The chromatin regulator BRPF3 preferentially activates the HBO1 acetyltransferase but is dispensable for mouse development and survival [Gene Regulation]

December 16th, 2015 by

To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and PHD finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two PHD fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (a.k.a. KAT7) and two other subunits, but not with related acetyltransferases such as MOZ, MORF, TIP60 and hMOF (a.k.a. KAT6A, KAT6B, KAT5 and KAT8, respectively). We have also characterized a mutant mouse strain with a LacZ reporter inserted at the Brpf3 locus. Systematic analysis of β-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-qPCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The chromatin regulator BRPF3 preferentially activates the HBO1 acetyltransferase but is dispensable for mouse development and survival [Gene Regulation]

Modulation of potassium channels inhibits bunyavirus infection [Molecular Bases of Disease]

December 16th, 2015 by

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

NANOBODIES AS PROBES FOR PROTEIN DYNAMICS IN VITRO AND IN CELLS [Molecular Biophysics]

December 16th, 2015 by Dmitriev, O. Y., Lutsenko, S., Muyldermans, S.

Nanobodies are the recombinant antigen-recognizing domains of the minimalistic heavy-chain only antibodies produced by camels and llamas. Nanobodies can be easily generated, effectively optimized, and variously derivatized with standard molecular biology protocols. These properties have triggered the recent explosion in the nanobody use in basic and clinical research. This review focuses on the emerging use of nanobodies for understanding and monitoring protein dynamics on the scales ranging from isolated protein domains to live cells, from nanoseconds to hours. The small size and high solubility make nanobodies uniquely suited for studying protein dynamics by NMR. The ability to produce conformation-sensitive nanobodies in cells enables studies that link structural dynamics of a target protein to its cellular behavior. The link between in vitro and in-cell dynamics, afforded by nanobodies, brings the analysis of such important events as receptor signaling, membrane protein trafficking, and protein interactions to the next level of resolution.

Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology [Cell Biology]

December 16th, 2015 by

Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate ″indefinite″ ex vivo primary intestinal epithelial cultures, termed ″mini-intestines″. Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.

Deficiency of Neuronal p38{alpha}-MAPK Attenuates Amyloid Pathology in Alzheimer’s Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1 [Molecular Bases of Disease]

December 16th, 2015 by

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer's disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer's amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α-MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α-MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α-MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of p38α-MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α-MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Deficiency of Neuronal p38{alpha}-MAPK Attenuates Amyloid Pathology in Alzheimer’s Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1 [Molecular Bases of Disease]

A Unique Tool for Cellular Structural Biology: In-cell NMR [Cell Biology]

December 16th, 2015 by Luchinat, E., Banci, L.

Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When doing so, important structural and functional features of macromolecules may be lost, which depend on their native network of interactions within the cell. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches, and focus on the applications to protein folding, interactions and post-translational modifications.

Introduction: Modern Technologies for In-Cell Biochemistry [Protein Structure and Folding]

December 16th, 2015 by Lutsenko, S.

The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy, development of new fluorescent reagents, and genetic editing have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or non-tagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their action be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This Thematic Series summarizes recent studies that illustrate some first steps towards successfully answering these modern biochemical questions. The first Minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second Minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third Minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells.

Mutational constraints on local unfolding inhibit the rheological adaptation of von Willebrand factor [Molecular Bases of Disease]

December 16th, 2015 by

Unusually large von Willebrand factor (ULVWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease (VWD), two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to H1322 across the β2-β3 hairpin in the GPIbα binding interface which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes Both tRNASer and tRNALeu [RNA]

December 16th, 2015 by Ji, Q.-Q., Fang, Z.-P., Ye, Q., Ruan, Z.-R., Zhou, X.-L., Wang, E.-D.

Leucyl-tRNA synthetase (LeuRS) is a multi-domain enzyme that catalyzes Leu-tRNALeu formation and is classified into bacterial and archaeal/eukaryotic types, with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNALeu through different modes of interaction. In the human pathogen, Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNASer [CatRNASer(CAG)] in addition to its cognate CatRNALeu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNASer, suggesting the significance of their highly divergent CTDs in tRNASer recognition. The results of this study provided the first evidence of the indispensable function of eukaryotic LeuRS's CTD in recognizing non-cognate CatRNASer and cognate CatRNALeu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knockout strain.

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy [Glycobiology and Extracellular Matrices]

December 16th, 2015 by Kunze, G., Kohling, S., Vogel, A., Rademann, J., Huster, D.

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10-GAG interaction is unclear. In particular, experimental evidence about IL-10-GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Whereas no co-crystal structure of the IL-10-GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts (PCSs) of IL-10 and its heparin ligand as induced by a protein-attached lanthanide spin label provided structural restraints for the protein-ligand complex. Using these restraints, PCS-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines R102, R104, R106, R107 and lysines K117 and K119. This study represents the first experimental characterization of the IL-10-GAG complex structure and provides the starting point for revealing the biological significance of IL-10's interaction with GAGs.