Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.Extracellular matrix (ECM)¹ is a critical component of the tissue microenvironment. ECM plays a pivotal role in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years (7, 8). However, our ability to globally characterize ECM composition both in vitro and in vivo has been severely limited because of several unique attributes of ECM proteins such as high molecular weight glycans and the presence of covalent protein cross-links (6, 9, 10). Traditional proteomics approaches have proven to be ineffective for the identification of ECM proteins as demonstrated by the fact that collagens, despite being the most abundant protein in mammals, are significantly underrepresented in tissue-based proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for maximal and reproducible extraction and hence the accurate identification of small molecule and inorganic analytes (11). More recently, Capelo et al. (12) have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent mass spectrometry-based identification. Here we sought to determine whether this method could be optimized to prepare ECM samples for mass spectrometry-based analysis. For method development, we used rat tail collagen as a representative ECM protein for which current proteomics approaches have proven relatively unsuccessful. Type I collagen is defined as a right-handed triple helix heterotrimer comprising two identical α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion approach that improves our ability to perform proteomics analysis on a type I collagen preparation and then used this method to identify the protein composition of EHS murine chondrosarcoma matrix (10), herein referred to as Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion approach involves three cycles of ultrasonication for rapid initial trypsin digestion followed by overnight digestion using an acid-labile surfactant. This approach resulted in significant improvement in collagen peptide identification and the identification of numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The application of our ECM-optimized ultrasonic assisted trypsin digestion method is anticipated to significantly advance the identification of tissue- and disease state-specific ECM proteins.     

Receptor Specificity and Transmission of H2N2 Subtype Viruses Isolated from the Pandemic of 1957

Influenza viruses of the H2N2 subtype have not circulated among humans in over 40 years. The occasional isolation of avian H2 strains from swine and avian species coupled with waning population immunity to H2 hemagglutinin (HA) warrants investigation of this subtype due to its pandemic potential. In this study we examined the transmissibility of representative human H2N2 viruses, A/Albany/6/58 (Alb/58) and A/El Salvador/2/57 (ElSalv/57), isolated during the 1957/58 pandemic, in the ferret model. The receptor binding properties of these H2N2 viruses was analyzed using dose-dependent direct glycan array-binding assays. Alb/58 virus, which contains the 226L/228S amino acid combination in the HA and displayed dual binding to both alpha 2,6 and alpha 2,3 glycan receptors, transmitted efficiently to naïve ferrets by respiratory droplets. Inefficient transmission was observed with ElSalv/57 virus, which contains the 226Q/228G amino acid combination and preferentially binds alpha 2,3 over alpha 2,6 glycan receptors. However, a unique transmission event with the ElSalv/57 virus occurred which produced a 226L/228G H2N2 natural variant virus that displayed an increase in binding specificity to alpha 2,6 glycan receptors and enhanced respiratory droplet transmissibility. Our studies provide a correlation between binding affinity to glycan receptors with terminal alpha 2,6-linked sialic acid and the efficiency of respiratory droplet transmission for pandemic H2N2 influenza viruses.     

Metabolic connections during apoptotic cell engulfment

Billions of cells die via apoptosis every day and are swiftly removed. When a phagocyte engulfs an apoptotic cell, it essentially doubles its cellular contents, raising the question of how a phagocyte may manage the excess metabolic load. This Minireview discusses phagocyte cellular metabolism, the digestion of the ingested apoptotic cell, and the impact of these processes on engulfment.     

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.