Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome

<p>Abstract</p> <p>Background</p> <p>Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.</p> <p>Results</p> <p>We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCF^Skp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins <it>in vivo</it>. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.</p> <p>Conclusion</p> <p>SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.</p

Stimulation of DNA Glycosylase Activities by XPC Protein Complex: Roles of Protein-Protein Interactions

We showed that XPC complex, which is a DNA damage detector for nucleotide excision repair, stimulates activity of thymine DNA glycosylase (TDG) that initiates base excision repair. XPC appeared to facilitate the enzymatic turnover of TDG by promoting displacement from its own product abasic site, although the precise mechanism underlying this stimulation has not been clarified. Here we show that XPC has only marginal effects on the activity of E. coli TDG homolog (EcMUG), which remains bound to the abasic site like human TDG but does not significantly interacts with XPC. On the contrary, XPC significantly stimulates the activities of sumoylated TDG and SMUG1, both of which exhibit quite different enzymatic kinetics from unmodified TDG but interact with XPC. These results point to importance of physical interactions for stimulation of DNA glycosylases by XPC and have implications in the molecular mechanisms underlying mutagenesis and carcinogenesis in XP-C patients

Action of an endo-β-1,3(4)-glucanase on cellobiosyl unit structure in barley β-1,3:1,4-glucan.

β-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single β-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-β-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley β-1,3:1,4-glucan. To find the minor structure on which the endo-β-1,3(4)-glucanase acts, we prepared oligosaccharides from barley β-1,3:1,4-glucan by endo-β-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-β-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as β-Glc-1,3-β-Glc-1,4-β-Glc-1,3-β-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-β-1,3(4)-glucanase acts on the cellobiosyl units of barley β-1,3:1,4-glucan in an endo-manner.This work was supported in part by a grant-in-aid for Scientific Research to T. Kotake [Grant-in-Aid for Scientific Research no. 25514001] from Japan Society of the Promotion of Science; Y. Tsumuraya and T. Kotake [Grant-in-Aid for Scientific Research no. 24114006] from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Supports were also provided by BBSRC Sustainable Bioenergy Centre: Cell wall sugars program to P. Dupree [grant number BB/G016240/1].This is the final version of the article. It first appeared from Taylor & Francis via http://dx.doi.org/10.1080/09168451.2015.104636

Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.National Institutes of Health (U.S.) (Grant 5DP1-MH100706

Amphidinolide H, a Potent Cytotoxic Macrolide, Covalently Binds on Actin Subdomain 4 and Stabilizes Actin Filament

AbstractThe actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin

Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor Maelstrom

SummaryPIWI-interacting RNAs (piRNAs) protect the genome from transposons in animal gonads. Maelstrom (Mael) is an evolutionarily conserved protein, composed of a high-mobility group (HMG) domain and a MAEL domain, and is essential for piRNA-mediated transcriptional transposon silencing in various species, such as Drosophila and mice. However, its structure and biochemical function have remained elusive. Here, we report the crystal structure of the MAEL domain from Drosophila melanogaster Mael, at 1.6 Å resolution. The structure reveals that the MAEL domain has an RNase H-like fold but lacks canonical catalytic residues conserved among RNase H-like superfamily nucleases. Our biochemical analyses reveal that the MAEL domain exhibits single-stranded RNA (ssRNA)-specific endonuclease activity. Our cell-based analyses further indicate that ssRNA cleavage activity appears dispensable for piRNA-mediated transcriptional transposon silencing in Drosophila. Our findings provide clues toward understanding the multiple roles of Mael in the piRNA pathway

Structure of a tetrameric photosystem I from a glaucophyte alga Cyanophora paradoxa

Photosystem I (PSI) is one of the two photosystems functioning in light-energy harvesting, transfer, and electron transfer in photosynthesis. However, the oligomerization state of PSI is variable among photosynthetic organisms. We present a 3.8-angstrom resolution cryo-electron microscopic structure of tetrameric PSI isolated from the glaucophyte alga Cyanophora paradoxa, which reveals differences with PSI from other organisms in subunit composition and organization. The PSI tetramer is organized in a dimer of dimers with a C2 symmetry. Unlike cyanobacterial PSI tetramers, two of the four monomers are rotated around 90 degrees, resulting in a completely different pattern of monomer-monomer interactions. Excitation-energy transfer among chlorophylls differs significantly between Cyanophora and cyanobacterial PSI tetramers. These structural and spectroscopic features reveal characteristic interactions and excitation-energy transfer in the Cyanophora PSI tetramer, suggesting that the Cyanophora PSI could represent a turning point in the evolution of PSI from prokaryotes to eukaryotes

Mapping of histone-binding sites in histone replacement-completed spermatozoa

The majority of histones are replaced by protamines during spermatogenesis, but small amounts are retained in mammalian spermatozoa. Since nucleosomes in spermatozoa influence epigenetic inheritance, it is important to know how histones are distributed in the sperm genome. Conflicting data, which may result from different conditions used for micrococcal nuclease (MNase) digestion, have been reported: retention of nucleosomes at either gene promoter regions or within distal gene-poor regions. Here, we find that the swim-up sperm used in many studies contain about 10% population of sperm which have not yet completed the histone-to-protamine replacement. We develop a method to purify histone replacement-completed sperm (HRCS) and to completely solubilize histones from cross-linked HRCS without MNase digestion. Our results indicate that histones are retained at specific promoter regions in HRCS. This method allows the study of epigenetic status in mature sperm

Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K

AbstractThiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)4, like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase(+P15K)) possessed 0.86 Co atom/αβγ trimer and exhibited 78% of the activity of native SCNase. SCNase(+P15K) showed a UV–Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase(+P15K) had the γCys131-SO2H modification. These results indicate that SCNase(+P15K) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion