D Deheyn
Ferritin, a multimeric cage-like enzyme, is integral to iron metabolism across all phyla through the sequestration and storage of iron through efficient ferroxidase activity. While ferritin sequences from around 900 species have been identified, crystal structures from only 50 species have been reported, the majority from bacterial origin. We recently isolated a secreted ferritin from the marine invertebrate Chaetopterus sp. (parchment tubeworm), which resides in muddy coastal seafloors. Here, we present the first ferritin from a marine invertebrate to be crystallized, and its biochemical characterization...
November 10, 2017: Biochemical Journal
Ivan Petushkov, Daria Esyunina, Vladimir Mekler, Konstantin Severinov, Danil Pupov, Andrey Kulbachinsky
In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5'-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ(70) subunit of Escherichia coli RNAP...
November 3, 2017: Biochemical Journal
Alok K Mishra, Shivraj M Yabaji, Rikesh K Dubey, Ekta Dhamija, Kishore K Srivastava
The remarkable ability of M. tuberculosis ( Mtb ) to survive inside the human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belongs to an essential two-component system (TCS), PrrA/B; which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. Here, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein...
November 3, 2017: Biochemical Journal
Biao Dong, Xiaolu Zhang, Yafeng Fan, Songqiang Cao, Xuepei Zhang
The aim of this study was to investigate the effects and molecular mechanisms of GPR4 in cell apoptosis and renal ischemia-reperfusion (IR) injury in vivo and in vitroGPR4(-/-) mice and wild-type (WT) mice underwent renal IR or sham procedures. For hypoxia/reoxygenation (HR), human umbilical vein endothelial cells (HUVECs) were subjected to 4 h of hypoxia, followed by 6 h of reoxygenation. Renal histological changes were observed by periodic acid-schiff (PAS) staining and myeloperoxidase (MPO) activity. Apoptosis was detected by TUNEL staining...
October 31, 2017: Biochemical Journal
Juvid Aryaman, Iain G Johnston, Nick S Jones
Mitochondrial dysfunction is involved in a wide array of devastating diseases but the heterogeneity and complexity of these diseases' symptoms challenges theoretical understanding of their causation. With the explosion of -omics data, we have the unprecedented opportunity to gain deep understanding of the biochemical mechanisms of mitochondrial dysfunction. This goal raises the need to make these complex datasets interpretable. Quantitative modelling allows us to translate such datasets into intuition and suggest rational biomedical treatments...
October 27, 2017: Biochemical Journal
David R Owen, Jinjiang Fan, Enrico Campioli, Sathvika Venugopal, Andrew Midzak, Edward Daly, Aline Harlay, Leeyah Issop, Vincenzo Libri, Dimitra Kalogiannopoulou, Eduardo Oliver, Enrique Gallego-Colon, Alessandro Colasanti, Les Huson, Ilan Rabiner, Puvan Suppiah, Charles Essagian, Paul M Matthews, Vassilios Papadopoulos
The 18 kDa translocator protein (TSPO) is a ubiquitous conserved outer mitochondrial membrane protein implicated in numerous cell and tissue functions, including steroid hormone biosynthesis, respiration, cell proliferation and apoptosis.  TSPO binds with high affinity to cholesterol and numerous compounds, is expressed at high levels in steroid-synthesizing tissues, and mediates cholesterol import into mitochondria, which is the rate-limiting step in steroid formation.  In humans, the rs6971 polymorphism on the TSPO gene leads to an amino acid substitution in the fifth transmembrane loop of the protein, which is where the cholesterol-binding domain of TSPO is located, and this polymorphism has been associated with anxiety-related disorders...
October 26, 2017: Biochemical Journal
Michael Brad Strader, Rachel Bangle, Claire J Parker Siburt, Cornelius L Varnado, Jayashree Soman, Andres S Benitez Cardenas, Premila S Samuel, Eileen W Singleton, Alvin L Crumbliss, John S Olson, Abdu I Alayash
Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (βK82D) is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic crosslinking of Hb tetramers with and without the Hb Providence mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically crosslinked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer...
October 25, 2017: Biochemical Journal
Vanina Saraullo, Nicolas Di Siervi, Belen Jerez, Carlos Davio, Adolfo Zurita
Despite its importance in the regulation of growth and differentiation processes of a variety of organisms, the mechanism of synthesis and degradation of cAMP has not yet been described in Giardia lamblia In this work, we measured significant quantities of cAMP in trophozoites of G. lamblia incubated in vitro and later detected how it increases during the first hours of encystation, and how it then returns to basal levels at 24 h. Through an analysis of the genome of G. lamblia , we found sequences of three putative enzymes -one phosphodiesterase (gPDE) and two nucleotydyl cyclases (gNC1 and gNC2)- that should be responsible for the regulation of cAMP in G...
October 20, 2017: Biochemical Journal
Zimple Kurlawala, Rain Dunaway, Parag P Shah, Julie A Gosney, Leah J Siskind, Brian P Ceresa, Levi J Beverly
Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that mediates growth, proliferation and survival. Dysregulation of IGF pathway contributes to initiation, progression and metastasis of cancer and is also involved in diseases of glucose metabolism, such as Diabetes. We have identified Ubiquilin1 (UBQLN1) as a novel interaction partner of IGF1R, IGF2R and Insulin Receptor. UBQLN family of proteins have been studied primarily in the context of protein quality control and in the field of neurodegenerative disorders...
October 20, 2017: Biochemical Journal
Natália E Bernardes, Agnes Alessandra S Takeda, Thiago Revers Dreyer, Fernanda B Cupertino, Stela Virgilio, Nelly Pante, Maria Célia Bertolini, Marcos R M Fontes
The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism by N. crassa Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in this study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular and biophysical methods. A complemented strain that produced a sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus...
October 20, 2017: Biochemical Journal
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