Neural-tube specific paralogous genes and their upstream regulatory sequences

Ascidians are primitive chordates, and their critical evolutionary position expected to offer an excellent experimental system to understand the origin and the evolution of vertebrates (Satoh, 1994). Especially, neural tube is one of the characteristic features of chordates. In this study, we focused on the mechanism of the neural tube formation, and described the detailed expression profiles of two neural tube specific genes, CiNutl and CiNut2. Although the amount of CiNut2 expression is about 1/1000 compared to that of CiNutl, both two genes are expressed in the entire neural plate and neural tube during the course of the development. Both two genes are situated in an adjacent position of the chromosome in the same direction. Comparative analysis of the upstream regulatory sequence of two genes revealed the conserved sequences, which suggested having a role for the neural tube specific expression

Fusion protein analysis reveals the precise regulation between Hsp70 and Hsp100 during protein disaggregation

ClpB, a bacterial Hsp100, is a ring-shaped AAA+ chaperone that can reactivate aggregated proteins in cooperation with DnaK, a bacterial Hsp70, and its co-factors. ClpB subunits comprise two AAA+ modules with an interstitial rod-shaped M-domain. The M-domain regulates ClpB ATPase activity and interacts directly with the DnaK nucleotide-binding domain (NBD). Here, to clarify how these functions contribute to the disaggregation process, we constructed ClpB, DnaK, and aggregated YFP fusion proteins in various combinations. Notably, i) DnaK activates ClpB only when the DnaK substrate-binding domain (SBD) is in the closed conformation, affording high DnaK-peptide affinity; ii) although NBD alone can activate ClpB, SBD is required for disaggregation; and iii) tethering aggregated proteins to the activated ClpB obviates SBD requirements. These results indicate that DnaK activates ClpB only when the SBD tightly holds aggregated proteins adjacent to ClpB for effective disaggregation

The Structure of ClpB A Molecular Chaperone that Rescues Proteins from an Aggregated State

AbstractMolecular chaperones assist protein folding by facilitating their “forward” folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 Å long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation