Florey Department of Neuroscience and Mental Health - Research Publications
Permanent URI for this collection
Search Results
1 - 5 of 5
-
ItemConservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-06-25)Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.
-
ItemOncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Exosomal Proteins Following Epithelial-Mesenchymal Transition(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2013-08)Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.
-
ItemNo Preview AvailablePathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality( 2014)Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrPC, into the disease-associated isoform, PrPSc. Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrPC share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrPSc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrPSc, giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion.IMPORTANCEPrion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment.
-
ItemNo Preview AvailableIntercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms(NATL ACAD SCIENCES, 2014-03-04)Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.
-
ItemNo Preview AvailablePrion-infected cells regulate the release of exosomes with distinct ultrastructural features(WILEY, 2012-10)Exosomes are small membrane-bound vesicles released from cells and found in vivo in most biological fluids. Functions reported for exosomes include cell-cell communication, roles in modulating immune responses, and roles in the transfer of pathogens such as prions. Here we investigated the molecular characteristics of the structure of exosomes that harbor prion infectivity to determine the native structure of exosomes and whether infected exosomes have a distinct structure. Cryo-electron tomography revealed the previously unidentified ultrastructural detail of exosomes with high resolution. Exosomes were found to be naturally spherical in shape and to have a diverse population that varies in size and internal structure, such as differences in the number of membrane structures. Exosomes isolated from prion-infected cells contained a significantly different population of exosomes with distinct structural features compared to control vesicles from mock-infected cells. Exosomes are highly structured vesicles that can modify their structure on altering their protein cargo. This finding provides further insight into the role that the exosomal protein cargo plays on influencing the structure of the vesicles as well as highlighting the diversity of exosomes and their relationship to biological processes.