- by Spiewla, T., Grab, K., Depaix, A., Ziemkiewicz, K., Warminski, M., Jemielity, J., Kowalska, J.IFIT proteins (interferon-induced proteins with tetratricopeptide repeats) are key components of the innate immune response that bind to viral and cellular RNA targets to inhibit viral translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5' ends. IFIT1 preferably binds RNAs modified with the 7-methylguanosine (m7G) cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize non-canonical RNA 5' ends, including […]
- by Kletzien, O. A., Wuttke, D. S., Batey, R. T.The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ~100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U's RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively […]
- by Zhang, N., Wu, J., Gao, S., Peng, H., Li, H., Gibson, C., Wu, S., Zhu, J., Zheng, Q.Serotonylation has been identified as a novel protein post-translational modification (PTM) for decades, where an isopeptide bond is formed between the glutamine residue and serotonin through transamination. Transglutaminase 2 (also known as TGM2 or TGase2) was proven to act as the main writer enzyme for this PTM and a number of key regulatory proteins (including small GTPases, fibronectin, fibrinogen, serotonin transporter, and histone H3) have been characterized as the substrates of serotonylation. However, due to the lack of pan-specific antibody […]
- by Birtwistle, M. R., Huggins, J. R., Zadeh, C. O., Sarmah, D., Srikanth, S., Jones, B. K., Cascio, L. N., Dean, D. R.Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ~10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to […]
- by Bartholomew, B., Garcia, J., Paul, S., Shukla, S., Zhong, Y., Beauchemin, K.The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80s ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of […]
- by Percival, M., Zweckstetter, M.Membrane-less organelles are spatially heterogenous deposits of interacting macromolecules, often intrinsically disordered proteins and RNA, that form and dissolve in response to cellular stimuli. How membrane-less organelles control composition while maintaining stimuli-responsiveness in an environment with competitive interactions is not well understood. Here we demonstrate that natural polyamines, which are found in all living organisms and help in many biological processes, promote protein phase separation via attractive interactions with acidic disordered domains. We show that the abundant polyamine spermine promotes […]
- by Salter, T., Collinson, I., Allen, W. J.There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity; yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realise this potential. Here, we have adapted our recently-developed in vitro assay for Sec activity – based on […]
- by Norman, B. P., Dillon, J. P., Alkharabsheh, S. M., Wilson, P. J., Davison, A. S., Chapman, E., Baker, J., Coyle, S., Probert, C. S., Ranganath, L. R., Gallagher, J. A.Bone resorption involves dissolution of mineral and enzymatic degradation of bone matrix. The primary enzyme is cathepsin K but other proteases including matrix metalloproteinases are involved. Some cleavage products of cathepsin K have been partially identified, including crossed-linked telopeptides of type I collagen. However, the pathway of type I bone collagen degradation has not been fully elucidated. The aim of this study was to comprehensively characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions […]
- by Fiore, A., Yu, G., Northey, J. J., Patel, R., Ravenscroft, T. A., Ikegami, R., Kolkman, W., Kumar, P., Grimm, J. B., Dilan, T. L., Ruetten, V. M. S., Ahrens, M. B., Shroff, H., Lavis, L. D., Wang, S., Weaver, V. M., Pedram, K.All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that […]
- by Mamone, L., Tomas, R. S., Di Venosa, G., Gandara, L., Durantini, E., Buzzola, F., Casas, A.Photodynamic inactivation (PDI) utilizes a photosensitizer (PS) activated by visible light to generate reactive oxygen species (ROS), to kill bacteria. PDI is effective against planktonic microorganisms, but biofilms are less sensitive due to limited PS and oxygen penetration. Near-infrared treatment (NIRT), involve the use of near-infrared light to kill bacteria either via thermal effects or ROS production. Our objective was to enhance S. aureus biofilm's sensitivity to PDI by pre-treating with NIR irradiation before visible light exposure. In an in […]
- by Fu, Z., MacKinnon, R.In this study we used cryo-electron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C-terminus, which forms two helical layers linked by a {beta}-strand, and coiled-coil domains […]
- by Wilson, R. H., Chatterjee, S., Smithwick, E. R., Rama Damodaran, A., Bhagi-Damodaran, A.Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts are powerful tools for late-stage C-H functionalization, as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations. Here, we describe the ability of a non-heme iron halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid […]
- by Garg, A., Shang, R., Cvetanovic, T., Lai, E. C., Joshua-Tor, L.miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has structural plasticity to accommodate different pri-miRNAs. These also revealed key structural features of the 5' UG sequence motif, more comprehensively represented […]
- by Briola, G., Tehseen, M., Al-Amodi, A., Nguyen, P. Q., Savva, C. G., Hamdan, S. M., De Biasio, A.Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The clamp loader CTF18 interacts with the leading strand polymerase Pol {epsilon} and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18 complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18, which form the regulatory module interacting with Pol {epsilon}, are […]
- by North, B. J., Ohnstad, A. E., Ragusa, M. J., Shoemaker, C. J.During autophagy, potentially toxic cargo is enveloped by a newly formed autophagosome and trafficked to the lysosome for degradation. Ubiquitinated protein aggregates, a key target for autophagy, are identified by multiple autophagy receptors. NBR1 is an archetypal autophagy receptor and an excellent model for deciphering the role of the multivalent, heterotypic interactions made by cargo-bound receptors. Using NBR1 as a model, we find that three critical binding partners – ATG8-family proteins, FIP200, and TAX1BP1 – each bind to a short […]
- by Santos, T. M. A., Thomson, B. D., Marquez, M. D., Pan, L., Monfared, T. H., Kahne, D. E.The assembly of {beta}-barrel proteins into membranes is mediated by the evolutionarily conserved BAM complex. In Escherichia coli, BAM folds numerous substrates which vary considerably in size and shape. How BAM is able to efficiently fold such a diverse array of {beta}-barrel substrates is not clear. Here, we develop a disulfide crosslinking method to trap native substrates in vivo as they fold on BAM. By placing a cysteine within the luminal wall of the BamA barrel as well as in […]
- by Wang, H., Blind, R. D., Shears, S. B.Inositol polyphosphate multikinase (IPMK) is a ubiquitously expressed kinase that has been linked to several cancers. Here, we report 14 new co-crystal structures (1.7 [A] – 2.0 [A] resolution) of human IPMK complexed with various IPMK inhibitors developed by another group. The new structures reveal two ordered water molecules that participate in hydrogen-bonding networks, and an unoccupied pocket in the ATP-binding site of human IPMK. New Protein Data Bank (PDB) codes of these IPMK crystal structures are: 8V6W(1.95 [A]), 8V6X(1.75 […]
- by Tiku, V., Tatsuta, T., Jung, M., Rapaport, D., Dimmer, K. S.Mitochondria derive the majority of their lipids from other organelles through contact sites. These lipids, primarily phosphoglycerolipids, are the main components of mitochondrial membranes. In the cell, neutral lipids like triacylglycerides (TAGs) are stored in lipid droplets, playing an important role in maintaining cellular health. Enzymes like lipases mobilize these TAGs according to cellular needs. Neutral lipids have not yet been reported to play an important role in mitochondria so the presence of a putative TAG lipase – Tgl2, in […]
- by Kozlov, G., Jiang, J., Rutherford, T., Noronha, A., Wilds, C., Gehring, K.La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanine substitutions. Remarkably, multiple guanine substitutions did not further increase binding affinity, demonstrating […]
- by Roberts, J. R., Horibata, Y., Kwarcinski, F. E., Lam, V., Raczkowski, A. M., Hubbard, A., Sugimoto, H., White, B., Tall, G. G., Ohi, M. D., Maeda, S.Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) those are the main phospholipids in eukaryotic cells. Here we present the structure […]
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