Cofactor and Glycosylation Preferences for in vitro Prion Conversion are Predominantly Determined by Strain Conformation

Burke CM, Walsh DJ, Mark KMK, Deleault NR, Nishina KA, Agrimi U, Di Bari MA, Supattapone S

PLoS Pathog. 2020 Apr 15; 16(4):e1008495

Prion diseases are caused by the misfolding of a host-encoded glycoprotein, PrP^C, into a pathogenic conformer, PrP^Sc. Infectious prions can exist as different strains, composed of unique conformations of PrP^Sc that generate strain-specific biological traits, including distinctive patterns of PrP^Sc accumulation throughout the brain. Prion strains from different animal species display different cofactor and PrP^C glycoform preferences to propagate efficiently in vitro, but it is unknown whether these molecular preferences are specified by the amino acid sequence of PrP^C substrate or by the conformation of PrP^Sc seed. To distinguish between these two possibilities, we used bank vole PrP^C to propagate both hamster or mouse prions (which have distinct cofactor and glycosylation preferences) with a single, common substrate. We performed reconstituted sPMCA reactions using either (1) phospholipid or RNA cofactor molecules, or (2) di- or un-glycosylated bank vole PrP^C substrate. We found that prion strains from either species are capable of propagating efficiently using bank vole PrP^C substrates when reactions contained the same PrP^C glycoform or cofactor molecule preferred by the PrP^Sc seed in its host species. Thus, we conclude that it is the conformation of the input PrP^Sc seed, not the amino acid sequence of the PrP^C substrate, that primarily determines species-specific cofactor and glycosylation preferences. These results support the hypothesis that strain-specific patterns of prion neurotropism are generated by selection of differentially distributed cofactors molecules and/or PrP^C glycoforms during prion replication.

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 Jun 30;11(6):e1005017

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc 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 PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.

Prion protein glycosylation is not required for strain-specific neurotropism

Piro JR, Harris BT, Nishina K, Soto C, Morales R, Rees JR, Supattapone S

J Virol. 2009 Jun;83(11):5321-8

In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrP^Sc) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrP^C) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrP^Sc molecules. Both RML- and 301C-derived prions containing unglycosylated PrP^Sc molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrP^Sc deposition and neuronal vacuolation. These results show that PrP^Sc glycosylation is not necessary for strain-dependent prion neurotropism.

SarA of Staphylococcus aureus binds to the sarA promoter to regulate gene expression

Cheung AL, Nishina K, Manna AC

J Bacteriol. 2008 Mar;190(6):2239-43

The 375-bp sarA open reading frame is driven by three promoters, P1, P3, and P2. Using gel shift and DNase I footprinting assays, we found that SarA binds to two 26-bp sequences and one 31-bp sequence within the P1 and P3 promoters, respectively. Together with the results of transcription analyses, our data indicate that SarA binds to its own promoter to down-regulate sarA expression.

The SarA protein family of Staphylococcus aureus

Cheung AL, Nishina KA, Trotonda MP, Tamber S

Int J Biochem Cell Biol. 2008;40(3):355-61

Staphylococcus aureus is widely appreciated as an opportunistic pathogen, primarily in hospital-related infections. However, recent reports indicate that S. aureus infections can now occur in other wise healthy individuals in the community setting. The success of this organism can be attributed to the large array of regulatory proteins, including the SarA protein family, used to respond to changing microenvironments. Sequence alignment and structural data reveal that the SarA protein family can be divided into three subfamilies: 1) single domain proteins; 2) double domain proteins and 3) MarR homologs. Structural studies have also demonstrated that SarA, SarR, SarS, MgrA, and thus possibly all members of this protein family are winged helix proteins with minor variations. Mutagenesis studies of SarA disclose that the winged helix motifs are important for DNA binding and function. Recent progress concerning the functions and plausible mechanisms of regulation of SarA and its homologs are discussed.

Minocycline Modulates Neuroinflammation Independently of Its Antimicrobial Activity in Staphylococcus aureus-Induced Brain Abscess

Tammy Kielian, Nilufer Esen, Shuliang Liu, Nirmal K. Phulwani, Mohsin M. Syed, Napoleon Phillips, Koren Nishina†, Ambrose L. Cheung, Joseph D. Schwartzman, Jorg J. Ruhe

Am J Pathol. 2007 Oct;171(4):1199-214

Minocycline exerts beneficial immune modulatory effects in several noninfectious neurodegenerative disease models; however, its potential to influence the host immune response during central nervous system bacterial infections, such as brain abscess, has not yet been investigated. Using a minocycline-resistant strain of Staphylococcus aureus to dissect the antibiotic's bacteriostatic versus immune modulatory effects in a mouse experimental brain abscess model, we found that minocycline significantly reduced mortality rates within the first 24 hours following bacterial exposure. This protection was associated with a transient decrease in the expression of several proinflammatory mediators, including interleukin-1beta and CCL2 (MCP-1). Minocycline was also capable of protecting the brain parenchyma from necrotic damage as evident by significantly smaller abscesses in minocycline-treated mice. In addition, minocycline exerted anti-inflammatory effects when administered as late as 3 days following S. aureus infection, which correlated with a significant decrease in brain abscess size. Finally, minocycline was capable of partially attenuating S. aureus-dependent microglial and astrocyte activation. Therefore, minocycline may afford additional therapeutic benefits extending beyond its antimicrobial activity for the treatment of central nervous system infectious diseases typified by a pathogenic inflammatory component through its ability to balance beneficial versus detrimental inflammation.

Immunodetection of glycophosphatidylinositol-anchored proteins following treatment with phospholipase C

Nishina KA, Supattapone S

Anal Biochem. 2007 Apr 15;363(2):318-20

Many important regulatory proteins are attached to the extracellular leaflet of the plasma membrane by a glycophosphatidylinositol (GPI) anchor. One of the most common methods used to determine whether a protein contains a GPI anchor involves treating membranes with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC), followed by detection of the “released” candidate protein into the supernatant fraction by traditional Western blotting. Here, we report that the amounts of two representative GPI-anchored proteins, Thy-1 and the cellular prion protein (PrP^C), detected by Western blotting decreased >95% following PI-PLC treatment. The diminished signals appear to be caused by failure of the PI-PLC treated proteins to adhere to PVDF or nitrocellulose membranes, and could lead to false negative results in PI-PLC release assays. Detection of PI-PLC treated PrP^C molecules was successfully achieved by (1) slot blotting in the absence of SDS onto negatively charged Nylon membranes and (2) in-gel immunostaining.

The stoichiometry of host PrP^C glycoforms modulates the efficiency of PrP^Sc formation in vitro

Nishina KA, Deleault NR, Mahal SP, Baskakov I, Luhrs T, Riek R, Supattapone S

Biochemistry. 2006 Nov 28;45(47):14129-39

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrP^C, into a pathogenic isoform, PrP^Sc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrP^C molecules are required to propagate mouse RML prions, whereas diglycosylated PrP^C molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrP^C molecules. Thus, interactions between different PrP^C glycoforms appear to control the efficiency of prion formation in a species-specific manner.

Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions

Deleault NR, Geoghegan JC, Nishina K, Kascsak R, Williamson RA, Supattapone S

J Biol Chem. 2005 Jul 22;280(29):26873-9

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27–30 and PrP^C molecules at a molar ratio of 1:250 yielded ∼2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to ∼10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27–30, PrPC, and a stimulatory polyanionic compound.

Ionic strength and transition metals control PrP^Sc protease resistance and conversion-inducing activity

Nishina K, Jenks S, Supattapone S

J Biol Chem. 2004 Sep 24;279(39):40788-94

The essential component of infectious prions is a misfolded protein termed PrP^Sc, which is produced by conformational change of a normal host protein, PrP^C. It is currently unknown whether PrP^Sc molecules exist in a unique conformation or whether they are able to undergo additional conformational changes. Under commonly used experimental conditions, PrP^Sc molecules are characteristically protease-resistant and capable of inducing the conversion of PrP^C molecules into new PrP^Sc molecules. We describe the effects of ionic strength, copper, and zinc on the conformation-dependent protease resistance and conversion-inducing activity of PrP^Sc molecules in scrapie-infected hamster brains. In the absence of divalent cations, PrP^Sc molecules were >20-fold more sensitive to proteinase K digestion in low ionic strength buffers than in high ionic strength buffers. Addition of micromolar concentrations of copper or zinc ions restored the protease resistance of PrP^Sc molecules under conditions of low ionic strength. These transition metals also controlled the conformation of purified truncated PrP-(27–30) molecules at low ionic strength, confirming that the N-terminal octapeptide repeat region of PrP^Sc is not required for binding to copper or zinc ions. The protease-sensitive and protease-resistant conformations of PrP^Sc were reversibly interchangeable, and only the protease-resistant conformation of PrP^Sc induced by high ionic strength was able to induce the formation of new protease-resistant PrP (PrPres) molecules in vitro. These findings show that PrP^Sc molecules are structurally interconvertible and that only a subset of PrP^Sc conformations are able to induce the conversion of other PrP molecules.

In vitro prion protein conversion in detergent-solubilized membranes

Nishina K, Deleault NR, Lucassen RW, Supattapone S

Biochemistry. 2004 Mar 9;43(9):2613-21

A fundamental event in the pathogenesis of prion disease is the conversion of PrP^C, a normal glycophosphatidyl-anchored glycoprotein, into an infectious isoform designated PrP^Sc. In a modified version of the protein misfolding cyclic amplification (PMCA) technique [Saborio et al. (2001) Nature 411, 810−813], protease-resistant PrP^Sc-like molecules (PrPres) can be amplified in vitro in a species- and strain-specific manner from crude brain homogenates, providing a biochemical model of the prion conversion reaction [Lucassen et al. (2003) Biochemistry 42, 4127−4135]. In this study, we investigated the ability of enriched membrane subsets and detergent-solubilized membrane preparations to support PrPres amplification. Membrane fractionation experiments showed that purified synaptic plasma membrane preparations enriched in PrP^C but largely depleted of late endosomal and lysosomal markers were sufficient to support PrPres amplification. Detergent solubilization experiments showed that a small group of select detergents could be used to produce soluble preparations that contain PrP^C and fully support PrPres amplification. The stability of PrPres amplification ability in detergent-solubilized supernatants was dependent on detergent concentration. These results lead to the surprising conclusion that membrane attachment is not required for PrP^C to convert efficiently into PrPres in vitro and also indicate that biochemical purification of PrPres amplification factors from brain homogenates is a feasible approach.

Post-transcriptional suppression of pathogenic prion protein expression in Drosophila neurons

Deleault NR, Dolph PJ, Feany MB, Cook ME, Nishina K, Harris DA, Supattapone S

J Neurochem. 2003 Jun;85(6):1614-23

A wealth of evidence supports the view that conformational change of the prion protein, PrP^C, into a pathogenic isoform, PrP^Sc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrP^Sc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrP^Sc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.

In Vitro Amplification of Protease-Resistant Prion Protein Requires Free Sulfhydryl Groups

Ralf Lucassen, Koren Nishina, Surachai Supattapone

Biochemistry, 2003, 42 (14), pp 4127–4135

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP^Sc. Prion-infected neurons generate PrP^Sc from a host glycoprotein designated PrP^C through a process of induced conformational change, but the molecular mechanism by which PrP^C undergoes conformational change into PrP^Sc remains unknown. We employed an in vitro PrP^Sc amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP^Sc from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP^Sc amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP^Sc displays an optimum pH of ∼7, whereas amplification of CD-1 mouse RML PrP^Sc is optimized at pH ∼6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP^Sc amplification in vitro, indicating that the conformational change from PrP^C to PrP^Sc requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP^C to PrP^Sc.

Pharmacological approaches to prion research

Surachai Supattapone, Koren Nishina, Judy R. Rees

Volume 63, Issue 8, 15 April 2002, Pages 1383-1388

The “protein-only” mechanism by which infectious agents of prion diseases such as Creutzfeldt–Jakob disease and bovine spongiform encephalopathy replicate remains undetermined. The identification of several distinct classes of prion inhibitors has created an opportunity to investigate the mechanism of prion formation using pharmacological tools. These new inhibitors include substituted tricyclic derivatives, tetrapyrrole compounds, cysteine protease inhibitors, branched polyamines, and specific antibodies. Each inhibitor class contains at least one active compound that inhibits prion propagation in cell culture at sub-micromolar concentrations and several structurally related, inactive compounds. Work with branched polyamines and specific antibodies has already provided insight into the kinetics and cell biology of endogenous prion clearance mechanisms. Other anti-prion compounds do not appear to bind directly to the prion protein. Detailed investigation of the mechanism of drug action of these compounds may lead to the identification of novel prion propagation factors.

Identification and analysis of the polyhydroxyalkanoate-specific beta-ketothiolase and acetoacetyl coenzyme A reductase genes in the cyanobacterium Synechocystis sp. strain PCC6803

Taroncher-Oldenburg G, Nishina K, Stephanopoulos G

Appl Environ Microbiol. 2000 Oct;66(10):4440-8

Synechocystis sp. strain PCC6803 possesses a polyhydroxyalkanoate (PHA)-specific beta-ketothiolase encoded by phaA_Syn and an acetoacetyl-coenzyme A (CoA) reductase encoded by phaB_Syn. A similarity search of the entire Synechocystis genome sequence identified a cluster of two putative open reading frames (ORFs) for these genes, slr1993 and slr1994. Sequence analysis showed that the ORFs encode proteins having 409 and 240 amino acids, respectively. The two ORFs are colinear and most probably coexpressed, as revealed by sequence analysis of the promoter regions. Heterologous transformation of Escherichia coli with the two genes and the PHA synthase of Synechocystis resulted in accumulation of PHAs that accounted for up to 12.3% of the cell dry weight under high-glucose growth conditions. Targeted disruption of the above gene cluster in Synechocystis eliminated the accumulation of PHAs. ORFs slr1993 and slr1994 thus encode the PHA-specific beta-ketothiolase and acetoacetyl-CoA reductase of Synechocystis and, together with the recently characterized PHA synthase genes in this organism (S. Hein, H. Tran, and A. Steinbüchel, Arch. Microbiol. 170:162-170, 1998), form the first complete PHA biosynthesis pathway known in cyanobacteria. Sequence alignment of all known short-chain-length PHA-specific acetoacetyl-CoA reductases also suggests an extended signature sequence, VTGXXXGIG, for this group of proteins. Phylogenetic analysis further places the origin of phaA_Syn and phaB_Syn in the gamma subdivision of the division Proteobacteria.