Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis.

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.DOI: http://dx.doi.org/10.7554/eLife.13909.001

Aberrant Notch signaling is implicated in several cancers, including breast cancer. However, the mechanistic details of the specific receptors and function of ligand-mediated Notch signaling that promote breast cancer remains elusive. In our studies we show that DLL1, a Notch signaling ligand, is significantly overexpressed in ERα+ luminal breast cancer. Intriguingly, DLL1 overexpression correlates with poor prognosis in ERα+ luminal breast cancer, but not in other subtypes of breast cancer. In addition, this effect is specific to DLL1, as other Notch ligands (DLL3, JAGGED1, and JAGGED2) do not influence the clinical outcome of ERα+ patients. Genetic studies show that DLL1-mediated Notch signaling in breast cancer is important for tumor cell proliferation, angiogenesis, and cancer stem cell function. Consistent with prognostic clinical data, we found the tumor-promoting function of DLL1 is exclusive to ERα+ luminal breast cancer, as loss of DLL1 inhibits both tumor growth and lung metastasis of luminal breast cancer. Importantly, we find that estrogen signaling stabilizes DLL1 protein by preventing its proteasomal and lysososmal degradations. Moreover, estrogen inhibits ubiquitination of DLL1. Together, our results highlight an unexpected and novel subtype-specific function of DLL1 in promoting luminal breast cancer that is regulated by estrogen signaling. Our studies also emphasize the critical role of assessing subtype-specific mechanisms driving tumor growth and metastasis to generate effective subtype-specific therapeutics.

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Protein-protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein-protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein-protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α-Edr2 interaction was verified by co-localization analysis. Functionally, this p38α-Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α-MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)-MK5 and of the p38α-p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.

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12020 Background: Early referral to palliative care was associated with improved health-related quality of life (HRQL) and overall survival (OS) in a US phase 3 trial in lung cancer patients (pts). International studies in mixed cancer types have reported conflicting results. PEARL aimed to determine whether early referral to palliative care would improve HRQL, OS, and resource use in Australian pts with recently diagnosed, advanced thoracic cancers. Methods: Eligible participants (pts) in this unblinded, multi-centre, randomised, phase 3 trial had advanced thoracic cancers diagnosed within 60 days, and the ability to complete patient-rated outcome measures (PROMS). Pts were randomly allocated to early referral to palliative care (ER) or referral at clinician’s discretion (DR). All pts received standard oncological care. PROMS were completed at baseline, every 3-4 weeks for 6 months, then 6-8 weekly. The primary objective was to determine the frequency of sustained, substantial improvements in HRQL, defined as a 5-point improvement in the FACT-L Trial Outcome Index (TOI) maintained for at least 2 consecutive assessments. Secondary outcomes included OS, documentation of advanced care plan (ACP), PROM scores at 12 weeks, anxiety/depression (PROMIS-ED), lung cancer symptoms (FACT-L), global HRQL (ICECAP-SCM), carer-satisfaction and burden, and understanding of illness and prognosis. The accrual target of 200 gave 80% power (alpha 0.05) to detect an absolute improvement of 20% in the proportion of pts achieving the primary endpoint. Results: 113 pts and 78 carers were recruited when the trial closed for slow accrual. Pt characteristics were well balanced: 88 (75%) had NSCLC, 18 (16%) small cell and 7 (6%) mesothelioma. Median age was 69 (IQR 62-74), 63 (56%) were male; systemic anti-cancer therapy ongoing or planned in 88 (78%). Median follow-up was 30 months. First consultations with a palliative care specialist within 60 days of diagnosis occurred in more pts assigned ER vs DR (57% vs 3.5%). Sustained substantial improvements in FACT-L TOI were reported by similar numbers of pts assigned ER vs DR: 33% vs 32%, p = 0.9. OS was similar among those assigned ER versus DR (median 12 vs 18.4 months, p = 0.11). A similar % had a written advanced care plan at death: 15/40 (39%) vs 15/33 (47%). We found no important differences between arms in global HRQL (ICECAP-SCM), depression/anxiety (PROMIS-ED), lung cancer symptoms (FACT-L), carer satisfaction (FAMCARE-2), carer burden (CRA), or understanding of illness by carers or pts. Conclusions: Early referral to palliative care, compared with discretionary referral, did not improve important outcomes for Australian thoracic cancer pts or carers. Our findings suggest that the palliative care needs of such pts were addressed equally well by delayed referral when clinically indicated, resulting in reduced burden for resource-limited specialist palliative services. Clinical trial information: ACTRN12617000166370.