Limits...
A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus

Cofactor and protein-only PrPSc are stably propagating recombinant PrP conformers that differ in their ability to template the conversion of native PrPC.(A) Western blot showing three-round sPMCA reactions using recombinant PrP as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated. (B) Western blot showing three-round sPMCA reactions using normal mouse brain homogenate as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4488359&req=5

ppat.1005017.g001: Cofactor and protein-only PrPSc are stably propagating recombinant PrP conformers that differ in their ability to template the conversion of native PrPC.(A) Western blot showing three-round sPMCA reactions using recombinant PrP as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated. (B) Western blot showing three-round sPMCA reactions using normal mouse brain homogenate as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated.

Mentions: Cofactor and protein-only PrPSc are distinct misfolded recombinant PrP conformers that differ >105-fold in their specific infectivity for wild-type mice [10]. While both of these conformers demonstrate autocatalytic activity when used to seed sPMCA reactions containing recombinant PrP substrate (Fig 1A and [10]), only cofactor PrPSc also demonstrates autocatalysis when used to seed sPMCA reactions containing normal brain homogenate as the substrate (Fig 1B and [10]). The complete failure of protein-only PrPSc to function as a seed for conversion reactions containing native PrPC substrate (Fig 1B, left sample group) provides a logical explanation for this conformer’s lack of infectious activity in vivo, and may apply more generally to other recombinant PrPSc conformers which demonstrate low levels of specific infectivity in bioassays. Using cofactor PrPSc as a well-matched control, we therefore sought to gain structural and mechanistic insight into the substrate-dependence of protein-only PrPSc autocatalytic activity as a means to understand the structural and functional determinants of recombinant PrPSc infectivity.


A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers.

Noble GP, Wang DW, Walsh DJ, Barone JR, Miller MB, Nishina KA, Li S, Supattapone S - PLoS Pathog. (2015)

Cofactor and protein-only PrPSc are stably propagating recombinant PrP conformers that differ in their ability to template the conversion of native PrPC.(A) Western blot showing three-round sPMCA reactions using recombinant PrP as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated. (B) Western blot showing three-round sPMCA reactions using normal mouse brain homogenate as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488359&req=5

ppat.1005017.g001: Cofactor and protein-only PrPSc are stably propagating recombinant PrP conformers that differ in their ability to template the conversion of native PrPC.(A) Western blot showing three-round sPMCA reactions using recombinant PrP as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated. (B) Western blot showing three-round sPMCA reactions using normal mouse brain homogenate as the substrate and seeded with full-length or PK-digested cofactor and protein-only PrPSc, as indicated.
Mentions: Cofactor and protein-only PrPSc are distinct misfolded recombinant PrP conformers that differ >105-fold in their specific infectivity for wild-type mice [10]. While both of these conformers demonstrate autocatalytic activity when used to seed sPMCA reactions containing recombinant PrP substrate (Fig 1A and [10]), only cofactor PrPSc also demonstrates autocatalysis when used to seed sPMCA reactions containing normal brain homogenate as the substrate (Fig 1B and [10]). The complete failure of protein-only PrPSc to function as a seed for conversion reactions containing native PrPC substrate (Fig 1B, left sample group) provides a logical explanation for this conformer’s lack of infectious activity in vivo, and may apply more generally to other recombinant PrPSc conformers which demonstrate low levels of specific infectivity in bioassays. Using cofactor PrPSc as a well-matched control, we therefore sought to gain structural and mechanistic insight into the substrate-dependence of protein-only PrPSc autocatalytic activity as a means to understand the structural and functional determinants of recombinant PrPSc infectivity.

Bottom Line: Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163.Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers.Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Medicine, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.

ABSTRACT
Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.

No MeSH data available.


Related in: MedlinePlus