TDP-43 is a primarily nuclear RNA-binding protein, whose abnormal phosphorylation and cytoplasmic aggregation characterizes affected neurons in patients with amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that physiological nuclear TDP-43 in mouse and human brain forms homo-oligomers that are resistant to cellular stress. Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, as revealed by a 2.1 Å crystal structure in combination with nuclear magnetic resonance spectroscopy and electron microscopy. These head-to-tail TDP-43 oligomers are unique among known RNA-binding proteins and represent the functional form of the protein in vivo, since their destabilization results in loss of alternative splicing regulation of known neuronal RNA targets. Our findings indicate that N-terminal domain-driven oligomerization spatially separates the adjoining highly aggregation-prone, C-terminal low-complexity domains of consecutive TDP-43 monomers, thereby preventing low-complexity domain inter-molecular interactions and antagonizing the formation of pathologic aggregates.

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Highlights d Adaptation of Listeria phage serovar specificity through targeted RBP variations d High-resolution crystal structure of a Listeria phage receptor binding protein d Structure-guided design of RBP chimeras yields phages with predictable host ranges d Synthetic RBPs extend phage binding specificity (from SV 4b to 4a, 4b, 4d, 5, 6b)

Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.

BackgroundBiomedical knowledge bases (KB’s) have become important assets in life sciences. Prior work on KB construction has three major limitations. First, most biomedical KBs are manually built and curated, and cannot keep up with the rate at which new findings are published. Second, for automatic information extraction (IE), the text genre of choice has been scientific publications, neglecting sources like health portals and online communities. Third, most prior work on IE has focused on the molecular level or chemogenomics only, like protein-protein interactions or gene-drug relationships, or solely address highly specific topics such as drug effects.ResultsWe address these three limitations by a versatile and scalable approach to automatic KB construction. Using a small number of seed facts for distant supervision of pattern-based extraction, we harvest a huge number of facts in an automated manner without requiring any explicit training.We extend previous techniques for pattern-based IE with confidence statistics, and we combine this recall-oriented stage with logical reasoning for consistency constraint checking to achieve high precision. To our knowledge, this is the first method that uses consistency checking for biomedical relations. Our approach can be easily extended to incorporate additional relations and constraints.We ran extensive experiments not only for scientific publications, but also for encyclopedic health portals and online communities, creating different KB’s based on different configurations. We assess the size and quality of each KB, in terms of number of facts and precision. The best configured KB, KnowLife, contains more than 500,000 facts at a precision of 93% for 13 relations covering genes, organs, diseases, symptoms, treatments, as well as environmental and lifestyle risk factors.ConclusionKnowLife is a large knowledge base for health and life sciences, automatically constructed from different Web sources. As a unique feature, KnowLife is harvested from different text genres such as scientific publications, health portals, and online communities. Thus, it has the potential to serve as one-stop portal for a wide range of relations and use cases. To showcase the breadth and usefulness, we make the KnowLife KB accessible through the health portal (http://knowlife.mpi-inf.mpg.de).Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0549-5) contains supplementary material, which is available to authorized users.

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

Mitochondrial dysfunction has been implicated in many pathological conditions and diseases. The normal functioning of mitochondria relies on maintaining the inner mitochondrial membrane potential (also known as DJm) that is essential for ATP synthesis, Ca 2þ homeostasis, redox balance, and regulation of other key signaling pathways such as mitophagy and apoptosis. However, the detailed mechanisms by which DJm regulates cellular function remain incompletely understood, partially because of the difficulty of manipulating DJm with spatiotemporal resolution, reversibility, or cell type specificity. To address this need, we have developed a next generation optogenetic-based technique for controllable mitochondrial depolarization with light. We demonstrate successful targeting of the heterologous channelrhodopsin-2 fusion protein to the inner mitochondrial membrane and formation of functional cationic channels capable of light-induced selective DJm depolarization and mitochondrial autophagy. Importantly, we for the first time, to our knowledge, show that optogenetic-mediated mitochondrial depolarization can be well controlled to differentially influence the fate of cells expressing mitochondrial channelrhodopsin-2; whereas sustained moderate light illumination induces substantial apoptotic cell death, transient mild light illumination elicits cytoprotection via mitochondrial preconditioning. Finally, we show that Parkin overexpression exacerbates, instead of ameliorating, mitochondrial depolarization-mediated cell death in HeLa cells. In summary, we provide evidence that the described mitochondrial-targeted optogenetics may have a broad application for studying the role of mitochondria in regulating cell function and fate decision.

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