The UCMD-Causing COL6A1 (c:930 + 189C > T) Intron Mutation Leads to the Secretion and Aggregation of Single Mutated Collagen VI α1 Chains

Collagen VI is a unique member of the collagen family. Its assembly is a complex multistep process and the vulnerability of the process is manifested in muscular diseases. Mutations in COL6A1, COL6A2, and COL6A3 lead to the severe Ullrich Congenital Muscular Dystrophy (UCMD) and a spectrum of disease of varying severity including the milder Bethlem muscular dystrophy. The recently identified dominant intronic mutation in COL6A1 ( c . 930 + 189 C > T ) leads to the partial in-frame insertion of a pseudoexon between exon 11 and exon 12. The pseudoexon is translated into 24 amino acid residues in the N-terminal region of the triple helix and results in the interruption of the typical G-X-Y motif. This recurrent de novo mutation leads to UCMD with a severe progression within the first decade of life. Here, we demonstrate that a mutation-specific antibody detects the mutant chain colocalizing with wild type collagen VI in the endomysium in patient muscle. Surprisingly, in the cell culture of patient dermal fibroblasts, the mutant chain is secreted as a single α chain, while in parallel, normal collagen VI tetramers are assembled with the wild-type α1 chain. The mutant chain cannot be incorporated into collagen VI tetramers but forms large aggregates in the extracellular matrix that may retain the ability to interact with collagen VI and potentially with other molecules. Also, α1 chain-deficient WI-26 VA4 cells transfected with the mutant α1 chain do not assemble collagen VI tetramers but, instead, form aggregates. Interestingly, both the wild type and the mutant single α1 chains form amorphous aggregates when expressed in HEK293 cells in the absence of α2 and α3 chains. The detection of aggregated, assembly incompetent, mutant collagen VI α1 chains provides novel insights into the disease pathophysiology of UCMD patients with the COL6A1 ( c . 930 + 189 C > T ) mutation

It takes two transducins to activate the cGMP-phosphodiesterase 6 in retinal rods

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein alpha-subunits (G alpha*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction-diffusion simulations. G alpha* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of G alpha* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 G alpha*. PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that G alpha*. PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of G alpha* which binds with lower affinity, forming G alpha*. PDE6. G alpha*. Reaction-diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of G alpha* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated G alpha* fails to activate the effector enzyme.This work was funded by Deutsche Forschungsgemeinschaftthrough grant nos. SP 1130/1-1 and SFB 449 to M.H., K.P.H. andC.M.T.S., SFB 740 to F.N., M.H., K.P.H., T.M. and C.M.T.S., a EuropeanResearch Council starting grant (pcCell) to F.N. and a EuropeanResearch Council advanced grant (TUDOR) to K.P.H. E.B. holds aFreigeist-Fellowship from the Volkswagen Foundatio

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs

Dataset for: A marine cryptochrome with an inverse photo-oligomerization mechanism

<p>Raw data for all sequence analysis, SEC runs, and UV/Vis absorbance readings supporting the findings of the study 'A marine cryptochrome with an inverse photo-oligomerization mechanism'</p&gt

Dataset for: A marine cryptochrome with an inverse photo-oligomerization mechanism

<p>Raw data for all sequence analysis, SEC runs, MD simulations, and UV/Vis absorbance readings supporting the findings of the study 'A marine cryptochrome with an inverse photo-oligomerization mechanism'</p&gt

Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation

Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming nucleotide-binding oligomerization domain (NOD)-like receptors with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) mediate oligomerization into filamentous assemblies. We describe the cryo-electron microscopy (cryo-EM) structure of the human NLRP3(PYD) filament and identify a pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends designated as A- and B-ends. Coupling a titration polymerization assay to cryo-EM, we demonstrate that ASC adaptor protein elongation on NLRP3(PYD) nucleation seeds is unidirectional, associating exclusively to the B-end of the filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing a continuous transition between NLRP3 and ASC. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3, ASC, and Caspase-1 proteins

A marine cryptochrome with an inverse photo-oligomerization mechanism

Abstract Cryptochromes (CRYs) are a structurally conserved but functionally diverse family of proteins that can confer unique sensory properties to organisms. In the marine bristle worm Platynereis dumerilii, its light receptive cryptochrome L-CRY (PdLCry) allows the animal to discriminate between sunlight and moonlight, an important requirement for synchronizing its lunar cycle-dependent mass spawning. Using cryo-electron microscopy, we show that in the dark, PdLCry adopts a dimer arrangement observed neither in plant nor insect CRYs. Intense illumination disassembles the dimer into monomers. Structural and functional data suggest a mechanistic coupling between the light-sensing flavin adenine dinucleotide chromophore, the dimer interface, and the C-terminal tail helix, with a likely involvement of the phosphate binding loop. Taken together, our work establishes PdLCry as a CRY protein with inverse photo-oligomerization with respect to plant CRYs, and provides molecular insights into how this protein might help discriminating the different light intensities associated with sunlight and moonlight

Direct Interaction of Avian Cryptochrome 4 with a Cone Specific G-Protein

Background: Night-migratory birds sense the Earth's magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the alpha-subunit of the cone-secific heterotrimeric G protein from European robin. Methods: Protein-protein interaction studies include surface plasmon resonance, pulldown affinity binding and Forster resonance energy transfer. Results: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Forster resonance energy transfer. Conclusions: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception