Structural insights into thermophilic chaperonin complexes.

Group I chaperonins are dual heptamer protein complexes that play significant roles in protein homeostasis. The structure and function of the Escherichia coli chaperonin are well characterized. However, the dynamic properties of chaperonins, such as large ATPase-dependent conformational changes by binding of lid-like co-chaperonin GroES, have made structural analyses challenging, and our understanding of these changes during the turnover of chaperonin complex formation is limited. In this study, we used single-particle cryogenic electron microscopy to investigate the structures of GroES-bound chaperonin complexes from the thermophilic hydrogen-oxidizing bacteria Hydrogenophilus thermoluteolus and Hydrogenobacter thermophilus in the presence of ATP and AMP-PNP. We captured the structure of an intermediate state chaperonin complex, designated as an asymmetric football-shaped complex, and performed analyses to decipher the dynamic structural variations. Our structural analyses of inter- and intra-subunit communications revealed a unique mechanism of complex formation through the binding of a second GroES to a bullet-shaped complex.

Metal-binding and folding thermodynamics of Escherichia coli ribonuclease HI related to its catalytic function.

Escherichia coli ribonuclease HI (RNH) hydrolyzes the RNA strands of RNA/DNA hybrids in the presence of Mg2+ at the highest level, relative to other metal ions. The Mg2+ binding affinity was 8.39 × 103 M-1, which was lower than those of other metal ions. The low-affinity binder can express the maximum catalytic activity of RNH. The stability of RNH increased with increasing metal ion concentration, except for Zn2+. The thermodynamic origin for enhancing the stability of RNH with Mg2+ was more favorable entropy compared to those with other metal ions, indicating that Mg2+ binding changes the RNH structure while maintaining flexibility. Upon H124A mutation, the metal ion binding affinities decreased for Mn2+ and Zn2+ to a relatively large extent. The present thermodynamic analyses provide information on the structural dynamics of RNH with metal ion exchangeable binding, which can reasonably explain the metal-ion-dependent catalytic activity.

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography.

The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme-Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme-Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.

Plant G proteins interact with endoplasmic reticulum luminal protein receptors to regulate endoplasmic reticulum retrieval.

Maintaining endoplasmic reticulum (ER) homeostasis is essential for the production of biomolecules. ER retrieval, i.e., the retrograde transport of compounds from the Golgi to the ER, is one of the pathways that ensures ER homeostasis. However, the mechanisms underlying the regulation of ER retrieval in plants remain largely unknown. Plant ERD2-like proteins (ERD2s) were recently suggested to function as ER luminal protein receptors that mediate ER retrieval. Here, we demonstrate that heterotrimeric G protein signaling is involved in ERD2-mediated ER retrieval. We show that ERD2s interact with the heterotrimeric G protein Gα and Gγ subunits at the Golgi. Silencing of Gα, Gß, or Gγ increased the retention of ER luminal proteins. Furthermore, overexpression of Gα, Gß, or Gγ caused ER luminal proteins to escape from the ER, as did the co-silencing of ERD2a and ERD2b. These results suggest that G proteins interact with ER luminal protein receptors to regulate ER retrieval.

MicroRNA-320a downregulation mediates human liver cancer cell proliferation through the Wnt/ß-catenin signaling pathway.

MicroRNAs (miRs) have emerged as key epigenetic regulators involved in cancer progression. miR-320a has been demonstrated to be a novel tumor suppressive microRNA in several types of cancers. In the present study, the role of miR-320a in human hepatocellular carcinoma (HCC) was investigated. The expression levels of miR-320a and messenger RNA were determined by reverse transcription-quantitative polymerase chain reaction, while cell cycle and cell apoptosis were analyzed by flow cytometry. The cell proliferative ability was determined by Cell Counting Kit-8 assay and colony formation assay. The downstream target of miR-320a was confirmed by luciferase reporter assay, while the protein levels were measured by western blotting. The results revealed that miR-320a was inversely associated with HCC proliferation in HCC cell lines. Functional studies demonstrated that miR-320a significantly decreased the capability of cell proliferation and induced G0/G1 growth arrest in vitro. In addition, ß-catenin was identified as one of the direct targets of miR-320a, downregulating the expression level of ß-catenin, c-myc, cyclin D1 and dickkopf-1. In conclusion, miR-320a may act as a tumor-suppressive microRNA through targeting ß-catenin in HCC.