Chemical Acetylation of Ligands and Two-Step Digestion Protocol for Reducing Codigestion in Affinity Purification-Mass Spectrometry.

We present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC-MS analysis, improving sensitivity and quantitative accuracy.

High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Seasonal variation in UVA light drives hormonal and behavioural changes in a marine annelid via a ciliary opsin.

The right timing of animal physiology and behaviour ensures the stability of populations and ecosystems. To predict anthropogenic impacts on these timings, more insight is needed into the interplay between environment and molecular timing mechanisms. This is particularly true in marine environments. Using high-resolution, long-term daylight measurements from a habitat of the marine annelid Platynereis dumerilii, we found that temporal changes in ultraviolet A (UVA)/deep violet intensities, more than longer wavelengths, can provide annual time information, which differs from annual changes in the photoperiod. We developed experimental set-ups that resemble natural daylight illumination conditions, and automated, quantifiable behavioural tracking. Experimental reduction of UVA/deep violet light (approximately 370-430 nm) under a long photoperiod (16 h light and 8 h dark) significantly decreased locomotor activities, comparable to the decrease caused by a short photoperiod (8 h light and 16 h dark). In contrast, altering UVA/deep violet light intensities did not cause differences in locomotor levels under a short photoperiod. This modulation of locomotion by UVA/deep violet light under a long photoperiod requires c-opsin1, a UVA/deep violet sensor employing Gi signalling. C-opsin1 also regulates the levels of rate-limiting enzymes for monogenic amine synthesis and of several neurohormones, including pigment-dispersing factor, vasotocin (vasopressin/oxytocin) and neuropeptide Y. Our analyses indicate a complex inteplay between UVA/deep violet light intensities and photoperiod as indicators of annual time.

A ubiquitin-like domain controls protein kinase D dimerization and activation by trans-autophosphorylation.

Protein kinase D (PKD) is an essential Ser/Thr kinase in animals and controls a variety of diverse cellular functions, including vesicle trafficking and mitogenesis. PKD is activated by recruitment to membranes containing the lipid second messenger diacylglycerol (DAG) and subsequent phosphorylation of its activation loop. Here, we report the crystal structure of the PKD N terminus at 2.2 Å resolution containing a previously unannotated ubiquitin-like domain (ULD), which serves as a dimerization domain. A single point mutation in the dimerization interface of the ULD not only abrogated dimerization in cells but also prevented PKD activation loop phosphorylation upon DAG production. We further show that the kinase domain of PKD dimerizes in a concentration-dependent manner and autophosphorylates on a single residue in its activation loop. We also provide evidence that PKD is expressed at concentrations 2 orders of magnitude below the ULD dissociation constant in mammalian cells. We therefore propose a new model for PKD activation in which the production of DAG leads to the local accumulation of PKD at the membrane, which drives ULD-mediated dimerization and subsequent trans-autophosphorylation of the kinase domain.

A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription.

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.

Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.