Protein quality control: U-box-containing E3 ubiquitin ligases join the fold

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.     

Identification of a candidate proteomic signature to discriminate multipotent and non-multipotent stromal cells

Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.     

Mechanisms for quality control of misfolded transmembrane proteins

To prevent the accumulation of misfolded and aggregated proteins, the cell has developed a complex network of cellular quality control (QC) systems to recognize misfolded proteins and facilitate their refolding or degradation. The cell faces numerous obstacles when performing quality control on transmembrane proteins. Transmembrane proteins have domains on both sides of a membrane and QC systems in distinct compartments must coordinate to monitor the folding status of the protein. Additionally, transmembrane domains can have very complex organization and QC systems must be able to monitor the assembly of transmembrane domains in the membrane. In this review, we will discuss the QC systems involved in repair and degradation of misfolded transmembrane proteins. Also, we will elaborate on the factors that recognize folding defects of transmembrane domains and what happens when misfolded transmembrane proteins escape QC and aggregate. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Stimulation of the autonomic nervous system in colorectal surgery: a study protocol for a randomized controlled trial

In this study we describe the sociodemographic characteristics of people participating in a clinical trial on the safety and immunogenicity of a H5N1 influenza vaccine and we identify the main motivations for joining it.     

Nitrogen dynamics of a boreal black spruce wildfire chronosequence

This study examined the nitrogen (N) dynamics of a black spruce (Picea mariana (Mill.) BSP)-dominated chronosequence in Manitoba, Canada. The seven sites studied each contained separate well- and poorly drained stands, originated from stand-killing wildfires, and were between 3 and 151 years old. Our goals were to (i) measure total N concentration ([N]) of all biomass components and major soil horizons; (ii) compare N content and select vegetation N cycle processes among the stands; and (iii) examine relationships between ecosystem C and N cycling for these stands. Vegetation [N] varied significantly by tissue type, species, soil drainage, and stand age; woody debris [N] increased with decay state and decreased with debris size. Soil [N] declined with horizon depth but did not vary with stand age. Total (live + dead) biomass N content ranged from 18.4 to 99.7 g N m⁻² in the well-drained stands and 37.8–154.6 g N m⁻² in the poorly drained stands. Mean soil N content (380.6 g N m⁻²) was unaffected by stand age. Annual vegetation N requirement (5.9 and 8.4 g N m⁻² yr⁻¹ in the middle-aged well- and poorly drained stands, respectively) was dominated by trees and fine roots in the well-drained stands, and bryophytes in the poorly drained stands. Fraction N retranslocated was significantly higher in deciduous than evergreen tree species, and in older than younger stands. Nitrogen use efficiency (NUE) was significantly lower in bryophytes than in trees, and in deciduous than in evergreen trees. Tree NUE increased with stand age, but overall stand NUE was roughly constant (∼ ∼150 g g⁻¹ N) across the entire chronosequence.     

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl⁻ channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRΔF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.     

The effects of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss): exposure duration and contribution of suspended particles

Municipal sewage effluents are complex mixtures of contaminants known to disrupt both immune and endocrine functions in aquatic organisms. The present study sought to determine the impacts of municipal effluent on the immune systems of juvenile rainbow trout (Oncorhynchus mykiss), by exposing specimens to low concentrations (0.01%, 0.1%, 1% or 10%) of sewage effluent for periods of 28 or 90 days. The soluble and insoluble fractions of the effluent were also studied to assess the contribution of fractions rich in microorganisms and particles on fish immune systems. To this end, the trout were also exposed to soluble and insoluble fractions of the effluent for a period of 28 days. Immunocompetence was assessed by the following three parameters: phagocytosis, natural cytotoxic cells (NCC) and blastogenesis of lymphocytes under mitogen stimulation. Fish exposed to the 1% sewage effluent concentration for 28 days had enhanced phagocytic activity; at 90 days, phagocytic activity was reduced. T and B lymphocyte proliferation in fish from both groups was similarly stimulated. Phagocytosis and NCC activities were influenced more by the insoluble fraction than the soluble fraction of the effluent. Conversely, mitogen-stimulated T lymphocyte proliferation was enhanced in cells of fish exposed to the soluble fraction of the effluents, with a dampening effect on the insoluble (particulate) fraction of the effluent. In conclusion, the effects of the effluent and its fractions were higher at the cellular-mediated immunity level than at the acquired immunity level. Immunotoxicity data on the soluble fraction of the effluent were more closely associated to data on the unfractionated effluent, but the contribution of the particulate fraction could not be completely ignored for phagocytosis and B lymphocyte proliferation.     

Swapping nucleotides, tuning Hsp70

Molecular chaperones such as heat shock protein 70 (Hsp70) are crucial for protein folding. Crystal structures of Hsp70 in a complex with the nucleotide exchange factor (NEF) Hsp110 reported in this issue of Cell (Polier et al., 2008) and in Molecular Cell (Schuermann et al., 2008) provide new insights into how NEF action specifies Hsp70 cellular function.