Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

PRP and BMAC for Musculoskeletal Conditions via Biomaterial Carriers.

Platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) are orthobiologic therapies considered as an alternative to the current therapies for muscle, bone and cartilage. Different formulations of biomaterials have been used as carriers for PRP and BMAC in order to increase regenerative processes. The most common biomaterials utilized in conjunction with PRP and BMAC clinical trials are organic scaffolds and natural or synthetic polymers. This review will cover the combinatorial strategies of biomaterial carriers with PRP and BMAC for musculoskeletal conditions (MsCs) repair and regeneration in clinical trials. The main objective is to review the therapeutic use of PRP and BMAC as a treatment option for muscle, bone and cartilage injuries

IVIG Delays Onset in a Mouse Model of Gerstmann-Sträussler-Scheinker Disease

Our previous studies showed that intravenous immunoglobulin (IVIG) contained anti-Aβ autoantibodies that might be able to treat Alzheimer's disease (AD). Recently, we identified and characterized naturally occurring autoantibodies against PrP from IVIG. Although autoantibodies in IVIG blocked PrP fibril formation and PrP neurotoxicity in vitro, it remained unknown whether IVIG could reduce amyloid plaque pathology in vivo and be used to effectively treat animals with prion diseases. In this study, we used Gerstmann-Sträussler-Scheinker (GSS)-Tg (PrP-A116V) transgenic mice to test IVIG efficacy since amyloid plaque formation played an important role in GSS pathogenesis. Here, we provided strong evidence that demonstrates how IVIG could significantly delay disease onset, elongate survival, and improve clinical phenotype in Tg (PrP-A116V) mice. Additionally, in treated animals, IVIG could markedly inhibit PrP amyloid plaque formation and attenuate neuronal apoptosis at the age of 120 days in mice. Our results indicate that IVIG may be a potential, effective therapeutic treatment for GSS and other prion diseases

Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms

Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogenous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP+90R) and completely abolished (PrP+90R versus PrP+90R) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases

Some are Punished and Some are Rewarded: A Study of the Impact of Performance Pay on Job Satisfaction

Using an econometric procedure that corrects for both self-selection of individuals into their preferred compensation scheme and wage endogeneity, this study investigates whether significant differences exist in the job satisfaction of individuals receiving performance-related pay (PRP) compared to those on alternative compensation plans. Using data from four waves of the British Household Panel Survey (BHPS), it is found that PRP exerts a positive effect on the mean job satisfaction of (very) high-paid workers only. A potential explanation for this pattern could be that for lower-paid employees PRP is perceived to be controlling, whereas higher-paid workers derive a utility benefit from what they regard as supportive reward schemes. Using PRP as an incentive device in the UK could therefore be counterproductive in the long run for certain low-paid occupations.European Commissio

High prevalence of scrapie in a dairy goat herd: tissue distribution of disease-associated PrP and effect of PRNP genotype and age

Following a severe outbreak of clinical scrapie in 2006–2007, a large dairy goat herd was culled and 200 animals were selected for post-mortem examinations in order to ascertain the prevalence of infection, the effect of age, breed and PRNP genotype on the susceptibility to scrapie, the tissue distribution of diseaseassociated PrP (PrP$^{\rm d}$), and the comparative efficiency of different diagnostic methods. As determined by immunohistochemical (IHC) examinations with Bar224 PrP antibody, the prevalence of preclinical infection was very high (72/200; 36.0%), with most infected animals being positive for PrP$^{\rm d}$ in lymphoreticular system (LRS) tissues (68/72; 94.4%) compared to those that were positive in brain samples (38/72; 52.8%). The retropharyngeal lymph node and the palatine tonsil showed the highest frequency of PrP$^{\rm d}$ accumulation (87.3% and 84.5%, respectively), while the recto-anal mucosa-associated lymphoid tissue (RAMALT) was positive in only 30 (41.7%) of the infected goats. However, the efficiency of rectal and palatine tonsil biopsies taken shortly before necropsy was similar. The probability of brain and RAMALT being positive directly correlated with the spread of PrP$^{\rm d}$ within the LRS. The prevalence of infection was influenced by PRNP genetics at codon 142 and by the age of the goats: methionine carriers older than 60 months showed a much lower prevalence of infection (12/78; 15.4%) than those younger than 60 months (20/42; 47.6%); these last showed prevalence values similar to isoleucine homozygotes of any age (40/80; 50.0%). Two of seven goats with definite signs of scrapie were negative for PrP$^{\rm d}$ in brain but positive in LRS tissues, and one goat showed biochemical and IHC features of PrP$^{\rm d}$ different from all other infected goats. The results of this study have implications for surveillance and control policies for scrapie in goats

Prion protein interacts with bace1 and differentially regulates its activity towards wild type and swedish mutant amyloid precursor protein

In Alzheimer disease amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of Aβ. We have reported previously that the cellular prion protein (PrP(C)) inhibited the action of BACE1 toward human wild type APP (APP(WT)) in cellular models and that the levels of endogenous murine Aβ were significantly increased in PrP(C)-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrP(C) interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APP(WT) but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP(Swe)). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APP(Swe,Ind)), PrP(C) deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrP(C) inhibited the action of BACE1 on APP(WT), it did not inhibit BACE1 activity toward APP(Swe). The differential subcellular location of the BACE1 cleavage of APP(Swe) relative to APP(WT) provides an explanation for the failure of PrP(C) deletion to affect Aβ accumulation in APP(Swe,Ind) mice. Thus, although PrP(C) exerts no control on cleavage of APP(Swe) by BACE1, it has a profound influence on the cleavage of APP(WT), suggesting that PrP(C) may be a key protective player against sporadic Alzheimer disease

Molecular dynamics as an approach to study prion protein misfolding and the effect of pathogenic mutations

Computer simulation of protein dynamics offers unique high-resolution information that complements experiment. Using experimentally derived structures of the natively folded prion protein (PrP), physically realistic dynamics and conformational changes can be simulated, including the initial steps of misfolding. By introducing mutations in silico, the effect of pathogenic mutations on PrP conformation and dynamics can be assessed. Here, we briefly introduce molecular dynamics methods and review the application of molecular dynamics simulations to obtain insight into various aspects of the PrP, including the mechanism of misfolding, the response to changes in the environment, and the influence of disease-related mutations

Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway

Targeting of the prion protein to the cytosol: mechanisms and consequences

Prion diseases are characterized by the conformational transition of the cellular prion protein (PrPC) into an aberrant protein conformer, designated scrapie-prion protein (PrPSc). A causal link between protein misfolding and neurodegeneration has been established for a variety of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and polyglutamine diseases, but there is an ongoing debate about the nature of the neurotoxic species and how non-native conformers can damage neuronal populations. PrP is normally imported into the endoplasmic reticulum (ER) and targeted to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, several conditions, such as ER stress or some pathogenic mutations in the PrP gene, can induce the mislocalization of PrP in the cytosol, where it has a neurotoxic potential as demonstrated in cell culture and transgenic mouse models. In this review we focus on intrinsic factors and cellular pathways implicated in the import of PrP into the ER and its mistargeting to the cytosol. The findings summarized here not only reveal a complex regulation of the biogenesis of PrP, but also provide interesting new insight into toxic activities of pathogenic protein conformers and quality control pathways of ER-targeted proteins