The RNA helicase ​Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes.

Aquarius is a multifunctional putative RNA helicase that binds precursor-mRNA introns at a defined position. Here we report the crystal structure of human Aquarius, revealing a central RNA helicase core and several unique accessory domains, including an ARM-repeat domain. We show that Aquarius is integrated into spliceosomes as part of a pentameric intron-binding complex (IBC) that, together with the ARM domain, cross-links to U2 snRNP proteins within activated spliceosomes; this suggests that the latter aid in positioning Aquarius on the intron. Aquarius's ARM domain is essential for IBC formation, thus indicating that it has a key protein-protein-scaffolding role. Finally, we provide evidence that Aquarius is required for efficient precursor-mRNA splicing in vitro. Our findings highlight the remarkable structural adaptations of a helicase to achieve position-specific recruitment to a ribonucleoprotein complex and reveal a new building block of the human spliceosome

Influence of laser polishing on the material properties of aluminium L-PBF components

In this study, the influence of laser polishing on the microstructural and mechanical properties of additively manufactured aluminium AlSi10Mg Laser Powder Bed Fusion (L-PBF) parts is analysed. The investigation is carried out on a 5-axis laser cell equipped with 1D Scanner optics driven by a solid-state disc laser at a wavelength of 1030 nm. Laser polishing is performed with pulsed or continuous laser radiation on samples in the initial L-PBF state or after stress relief treatment in a furnace. The metallurgical investigation of the remelting zone with a depth of 101–237 µm revealed an unchanged and homogeneous chemical composition, with a coarsened α-phase and a changed grain structure. The hardness within the remelting zone is reduced to 102–104 HV 0.1 compared to 146 HV 0.1 at the L-PBF initial state. Below the remelting zone, within the heat affected zone, a reduced microhardness, which can reach a thickness up to 1.5 mm, occurs. Laser polishing results in a reduction in residual stresses and resulting distortions compared to the L-PBF initial state. Nevertheless, the re-solidification shrinkage of the polished surface layer introduces additional tensions, resulting in sample distortions well above ones remaining after a stress relieve heat treatment of the initial state. The mechanical properties, analysed on laser polished flat tensile specimens, revealed an increase in the ultimate elongation from 4.5% to 5.4–10.7% and a reduction in the tensile strength from 346 N/mm2 to 247–271 N/mm2 through laser polishing. Hence, the strength resulting from this is comparable to the initial L-PBF specimens after stress relieve heat treatment

Probing the kinetics of quantum dot-based proteolytic sensors.

As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Forster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k (cat)/K (m) ratios. The range of k (cat)/K-m values derived is 0.5-1.1 mM(-1) s(-1) for the collagenase sensor and 3.7-4.2 mM(-1) s(-1) for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization

Part II: α-synuclein and its molecular pathophysiological role in neurodegenerative disease

α-Synuclein (αSN) brain pathology is a conspicuous feature of several neurodegenerative diseases. These include prevalent conditions such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and the Lewy body variant of Alzheimer’s disease (LBVAD), as well as rarer conditions including multiple systems atrophy (MSA), and neurodegeneration with brain iron accumulation type-1 (NBIA-1). Common in these diseases, some referred to as α-synucleinopathies, are microscopic proteinaceous insoluble inclusions in neurons and glia that are composed largely of fibrillar aggregates of αSN. This molecular form of αSN contrasts sharply with normal αSN, which is an abundant soluble presynaptic protein in brain neurons. αSN is a highly conserved protein in vertebrates and only seven of its 140 amino acids differ between human and mouse. Flies lack an αSN gene. Implicated in neurotoxicity are two αSN mutants (A53T and A30P) that cause extremely rare familial forms of PD, αSN fibrils and protofibrils, soluble protein complexes of αSN with 14-3-3 protein, and phosphorylated, nitrosylated, and ubiquitylated αSN species. Unlike rare forms of fPD caused by mutations in αSN, disease mechanisms in most α-synucleinopathies implicate wildtype αSN and seem to converge around oxidative damage and impairments in protein catabolism. It is not known whether these causalities involve αSN from the beginning, but defects in the handling of this protein seem to contribute to disease progression because accumulation of toxic αSN forms damage neurons. Here, we summarize the main structural features of αSN and its functions, and discuss the molecular αSN species implicated in human disease and transgenic animal models of α-synucleinopathy in fly and rodents

Tau stabilizes microtubules by binding at the interface between tubulin heterodimers.

The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer’s disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau–microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology

Dithiothreitol (DTT) acts as a specific, UV-inducible cross-linker in elucidation of protein-RNA interactions.

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs). The mass spectrometric analysis of such peptide-RNA cross-links provides high resolution structural data to the point of mapping protein-RNA interactions to specific peptides or even amino acids. However, the approach suffers from the low yield of cross-linking products, which can be addressed by improving enrichment and analysis methods. In the present article, we introduce dithiothreitol (DTT) as a potent protein-RNA cross-linker. In order to evaluate the efficiency and specificity of DTT, we used two systems, a small synthetic peptide from smB protein incubated with U1 snRNA oligonucleotide and native ribonucleoprotein complexes from S. cerevisiae. Our results unambiguously show that DTT covalently participates in cysteine-uracil crosslinks, which is observable as a mass increment of 151.9966 Da (C4H8S2O2) upon mass spectrometric analysis. DTT presents advantages for cross-linking of cysteine containing regions of proteins. This is evidenced by comparison to experiments where (tris(2-carboxyethyl)phosphine) is used as reducing agent, and significantly less cross-links encompassing cysteine residues are found. We further propose insertion of DTT between the cysteine and uracil reactive sites as the most probable structure of the cross-linking products