The transcriptional regulator GalR self-assembles to form highly regular tubular structures

The Gal repressor regulates transport and metabolism of D-galactose in Escherichia coli and can mediate DNA loop formation by forming a bridge between adjacent or distant sites. GalR forms insoluble aggregates at lower salt concentrations in vitro, which can be solubilized at higher salt concentrations. Here, we investigate the assembly and disassembly of GalR aggregates. We find that a sharp transition from aggregates to soluble species occurs between 200 and 400 mM NaCl, incompatible with a simple salting-in effect. The aggregates are highly ordered rod-like structures, highlighting a remarkable ability for organized self-assembly. Mutant studies reveal that aggregation is dependent on two separate interfaces of GalR. The highly ordered structures dissociate to smaller aggregates in the presence of D-galactose. We propose that these self-assembled structures may constitute galactose-tolerant polymers for chromosome compaction in stationary phase cells, in effect linking self-assembly with regulatory function

Alpha-Synuclein-Specific Naturally Occurring Antibodies Inhibit Aggregation In Vitro and In Vivo

Parkinson’s disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology

Structural Characterization of the Saccharomyces cerevisiae THO Complex by Small-Angle X-Ray Scattering

The THO complex participates during eukaryotic mRNA biogenesis in coupling transcription to formation and nuclear export of translation-competent messenger ribonucleoprotein particles. In Saccharomyces cerevisiae, THO has been defined as a heteropentamer composed of the Tho2p, Hpr1p, Tex1p, Mft1p, and Thp2p subunits and the overall three-dimensional shape of the complex has been established by negative stain electron microscopy. Here, we use small-angle X-ray scattering measured for isolated THO components (Mft1p and Thp2p) as well as THO subcomplexes (Mft1p-Thp2p and Mft1p-Thp2p-Tho2p) to construct structural building blocks that allow positioning of each subunit within the complex. To accomplish this, the individual envelopes determined for Mft1p and Thp2p are first fitted inside those of the Mft1p-Thp2p and Mft1p-Thp2p-Tho2p complexes. Next, the ternary complex structure is placed in the context of the five-component electron microscopy structure. Our model reveals not only the position of each protein in the THO complex relative to each other, but also shows that the pentamer is likely somewhat larger than what was observed by electron microscopy