The Region II CEE represents an OGT-binding sequence.

<p>(A) Region II enhances OGT–HCF-1rep1 binding. Full-length (FL) and deletion HCF-1rep1 constructs were tested for OGT binding in the presence of UDP-GlcNAc using an <i>in vitro</i> OGT-directed pull-down assay. Detection of OGT and HCF-1rep1 was performed, using the indicated antibodies. Shown are 100% of OGT pull-down (panels a and b) and 11% of the input (panels c and d). *, IgG heavy chain. (B) HCF-1_PRO-repeat-independent OGT–Region II binding. (Left) Schematics of the HCF-1 constructs used in this experiment. (Right) HCF-1rep1 containing Region II and an OGT-binding defective HCF-1_PRO repeat (+II_T17–22A), or GST-fusion constructs containing Region II (wild-type or scrambled) alone or Region III alone (II_alone, II_scramb_alone, III_alone) were tested for binding with wild-type (WT) (left panel) or 5N-5A mutant (right panel) OGT. HCF-1 binding was detected as in (A). In (A) and (B), weak (⭕) and effective (●) OGT binding is indicated.</p

Region II CEE <i>O</i>-GlcNAcylation and HCF-1_PRO-repeat proteolysis are independent OGT activities.

<p>(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for <i>O</i>-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 <i>O</i>-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) <i>O</i>-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136636#pone.0136636.ref009" target="_blank">9</a>]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different <i>O</i>-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached <i>O</i>-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1_PRO repeats, or containing a deletion of the HCF-1_PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 <i>O</i>-GlcNAcylation is not fundamental for HCF-1_PRO-repeat cleavage. <i>In vitro</i> cleavage activities of wild-type OGT (WT) and an <i>O</i>-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and <i>O</i>-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D <i>O</i>-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.</p

HCF-1_PRO-repeat cleavage enhancement by a sequence nearby the HCF-1_PRO repeat 1.

<p>(A) HCF-1_PRO-repeat cleavage is context dependent <i>in vivo</i>. (Top) Schematic of the HCF-1rep1 precursor subdivided into Region I (25 residues, blue), Region II (58 residues, pink), and Region III (60 residues, gray). (Bottom) HEK 293 cells were transfected with expression vectors encoding HCF-1rep1 FL or deletion constructs, either containing or lacking Regions I, II or III. Proteins were immunoprecipitated by an N-terminal HA-tag and assayed for cleavage by visualization by α-HA-tag immunoblot. *, C-terminal precursor truncations. (B) Region II enhances HCF-1_PRO-repeat cleavage <i>in vitro</i>. Cleavage efficiency during an <i>in vitro</i> cleavage assay time course of selected HCF-1rep1 constructs. HCF-1rep1 constructs were incubated with OGT for 0 to 8 h and precursor and resulting N-terminal cleavage products were analyzed for cleavage by α-GST-immunoblot. Uncleaved and cleaved products were quantified and cleavage efficiencies determined as cleaved products over total. Shown are the means and standard deviations of three independent experiments. (C) Region II cleavage-enhancement activity is sequence specific. <i>In vitro</i> cleavage assay of HCF-1rep1 FL and Region II constructs containing a scrambled Region II sequence (+II_scrambled) or an inactive HCF-1_PRO repeat (+II_T17–22A). Resulting precursor and N-terminal cleavage products were analyzed for cleavage with the indicated antibodies. (D) Region II activates the inactive POUrep2 construct for cleavage. (Left) Schematic of the GST-fusion construct POUrep2 containing HCF-1_PRO repeat 2 (rep2), embedded in between the POU-specific (POU_S) and POU-homeo domains (POU_H) of Oct-1. Region II or Region III were inserted N-terminal of rep2, respectively. (Right) <i>In vivo</i> cleavage activities in HEK 293 cells, transiently transfected with transfection medium (mock) or POUrep2 encoding plasmids. Precursors and cleaved fragments were purified via immunoprecipitation of an N-terminal HA-tag and cleavage assayed using the indicated antibody. In (A), (C) and (D), prominent (●) and faint (⭕) cleavage products are indicated.</p

Ohjaamon pulpettilaitteiden, kattokonsolien ja näyttöjen keskitetyn himmennysjärjestelmän toteutusvaihtoehtojen kartoitus ja kehitys

Työn tarkoituksena kehittää laivan ohjaamoon keskitetty himmennysjärjestelmä. Himmennysjärjestelmään tulisi liittää pulpettilaitteita, kattokonsoleita ja näyttöjä. Esimerkki laivaksi otettiin NB 1378, Namibiaan toimitettava kalantutkimukseen menevä alus. Työ aloitettiin selvittämällä mitä laitteita aluksen ohjaamo sisältää ja tutkimalla niiden layout-piirustuksia, samalla selvittäen miten laitteet olisi mahdollista liittää uuteen himmennysjärjestelmään.The purpose of this thesis is the development of centralized dimming system for a ship. Dimming system should be connected to booth equipment, roof consoles and monitors. The example ship used in the thesis is NB 1378, fish research vessel. The work began by identifying the equipment that is located in the ship's bridge and deck and studying their layout drawings

Figure S2 from Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats

O-linked <i>N</i>-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site