ABSTRACT
Protein-folding stress is characteristic of specialized secretory cells and plays a dominant role in a multitude of diseases. The unfolded protein response (UPR) thus triggered is a proteostatic signaling network that adapts the protein-folding capacity of the endoplasmic reticulum to the cellular demands. We have measured the binding affinities between human GRP78, an essential chaperone located in ER, and two transmembrane UPR sensors (human PERK and Ire1α), with or without the addition of an unfolded protein client. We reveal distinct binding affinities between the binary and ternary complexes thus formed, that suggest a preference for the PERK signaling branch under stress, and a predilection for the GRP78-UPR sensor complex formation upon stressor removal. These results imply a gated UPR mechanism that tunes the overall cellular behavior to the accumulation of unfolded proteins.
Subject(s)
Endoribonucleases/chemistry , Heat-Shock Proteins/chemistry , Molecular Docking Simulation , Protein Serine-Threonine Kinases/chemistry , Unfolded Protein Response , eIF-2 Kinase/chemistry , Binding Sites , Endoplasmic Reticulum Chaperone BiP , Endoribonucleases/ultrastructure , Heat-Shock Proteins/ultrastructure , Humans , Models, Chemical , Protein Binding , Protein Serine-Threonine Kinases/ultrastructure , eIF-2 Kinase/ultrastructureABSTRACT
The greater resistance of HCV genotype 1 infection to IFN therapy has been partially attributed to functional inhibition of the type 1 interferon induced anti-viral protein PKR in vitro. Whether PKR has antiviral activity against HCV in vivo is unknown. Whilst the ultra-structural localisation of PKR is known in vitro, it is not defined in chronic hepatitis C disease. Using a novel immuno-gold technique we characterised the expression of intrahepatic PKR protein at the ultra-structural level in four patients with chronic HCV disease compared to normal human PBMCs, HepG2 cells and a normal human liver biopsy. All four HCV patients labelled for PKR protein, localising to the nucleus, nucleolus and cytoplasm. Nuclear labelling was confined mainly to the nucleolus and euchromatin. Cytoplasmic labelling was evident within smooth vesicles. Strong immunogold labelling was also evident within the cisternae of the rough endoplasmic reticulum. A similar pattern of ultra-structural nuclear and cytoplasmic PKR protein labelling was seen in PBMCs from healthy donors, HepG2 cells and a normal liver biopsy. The mean nuclear and cytoplasmic count for PKR protein in the HCV group was 21 +/- 4 and 18 +/- 3 gold particles/microm(2), respectively. This represented an increase, though not statistically significant, in nuclear and cytoplasmic labelling for PKR protein in HCV biopsies relative to normal liver tissue.
Subject(s)
Hepatitis C, Chronic/enzymology , Hepatitis C, Chronic/virology , Hepatocytes/enzymology , Hepatocytes/ultrastructure , Immunohistochemistry/methods , eIF-2 Kinase/metabolism , eIF-2 Kinase/ultrastructure , Adult , Cell Nucleus/drug effects , Cell Nucleus/enzymology , Cell Nucleus/ultrastructure , Female , Hep G2 Cells , Hepatitis C, Chronic/pathology , Hepatocytes/drug effects , Hepatocytes/pathology , Humans , Interferon-alpha/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/ultrastructure , Liver/enzymology , Liver/pathology , Liver/ultrastructure , Liver/virology , Male , Middle Aged , Protein Transport/drug effects , Staining and LabelingABSTRACT
Specific factors that affect the resolution of single-particle reconstructions are discussed. We present reconstructions of six particles (DNA-dependent protein kinase catalytic subunit, alphaB-crystallin, the ribonucleoprotein vault, hepatitis A virus, adenovirus type 2, and the adenovirus type 12/alpha(v)beta5 integrin complex), which have a variety of symmetries (asymmetric to 60-fold) and a wide range of molecular masses (470 kDa to 150 MDa). In the case of icosahedral viruses, we have found that applying a "soft" mask to remove regions of disordered density improves the resolution given by the Fourier shell correlation 0.5 criterion. This masking procedure is also useful during refinement to improve the quality of the reference model and thus aid in precise alignment of the particle images. For asymmetric particles, we note that image classification, although often a necessary step to generate a first reconstruction, can limit the achievable resolution. The diameter of the particle and the available computational power can also affect the resolution, as can structural variability within the particle.
