Keratin 6a reorganization for ubiquitin-proteasomal processing is a direct antimicrobial response.

Skin and mucosal epithelia deploy antimicrobial peptides (AMPs) to eliminate harmful microbes. We reported that the intermediate filament keratin 6a (K6a) is constitutively processed into antimicrobial fragments in corneal epithelial cells. In this study, we show that K6a network remodeling is a host defense response that directly up-regulates production of keratin-derived AMPs (KAMPs) by the ubiquitin-proteasome system (UPS). Bacterial ligands trigger K6a phosphorylation at S19, S22, S37, and S60, leading to network disassembly. Mutagenic analysis of K6a confirmed that the site-specific phosphorylation augmented its solubility. K6a in the cytosol is ubiquitinated by cullin-RING E3 ligases for subsequent proteasomal processing. Without an appreciable increase in K6a gene expression and proteasome activity, a higher level of cytosolic K6a results in enhanced KAMP production. Although proteasome-mediated proteolysis is known to produce antigenic peptides in adaptive immunity, our findings demonstrate its new role in producing AMPs for innate immune defense. Manipulating K6a phosphorylation or UPS activity may provide opportunities to harness the innate immunity of epithelia against infection.

Complete Genome Sequence Analysis of Bacillus subtilis T30.

The complete genome sequence of Bacillus subtilis T30 was determined by SMRT sequencing. The entire genome contains 4,138 predicted genes. The genome carries one intact prophage sequence (37.4 kb) similar to Bacillus phage SPBc2 and one incomplete prophage genome of 39.9 kb similar to Bacillus phage phi105.

Slippery substrates impair function of a bacterial protease ATPase by unbalancing translocation versus exit.

BACKGROUND: ATP-dependent proteases translocate and unfold their substrates. RESULTS: A human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure. CONCLUSION: Sequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains. SIGNIFICANCE: Compartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.

Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs.

Type IIS restriction endonuclease BtsCI (GGATG 2/0) is a neoschizomer of FokI (GGATG 9/13) and cleaves closer to the recognition sequence. Although M.BtsCI shows 62% amino acid sequence identity to M.FokI, BtsCI and FokI restriction endonucleases do not share significant amino acid sequence similarity. BtsCI belongs to a group of Type IIS restriction endonucleases, BsmI, Mva1269I and BsrI, that carry two different catalytic sites in a single polypeptide. By inactivating one of the catalytic sites through mutagenesis, we have generated nicking variants of BtsCI that specifically nick the bottom-strand or the top-strand of the target site. By treating target DNA sequentially with the appropriate combinations of FokI and BtsCI nicking variants, we are able to generate long overhangs suitable for fluorescent labeling through end-filling or other techniques based on annealing of complementary DNA sequences.

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA.

BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5'GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).

The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome.

Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by enzymatically depurinating a specific adenine residue at the sarcin-ricin loop of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation centre of the ribosome. Here, we present the 2.2 A crystal structure of trichosanthin (TCS) complexed to the peptide SDDDMGFGLFD, which corresponds to the conserved C-terminal elongation factor binding domain of the ribosomal P protein. The N-terminal region of this peptide interacts with Lys173, Arg174 and Lys177 in TCS, while the C-terminal region is inserted into a hydrophobic pocket. The interaction with the P protein contributes to the ribosome-inactivating activity of TCS. This 11-mer C-terminal P peptide can be docked with selected important plant and bacterial RIPs, indicating that a similar interaction may also occur with other RIPs.

Targeting DNA 5mCpG sites with chimeric endonucleases.

Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the (5)mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C(5)mCGG)-and M.SssI ((5)mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA ((5)mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified lambda DNA indicates that cleavage takes place outside the (5)mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N(4-17)(5)mCpG, where / indicates the cleavage site). Such (5)mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.