Identification of novel PANDAR protein interaction partners involved in splicing regulation

Interactions of long non-coding RNAs (lncRNA) with proteins play important roles in the regulation of many cellular processes. PANDAR (Promotor of CDKN1A Antisense DNA damage Activated RNA) is a lncRNA that is transcribed in a p53-dependent manner from the CDKN1A promoter and is involved in the regulation of proliferation and senescence. Overexpression of PANDAR has been observed in several tumor species and correlated with a poor prognosis for patient survival rate. Depending on the cellular state, PANDAR is known to interact with proteins such as the nuclear transcription factor Y subunit A (NF-YA) and the scaffold attachment factor A (SAF-A). However, a comprehensive analysis of the PANDAR interactome was missing so far. Therefore, we applied peptide nucleic acid (PNA)-based pull-downs combined with quantitative mass spectrometry to identify new protein binding partners. We confirmed potential candidates like U2AF65 and PTBP1, known to be involved in RNA processing. Furthermore, we observed that overexpression of PANDAR leads to a reduced level of the short proapoptotic BCL-X splice variant (BCL-XS) which is regulated by PTBP1. Simultaneous overexpression of PTBP1 was able to rescue this effect. Overall, our data suggest a role for PANDAR in the regulation of splicing events via its interaction partner PTBP1

Cell permeable stapled peptide inhibitor of Wnt signaling that targets β-catenin protein‒protein interactions

The Wnt signaling pathway plays a critical role in cell proliferation and differentiation, thus it is often associated with diseases such as cancers. Unfortunately, although attractive, developing anti-cancer strategy targeting Wnt signaling has been challenging given that the most attractive targets are involved in protein-protein interactions (PPIs). Here, we develop a stapled peptide inhibitor that targets the interaction between β-catenin and T cell factor/lymphoid enhancer-binding factor transcription factors, which are crucially involved in Wnt signaling. Our integrative approach combines peptide stapling to optimize proteolytic stability, with lessons learned from cell-penetrating peptide (CPP) design to maximize cellular uptake resulting in NLS-StAx-h, a selective, cell permeable, stapled peptide inhibitor of oncogenic Wnt signaling that efficiently inhibits β-catenin-transcription factor interactions. We expect that this type of integrative strategy that endows stapled peptides with CPP features will be generally useful for developing inhibitors of intracellular PPIs

Proteomic snapshot of the EGF-induced ubiquitin network

In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways

Rates and regulation of nitrogen cycling in seasonally hypoxic sediments during winter (Boknis Eck, SW Baltic Sea): Sensitivity to environmental variables

This study investigates the biogeochemical processes that control the benthic fluxes of dissolved nitrogen (N) species in Boknis Eck - a 28 m deep site in the Eckernförde Bay (southwestern Baltic Sea). Bottom water oxygen concentrations (O2-BW) fluctuate greatly over the year at Boknis Eck, being well-oxygenated in winter and experiencing severe bottom water hypoxia and even anoxia in late summer. The present communication addresses the winter situation (February 2010). Fluxes of ammonium (NH4+), nitrate (NO3-) and nitrite (NO2-) were simulated using a benthic model that accounted for transport andbiogeochemical reactions and constrained with ex situ flux measurements and sediment geochemical analysis. The sediments were a net sink for NO3- (-0.35 mmol m-2 d-1 of NO3-), of which 75% was ascribed to dissimilatory reduction of nitrate to ammonium (DNRA) by sulfide oxidizing bacteria, and 25% to NO3- reduction to NO2- by denitrifying microorganisms. NH4+ fluxes were high (1.74 mmol m-2d-1 of NH4+), mainly due to the degradation of organic nitrogen, and directed out of the sediment. NO2-fluxes were negligible. The sediments in Boknis Eck are, therefore, a net source of dissolved inorganic nitrogen(DIN = NO3- + NO2- + NH4+) during winter. This is in large part due to bioirrigation, which accounts for 76% of the benthic efflux of NH4+, thus reducing the capacity for nitrification of NH4+. The combined rate of fixed N loss by denitrification and anammox was estimated at 0.08 mmol m-2 d-1 of N2, which is at the lower end of previously reported values. A systematic sensitivity analysis revealed that denitrification and anammox respond strongly and positively to the concentration of NO3- in the bottomwater (NO3-BW).Higher O2-BW decreases DNRA and denitrification but stimulates both anammox and the contribution ofanammox to total N2 production (%Ramx). A complete mechanistic explanation of these findings is provided. Our analysis indicates that nitrification is the geochemical driving force behind the observed correlation between %Ramx and water depth in the seminal study of Dalsgaard et al. (2005). Despite remaining uncertainties, the results provide a general mechanistic framework for interpreting the existing knowledge of N-turnover processes and fluxes in continental margin sediments, as well as predicting the types of environment where these reactions are expected to occur prominently

Sleep-wake cycles drive daily dynamics of synaptic phosphorylation

The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures

In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface-exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability