Inhibition of Deubiquitinase Activity and Ubiquitination of Jak2 Blocks Cytokine Signaling and Induces Tumor Cell Apoptosis

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid ubiquitination and subsequent re-localization of Jak2 into signaling incompetent aggresomes. In addition to Jak2, WP1130 also induces accumulation of other ubiquitinated proteins without inhibiting 20S proteasome activity. Further analysis of the mechanism of action of WP1130 revealed that WP1130 acts as a partly selective DUB inhibitor. It specifically inhibits the deubiquitinase activity of USP9x, USP5, USP14 and UCH37. WP1130 mediated inhibition of tumor-associated DUBs resulted in down-regulation of anti-apoptotic and up-regulation of pro-apoptotic proteins, such as MCL-1 and p53 respectively. Our results demonstrate that chemical modification of a previously described Jak2 inhibitor results in the unexpected discovery of a novel compound which acts as a DUB inhibitor, suppressing Jak-Stat signaling by a novel mechanism

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT

The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities

O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1 <sub>PRO</sub> repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1 <sub>PRO</sub> repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1 <sub>PRO</sub> -repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor R <sub>p</sub> -αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1 <sub>PRO</sub> -repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1 <sub>PRO</sub> -repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies

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

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