New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors
We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific...
Source: Biochemical Journal - August 14, 2018 Category: Biochemistry Authors: Byrne, D. P., Li, Y., Ramakrishnan, K., Barsukov, I. L., Yates, E. A., Eyers, C. E., Papy-Garcia, D., Chantepie, S., Pagadala, V., Liu, J., Wells, C., Drewry, D. H., Zuercher, W. J., Berry, N. G., Fernig, D. G., Eyers, P. A. Tags: Research Articles Source Type: research
Crystal structure and pH-dependent allosteric regulation of human {beta}-ureidopropionase, an enzyme involved in anticancer drug metabolism
β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombin...
Source: Biochemical Journal - July 31, 2018 Category: Biochemistry Authors: Maurer, D., Lohkamp, B., Krumpel, M., Widersten, M., Dobritzsch, D. Tags: Research Articles Source Type: research
Binding mode of AIF(370-394) peptide to CypA: insights from NMR, label-free and molecular docking studies
The complex formation between the proteins apoptosis-inducing factor (AIF) and cyclophilin A (CypA) following oxidative stress in neuronal cells has been suggested as a main target for reverting ischemia-stroke damage. Recently, a peptide encompassing AIF residues 370–394 has been developed to target the AIF-binding site on CypA, to prevent the association between the two proteins and suppress glutamate-induced cell death in neuronal cells. Using a combined approach based on NMR spectroscopy, synthesis and in vitro testing of all Ala-scan mutants of the peptide and molecular docking/molecular dynamics, we have genera...
Source: Biochemical Journal - July 31, 2018 Category: Biochemistry Authors: Farina, B., Sturlese, M., Mascanzoni, F., Caporale, A., Monti, A., Di Sorbo, G., Fattorusso, R., Ruvo, M., Doti, N. Tags: Research Articles Source Type: research
Interaction of the cryptic fragment of myelin basic protein with mitochondrial voltage-dependent anion-selective channel-1 affects cell energy metabolism
In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84–104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down assay, mass spectrometry and molecular modeling studies suggested that, similar with many other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anio...
Source: Biochemical Journal - July 31, 2018 Category: Biochemistry Authors: Remacle, A. G., Hullugundi, S. K., Dolkas, J., Angert, M., Cieplak, P., Scott, D., Chernov, A. V., Shubayev, V. I., Strongin, A. Y. Tags: Research Articles Source Type: research
Cilium structure, assembly, and disassembly regulated by the cytoskeleton
The cilium, once considered a vestigial structure, is a conserved, microtubule-based organelle critical for transducing extracellular chemical and mechanical signals that control cell polarity, differentiation, and proliferation. The cilium undergoes cycles of assembly and disassembly that are controlled by complex inter-relationships with the cytoskeleton. Microtubules form the core of the cilium, the axoneme, and are regulated by post-translational modifications, associated proteins, and microtubule dynamics. Although actin and septin cytoskeletons are not major components of the axoneme, they also regulate cilium organi...
Source: Biochemical Journal - July 31, 2018 Category: Biochemistry Authors: Mirvis, M., Stearns, T., James Nelson, W. Tags: Review Articles Source Type: research
The role of metabolism and tunneling nanotube-mediated intercellular mitochondria exchange in cancer drug resistance
Intercellular communications play a major role in tissue homeostasis. In pathologies such as cancer, cellular interactions within the tumor microenvironment (TME) contribute to tumor progression and resistance to therapy. Tunneling nanotubes (TNTs) are newly discovered long-range intercellular connections that allow the exchange between cells of various cargos, ranging from ions to whole organelles such as mitochondria. TNT-transferred mitochondria were shown to change the metabolism and functional properties of recipient cells as reported for both normal and cancer cells. Metabolic plasticity is now considered a hallmark ...
Source: Biochemical Journal - July 31, 2018 Category: Biochemistry Authors: Hekmatshoar, Y., Nakhle, J., Galloni, M., Vignais, M.-L. Tags: Review Articles Source Type: research
Interplay between negative and positive design elements in G{alpha} helical domains of G proteins determines interaction specificity toward RGS2
Regulators of G protein signaling (RGS) proteins inactivate Gα subunits, thereby controlling G protein-coupled signaling networks. Among all RGS proteins, RGS2 is unique in interacting only with the Gαq but not with the Gαi subfamily. Previous studies suggested that this specificity is determined by the RGS domain and, in particular, by three RGS2-specific residues that lead to a unique mode of interaction with Gαq. This interaction was further proposed to act through contacts with the Gα GTPase domain. Here, we combined energy calculations and GTPase activity measurements to determine which G...
Source: Biochemical Journal - July 26, 2018 Category: Biochemistry Authors: Kasom, M., Gharra, S., Sadiya, I., Avital-Shacham, M., Kosloff, M. Tags: Research Articles Source Type: research
The chromatin nuclear protein NUPR1L is intrinsically disordered and binds to the same proteins as its paralogue
NUPR1 is a protumoral multifunctional intrinsically disordered protein (IDP), which is activated during the acute phases of pancreatitis. It interacts with other IDPs such as prothymosin α, as well as with folded proteins such as the C-terminal region of RING1-B (C-RING1B) of the Polycomb complex; in all those interactions, residues around Ala33 and Thr68 (the ‘hot-spot’ region) of NUPR1 intervene. Its paralogue, NUPR1L, is also expressed in response to DNA damage, it is p53-regulated, and its expression down-regulates that of the NUPR1 gene. In this work, we characterized the conformational preferences o...
Source: Biochemical Journal - July 26, 2018 Category: Biochemistry Authors: Neira, J. L., Lopez, M. B., Sevilla, P., Rizzuti, B., Camara-Artigas, A., Vidal, M., Iovanna, J. L. Tags: Research Articles Source Type: research
Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5K{gamma}
Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5K. So far, the exact role of this aspartate residue has yet to be elucidated. In this work, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site. Compared with th...
Source: Biochemical Journal - July 26, 2018 Category: Biochemistry Authors: Zeng, X., Uyar, A., Sui, D., Donyapour, N., Wu, D., Dickson, A., Hu, J. Tags: Research Articles Source Type: research
Characterization of the PLP-dependent transaminase initiating azasugar biosynthesis
Biosynthesis of the azasugar 1-deoxynojirimycin (DNJ) critically involves a transamination in the first committed step. Here, we identify the azasugar biosynthetic cluster signature in Paenibacillus polymyxa SC2 (Ppo), homologous to that reported in Bacillus amyloliquefaciens FZB42 (Bam), and report the characterization of the aminotransferase GabT1 (named from Bam). GabT1 from Ppo exhibits a specific activity of 4.9 nmol/min/mg at 30°C (pH 7.5), a somewhat promiscuous amino donor selectivity, and curvilinear steady-state kinetics that do not reflect the predicted ping-pong behavior typical of aminotransferases. A...
Source: Biochemical Journal - July 17, 2018 Category: Biochemistry Authors: Arciola, J. M., Horenstein, N. A. Tags: Research Articles Source Type: research
Systems analysis of metabolism in platelet concentrates during storage in platelet additive solution
Platelets (PLTs) deteriorate over time when stored within blood banks through a biological process known as PLT storage lesion (PSL). Here, we describe the refinement of the biochemical model of PLT metabolism, iAT-PLT-636, and its application to describe and investigate changes in metabolism during PLT storage. Changes in extracellular acetate and citrate were measured in buffy coat and apheresis PLT units over 10 days of storage in the PLT additive solution T-Sol. Metabolic network analysis of these data was performed alongside our prior metabolomics data to describe the metabolism of fresh (days 1–3), intermediate...
Source: Biochemical Journal - July 17, 2018 Category: Biochemistry Authors: Johannsson, F., Guthmundsson, S., Paglia, G., Guthmundsson, S., Palsson, B., Sigurjonsson, O. E., Rolfsson, O. Tags: Research Articles Source Type: research
Crystal structures of the kinase domain of PpkA, a key regulatory component of T6SS, reveal a general inhibitory mechanism
The type VI secretion system (T6SS) is a versatile and widespread export system found in many Gram-negative bacteria that delivers effector proteins into target cells. The functions of T6SSs are tightly regulated by diverse mechanisms at multiple levels, including post-translational modification through threonine phosphorylation via the Ser/Thr protein kinase (STPK) PpkA. Here, we identified that PpkA is essential for T6SS secretion in Serratia marcescens since its deletion eliminated the secretion of haemolysin co-regulated protein, while the periplasmic and transmembrane portion of PpkA was found to be disposable for T6S...
Source: Biochemical Journal - July 17, 2018 Category: Biochemistry Authors: Li, P., Xu, D., Ma, T., Wang, D., Li, W., He, J., Ran, T., Wang, W. Tags: Research Articles Source Type: research
Plant mitochondrial protein import: the ins and outs
The majority of the mitochondrial proteome, required to fulfil its diverse range of functions, is cytosolically synthesised and translocated via specialised machinery. The dedicated translocases, receptors, and associated proteins have been characterised in great detail in yeast over the last several decades, yet many of the mechanisms that regulate these processes in higher eukaryotes are still unknown. In this review, we highlight the current knowledge of mitochondrial protein import in plants. Despite the fact that the mechanisms of mitochondrial protein import have remained conserved across species, many unique feature...
Source: Biochemical Journal - July 17, 2018 Category: Biochemistry Authors: Ghifari, A. S., Gill-Hille, M., Murcha, M. W. Tags: Review Articles Source Type: research
Global conformational changes in IgG-Fc upon mutation of the FcRn-binding site are not associated with altered antibody-dependent effector functions
Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding si...
Source: Biochemical Journal - July 5, 2018 Category: Biochemistry Authors: Burvenich, I. J. G., Farrugia, W., Liu, Z., Makris, D., King, D., Gloria, B., Perani, A., Allan, L. C., Scott, A. M., Ramsland, P. A. Tags: Research Articles Source Type: research
Probing the specificity of CYP112 in bacterial gibberellin biosynthesis
Biosynthesis of the gibberellin A (GA) plant hormones evolved independently in plant-associated fungi and bacteria. While the relevant enzymes have distinct evolutionary origins, the pathways proceed via highly similar reactions. One particularly complex transformation involves combined demethylation and -lactone ring formation, catalyzed in bacteria by the cytochrome P450 CYP112 in three individual steps, which involves large structural changes in the transition from substrate to product, with further divergence in the recently demonstrated use of two separate mechanistic routes. Here, the substrate specificity of the iso...
Source: Biochemical Journal - July 5, 2018 Category: Biochemistry Authors: Nagel, R., Peters, R. J. Tags: Research Articles Source Type: research
