Online ISSN: X. Expression of a particular transmembrane form produced neurodegenerative changes in mice similar to those of some genetic prion diseases.Thus, aberrant regulation of protein biogenesis and topology at the endoplasmic reticulum can result in neurodegeneration.More enigmatic at the present time is our understanding of the biochemical and cell biological events that form the basis for the pathophysiological progression of neurodegeneration in prion diseases.One form appears to be fully translocated into the ER lumen and hence is termed the secretory form. Topology of a protein can be assessed by determining whether any regions of the molecule are accessible to proteases added to the outside of the membrane vesicles.Full protection from exogenous protease indicates complete translocation into the ER lumen.Conversely, digestion of certain domains to yield discrete proteaseprotected fragments indicates a membranespanning topology, the exact orientation of which can be clarif ied by identif ication of the protected fragments with epitopespecif ic antibodies.This use of proteases as a probe of topology is distinctly different from the use of proteases as probes of protein conformation. Because the topology assay is carried out in the absence of detergent, the protection from protease is due to an intact membrane barrier.To explore this hypothesis, wefirst identif ied four mutations within STETM that greatly alter the ratio of the topological forms when assayed by cellfree translation.The approximate sizes of the fragments generated from each form are indicated above the diagram.After translation, samples were either left untreated or digested with PK in the absence or presence of. All transgenic mice spanning three Naloxone hydrochloride generations developed clinical signs of prion disease, including Honokiol ataxia and paresis, the average age of onset was days, with the earliest development of symptoms at days. In contrast, none of the nontransgenic littermates exhibited any signs of illness.Numerals below some symbols indicate multiple individuals represented by that symbol.PrP in varying amounts of brain hoimmersionfixed brain section with antibodies to glial fibrillary acidic protein mogenate, demonstrating reactive astrocytic gliosis.While still in the ER, carbohydrates can be removed eff iciently with the enzyme endoglycosidase H. As a control, the resident ER protein GRP, which would not be expected to acquire endo H resistance, was also examined.Complete protection of GRP in the absence of detergent in each instance indicates that the PK had access to only the outside of the vesicles.H transgenic mouse were denatured in SDS and digested with either endo H, and the samples were analyzed by immunonormalized by adjusting the exposure time to the film.We explored this idea byfirst identifying a transgenic line of mice expressing the KH II mutation at low levels.First, this mutation lies in the hydrophobic domain. Unfortunately, assay of protein topology in human brain is problematic because fresh tissue suitable for subcellular fractionation is not readily available.Moreover, frozen specimens do not maintain adequate subcellular architecture, posing yet another obstacle to isolation of intact intracellular membranes that would be suitable for topologic studies.Such differences in conformation may be maintained in extracts from frozen brain and would be lost completely only upon denaturation.

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