Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Evaluation of exercise on individuals with dementia and their carers: a randomised controlled trial

Background Almost all of the 820,000 people in the UK with dementia will experience Behavioural and Psychological Symptoms of Dementia (BPSD). However, research has traditionally focused on treating cognitive symptoms, thus neglecting core clinical symptoms that often have a more profound impact on living with dementia. Recent evidence (Kales et al, 2007; Ballard et al, 2009) indicates that the popular approach to managing BPSD - prescription of anti-psychotic medication - can increase mortality and the risk of stroke in people with dementia as well as impair quality of life and accelerate cognitive decline. Consequently, there is a need to evaluate the impact that non-pharmacological interventions have on BPSD; we believe physical exercise is a particularly promising approach. Methods/Design We will carry out a pragmatic, randomised, single-blind controlled trial to evaluate the effectiveness of exercise (planned walking) on the behavioural and psychological symptoms of individuals with dementia. We aim to recruit 146 people with dementia and their carers to be randomized into two groups; one will be trained in a structured, tailored walking programme, while the other will continue with treatment as usual. The primary outcome (BPSD) will be assessed with the Neuropsychiatric Inventory (NPI) along with relevant secondary outcomes at baseline, 6 and 12 weeks. Discussion Designing this study has been challenging both ethically and methodologically. In particular to design an intervention that is simple, measurable, safe, non-invasive and enjoyable has been testing and has required a lot of thought. Throughout the design, we have attempted to balance methodological rigour with study feasibility. We will discuss the challenges that were faced and overcome in this paper

BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance

<p>Abstract</p> <p>Background</p> <p>The <it>BCR-ABL1 </it>translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). The advent of tyrosine kinase inhibitors (TKI) has fundamentally changed the treatment of CML. However, TKI are not equally effective for treating ALL. Furthermore, <it>de novo </it>or <it>secondary </it>TKI-resistance is a significant problem in CML. We screened a panel of <it>BCR-ABL1 </it>positive ALL and CML cell lines to find models for imatinib-resistance.</p> <p>Results</p> <p>Five of 19 <it>BCR-ABL1 </it>positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of <it>BCR-ABL1 </it>and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are <it>BCR-ABL1 </it>downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than <it>BCR-ABL1 </it>might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.</p> <p>Conclusion</p> <p>We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of <it>BCR-ABL1 </it>or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in <it>PIK3CA</it>, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.</p

Differences in pre-sleep activity and sleep location are associated with variability in daytime/nighttime sleep electrophysiology in the domestic dog

The domestic dog (Canis familiaris) is a promising animal model. Yet, the canine neuroscience literature is predominantly comprised of studies wherein (semi-)invasive methods and intensive training are used to study awake dog behavior. Given prior findings with humans and/or dogs, our goal was to assess, in 16 family dogs (1.5–7 years old; 10 males; 10 different breeds) the effects of pre-sleep activity and timing and location of sleep on sleep electrophysiology. All three factors had a main and/or interactive effect on sleep macrostructure. Following an active day, dogs slept more, were more likely to have an earlier drowsiness and NREM, and spent less time in drowsiness and more time in NREM and REM. Activity also had location- and time of day-specific effects. Time of day had main effects; at nighttime, dogs slept more and spent less time in drowsiness and awake after first drowsiness, and more time in NREM and in REM. Location had a main effect; when not at home, REM sleep following a first NREM was less likely. Findings are consistent with and extend prior human and dog data and have implications for the dog as an animal model and for informing future comparative research on sleep