Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies
In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibit...
Source: Biochemical Journal - August 8, 2019 Category: Biochemistry Authors: Scull, C. E., Zhang, Y., Tower, N., Rasmussen, L., Padmalayam, I., Hunter, R., Zhai, L., Bostwick, R., Schneider, D. A. Tags: Research Articles Source Type: research
Gelatinase B/matrix metalloproteinase-9 and other neutrophil proteases switch off interleukin-2 activity
Interleukin 2 (IL-2) is critical for T cell development and homeostasis, being a key regulator of adaptive immune responses in autoimmunity, hypersensitivity reactions and cancer. Therefore, its abundance in serum and peripheral tissues needs tight control. Here, we described a new mechanism contributing to the immunobiology of IL-2. We demonstrated, both in biochemical and cell-based assays, that IL-2 is subject to proteolytic processing by neutrophil matrix metalloproteinase-9 (MMP-9). IL-2 fragments produced after cleavage by MMP-9 remained linked by a disulfide bond and displayed a reduced affinity for all IL-2 recepto...
Source: Biochemical Journal - August 8, 2019 Category: Biochemistry Authors: Rybakin, V., Stas, M., Ugarte-Berzal, E., Noppen, S., Vandooren, J., Van Aelst, I., Liekens, S., Proost, P., Opdenakker, G. Tags: Research Articles Source Type: research
Dissecting the cytochrome c2-reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy
We examined the interaction of two components of bacterial photosynthesis, cytochrome c2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC–LH1–PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c2 molecules. Microscale patterns of cytochrome c2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c2. Followi...
Source: Biochemical Journal - August 8, 2019 Category: Biochemistry Authors: Vasilev, C., Mayneord, G. E., Brindley, A. A., Johnson, M. P., Hunter, C. N. Tags: Research Articles Source Type: research
A biochemical comparison of fungal GH6 cellobiohydrolases
Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investiga...
Source: Biochemical Journal - August 4, 2019 Category: Biochemistry Authors: Christensen, S. J., Krogh, K. B. R. M., Spodsberg, N., Borch, K., Westh, P. Tags: Research Articles Source Type: research
Large-scale chromatin organisation in interphase, mitosis and meiosis
The spatial configuration of chromatin is fundamental to ensure any given cell can fulfil its functional duties, from gene expression to specialised cellular division. Significant technological innovations have facilitated further insights into the structure, function and regulation of three-dimensional chromatin organisation. To date, the vast majority of investigations into chromatin organisation have been conducted in interphase and mitotic cells leaving meiotic chromatin relatively unexplored. In combination, cytological and genome-wide contact frequency analyses in mammalian germ cells have recently demonstrated that ...
Source: Biochemical Journal - August 4, 2019 Category: Biochemistry Authors: MacGregor, I. A., Adams, I. R., Gilbert, N. Tags: Review Articles Source Type: research
Structural insights into SUMO E1-E2 interactions in Arabidopsis uncovers a distinctive platform for securing SUMO conjugation specificity across evolution
SUMOylation of proteins involves the concerted action of the E1-activating enzyme, E2-conjugating enzyme and E3-ligases. An essential discrimination step in the SUMOylation pathway corresponds to the initial interaction between E1 ubiquitin-fold domain (UFD) and E2 enzymes. Although E2 orthologs possess high sequence identity, the E2 binding region of the UFD domains has diverged across evolution. Moreover, in reciprocal in vitro conjugation reactions Arabidopsis E1 and E2 SCE1 fail to interact efficiently with cognate human E2 Ubc9 and E1 partners, respectively. To gain more insights into the properties of this interface ...
Source: Biochemical Journal - July 30, 2019 Category: Biochemistry Authors: Liu, B., Lois, L. M., Reverter, D. Tags: Research Articles Source Type: research
Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism
In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.2 and BvHb2. Their affinities for oxygen, CO, and hexacoordination were determined. Their role in nitrogen metabolism was studied by assessing their ability to bind NO, to reduce nitrite (NiR, nitrite reductase), and to form nitrate (NOD, NO dioxygenase). Results show that BvHb1.2 has high NOD-like activity, in agreement with the high nitrate levels found in seeds where t...
Source: Biochemical Journal - July 30, 2019 Category: Biochemistry Authors: Leiva Eriksson, N., Reeder, B. J., Wilson, M. T., Bülow, L. Tags: Research Articles Source Type: research
The role of PTB domain containing adaptor proteins on PICALM-mediated APP endocytosis and localization
One hallmark of Alzheimer's disease (AD) is the presence of amyloid plaques, which mainly consist of the amyloid precursor protein (APP) cleavage product amyloid β (Aβ). For cleavage to occur, the APP must be endocytosed from the cell surface. The phosphatidylinositol binding clathrin assembly protein (PICALM) is involved in clathrin-mediated endocytosis and polymorphisms in and near the gene locus were identified as genetic risk factors for AD. PICALM overexpression enhances APP internalization and Aβ production. Furthermore, PICALM shuttles into the nucleus, but its function within the nucleus is still unk...
Source: Biochemical Journal - July 30, 2019 Category: Biochemistry Authors: Merthan, L., Haller, A., Thal, D. R., von Einem, B., von Arnim, C. A. F. Tags: Research Articles Source Type: research
Structural basis of glycogen metabolism in bacteria
We describe and discuss the remarkable progress made in the understanding of the molecular mechanisms of substrate recognition and product release, allosteric regulation and catalysis of all those enzymes. (Source: Biochemical Journal)
Source: Biochemical Journal - July 30, 2019 Category: Biochemistry Authors: Cifuente, J. O., Comino, N., Trastoy, B., D'Angelo, C., Guerin, M. E. Tags: Review Articles Source Type: research
Biochemical features of primary cells from a pediatric patient with a gain-of-function ODC1 genetic mutation
In conclusion, our patient and potentially other patients that carry a similar ODC1 gain-of-function mutation might benefit from treatment with DFMO, a drug with a good safety profile, to suppress the exceptionally high ODC activity and putrescine levels in the body. (Source: Biochemical Journal)
Source: Biochemical Journal - July 23, 2019 Category: Biochemistry Authors: Schultz, C. R., Bupp, C. P., Rajasekaran, S., Bachmann, A. S. Tags: Research Articles Source Type: research
Comprehensive analysis of yeast ESCRT-III composition in single ESCRT-III deletion mutants
The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our finding...
Source: Biochemical Journal - July 23, 2019 Category: Biochemistry Authors: Heinzle, C., Mücke, L., Brune, T., Kölling, R. Tags: Research Articles Source Type: research
Less photoprotection can be good in some genetic and environmental contexts
Antioxidant systems modulate oxidant-based signaling networks and excessive removal of oxidants can prevent beneficial acclimation responses. Evidence from mutant, transgenic, and locally adapted natural plant systems is used to interpret differences in the capacity for antioxidation and formulate hypotheses for future inquiry. We focus on the first line of chloroplast antioxidant defense, pre-emptive thermal dissipation of excess absorbed light (monitored as nonphotochemical fluorescence quenching, NPQ) as well as on tocopherol-based antioxidation. Findings from NPQ-deficient and tocopherol-deficient mutants that exhibite...
Source: Biochemical Journal - July 17, 2019 Category: Biochemistry Authors: Demmig-Adams, B., Stewart, J. J., Adams, W. W. Tags: Review Articles Source Type: research
The Mycobacterium tuberculosis capsule: a cell structure with key implications in pathogenesis
Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the Mycobacterium tuberculosis (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes t...
Source: Biochemical Journal - July 17, 2019 Category: Biochemistry Authors: Kalscheuer, R., Palacios, A., Anso, I., Cifuente, J., Anguita, J., Jacobs, W. R., Guerin, M. E., Prados-Rosales, R. Tags: Review Articles Source Type: research
Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic {beta}-oxidation trifunctional enzyme complexes
The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid β-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl...
Source: Biochemical Journal - July 14, 2019 Category: Biochemistry Authors: Sah-Teli, S. K., Hynönen, M. J., Schmitz, W., Geraets, J. A., Seitsonen, J., Pedersen, J. S., Butcher, S. J., Wierenga, R. K., Venkatesan, R. Tags: Research Articles Source Type: research
Structural basis for histone H3K4me3 recognition by the N-terminal domain of the PHD finger protein Spp1
Saccharomyces cerevisiae Spp1, a plant homeodomain (PHD) finger containing protein, is a critical subunit of the histone H3K4 methyltransferase complex of proteins associated with Set1 (COMPASS). The chromatin binding affinity of the PHD finger of Spp1 has been proposed to modulate COMPASS activity. During meiosis, Spp1 plays another role in promoting programmed double-strand break (DSB) formation by binding H3K4me3 via its PHD finger and interacting with a DSB protein, Mer2. However, how the Spp1 PHD finger performs site-specific readout of H3K4me3 is still not fully understood. In the present study, we determined the cry...
Source: Biochemical Journal - July 14, 2019 Category: Biochemistry Authors: He, C., Liu, N., Xie, D., Liu, Y., Xiao, Y., Li, F. Tags: Research Articles Source Type: research
