Proteasome Activity Influences UV-Mediated Subnuclear Localization Changes of NPM

Peer reviewe

Optogenetic regulation of endogenous proteins

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets. Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.Peer reviewe

Identification of Novel p53 Pathway Activating Small-Molecule Compounds Reveals Unexpected Similarities with Known Therapeutic Agents

Peer reviewe

Nucleophosmin Phosphorylation by v-Cyclin-CDK6 Controls KSHV Latency

Nucleophosmin (NPM) is a multifunctional nuclear phosphoprotein and a histone chaperone implicated in chromatin organization and transcription control. Oncogenic Kaposi's sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). In the infected host cell KSHV displays two modes of infection, the latency and productive viral replication phases, involving extensive viral DNA replication and gene expression. A sustained balance between latency and reactivation to the productive infection state is essential for viral persistence and KSHV pathogenesis. Our study demonstrates that the KSHV v-cyclin and cellular CDK6 kinase phosphorylate NPM on threonine 199 (Thr199) in de novo and naturally KSHV-infected cells and that NPM is phosphorylated to the same site in primary KS tumors. Furthermore, v-cyclin-mediated phosphorylation of NPM engages the interaction between NPM and the latency-associated nuclear antigen LANA, a KSHV-encoded repressor of viral lytic replication. Strikingly, depletion of NPM in PEL cells leads to viral reactivation, and production of new infectious virus particles. Moreover, the phosphorylation of NPM negatively correlates with the level of spontaneous viral reactivation in PEL cells. This work demonstrates that NPM is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Nuclear actin is required for transcription during Drosophila Oogenesis

Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor (Mrtf), to chromatin remodelling and RNA polymerase (Pol) function. However, whether actin controls transcription of only specific genes or has a global role in gene expression has remained elusive. Our genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies majority of gene promoters together with Pol II, and on highly expressed genes these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies and manipulation of nuclear transport factors for actin leads to decreased expression of egg shell genes. Collectively, these results uncover a global role for actin in transcription, and demonstrate the in vivo importance of balanced nucleo-cytoplasmic shuttling of actin in transcriptional control of a developmental process.Peer reviewe

Ubiquitin recycling does not contribute to inhibition of NPM relocalization following UV radiation.

<p>U2OS cells were transfected with HA-tagged ubiquitin (<i>A</i>) or FLAG-tagged HAUSP (<i>B</i>). After 24 hours the cells were pretreated with MG132 followed by UV (35 J/m²) as shown and the cells were incubated for 6 hours. Cells were fixed and the expressed proteins were detected using HA- (<i>A</i>) or FLAG (<i>B</i>) -antibodies and co-stained for NPM. Nucleolar areas were quantified from three independent experiments. <i>C</i> U2OS cells stably expressing USP36-Flag were pretreated with MG132 followed by UV (35 J/m²) as shown and the cells were incubated for 3 hours. Cells were fixed and USP36 was detected using FLAG-antibody and cells were co-stained for NPM. Nucleolar areas were quantified. <i>D</i> U2OS cells were treated with UbE1 inhibitor (10 µM) or left untreated. After 24 hours the cells were exposed to UV (35 J/m²) and incubated for 3 hours. Cells were fixed and stained for NPM. Nucleolar areas were quantified from two independent experiments. Scale bars 20 µm.</p

UV-activated NPM relocalization is prevented by treatment with proteasome inhibitor.

<p><i>A</i> U2OS cells were treated with inhibitors targeting UV-activated cellular signaling (U0126 10 µM for MEK, SB203580 20 µM for p38 and SP600125 100 µM for JNK), DNA damage signaling (KU55933 10 µM for ATM, wortmannin 100 µM for ATM/ATR and NU7441 10 µM for DNA-PK) and proteasome (MG132 10 µM) or left untreated. One hour later the cells were exposed to UV radiation (35 J/m²) or left untreated. Cells were fixed after 3 hours and stained for NPM and UBF. Cells were imaged and intensities were quantified with Fiji-software using UBF as a nucleolar marker. The ratio of nucleolar and nucleoplasmic intensities was calculated from three independent experiments with two fields imaged per experiment. <i>P</i>-values were calculated using Student's T test, *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001. Error bars, SD. <i>N</i> ≥ 140 cells/analysis. <i>B</i> WS1 cells were treated with proteasome inhibitors MG132 (10 µM) or lactacystin (LC, 10 µM) for 1 hour prior to UV radiation (35 J/m²) or left untreated. The cells were fixed 6 hours later and stained for NPM. Scale bar 20 µm. <i>C</i> WS1 cells were treated with MG132 or left untreated. After 1 hour the cells were treated with UV radiation (35 J/m²) or left untreated. Cells were lysed 3 hours later into RIPA buffer. Equal amounts of total protein were separated by SDS-PAGE and immunoblotted for NPM. Tubulin was used as a loading control.</p

Nucleolar protein UV responses and proteasome inhibition are divergent and depend on the nucleolar subcompartment.

<p>WS1 cells were pretreated with MG132 followed by UV radiation (35 J/m²) as shown. Cells were fixed after 3 hours and stained for NCL and GNL3 (<i>A</i>), or FBL and UBF (<i>B</i>). Confocal images are shown for FBL and UBF (<i>B</i>). Scale bar 20 µm. <i>C</i> Western blotting analysis for the respective proteins. Equal amounts of total protein were separated by SDS-PAGE and immunoblotted for NCL, GNL3, FBL and UBF. Tubulin was used as a loading control.</p

Nucleolar mobility of NPM is altered after proteasome inhibition and UV damage.

<p>U2OS cells stably expressing NPM-ECGFP were treated either with MG132 (10 µM) for 4 hours, UV (35 J/m²), pretreated with MG132 for 1 hour followed by UV treatment (35 J/m²) and incubation for 3 hours, or left untreated (control). Averages of normalized intensities, mobile fractions (M_f) and recovery half-times (T_1/2) from at least three independent experiments for each treatment are shown. Error bars, SD. <i>N = </i>5–8 cells for each treatment.</p