Mechanistic insights into cellular alteration of prion by poly‐D‐lysine: the role of H2H3 domain

Abstract

Misfolding of the prion protein (PrP) is the central feature of prion diseases. The conversion of the normal α-helical PrP(C) into a pathological β-enriched PrP(Sc) constitutes an early event in the infectious process. Several hypotheses, involving different regions of the protein, endeavor to delineate the structural mechanism underlying this change of conformation. All current working hypotheses, however, are based on biophysical and modeling studies, the biological relevance of which still needs to be assessed. We have studied the effect of positively charged polymers on the conversion, using polylysine as a model system, and have investigated a possible mechanism of structural stabilization. We have shown that poly-D-lysine removes proteinase K-resistant PrP from prion-infected SN56 neuroblastoma cells without affecting PrP(C). The effect is enantiospecific since the levorotary isomer, poly-L-lysine, has a markedly weaker effect, likely because of its higher susceptibility to degradation. In vitro cross-linking and NMR studies confirm a direct interaction between polylysine and PrP, which mainly maps to the PrP region containing helices 2 and 3 (H2H3). Interaction prevents conformational conversion and protein aggregation. Our results establish a central role of H2H3 in PrP(Sc) amyloidogenesis and replication and provide biological relevance for the pathological misfolding of this domain.

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

“…As shown by the apparent contradiction between these models, a consensus has yet to be found that should be based on in vivo studies. In favor of the H2H3 hypothesis, we have previously shown that (i) H2H3 can be independently generated in an Escherichia coli system and retain its native secondary and tertiary structure (23), (ii) H2H3 is able to fibrillize and reproduce the oligomerization pathways of PrP (23,24), and (iii) in a cellular model of prion infection, polylysines are able to eliminate PK-resistant PrP through a direct interaction with H2H3 (25). These results led to the hypothesis that H2H3 could be the minimal region involved in the change of conformation of PrP.…”

Poly-L-histidine inhibits prion propagation in a prion-infected cell line

“…An assignment of NMR peaks indicated that most of the perturbed peaks (Δδ > 0.10) were mapped onto the loop region connecting Helix 2 and Helix 3 (residues 180-200) (Figure 4(b,c)). As indicated in the previous study [8], this loop region composes a negatively charged patch to serve as a binding surface for poly-L-Lys. These results indicated that poly-L-His and poly-L-Lys share a common mechanism in inhibiting prion propagation which involves direct binding to a negatively charged surface of PrP C .…”

“…This result ruled out the possibility that poly-L-His inhibits prion propagation through metal chelation. A previous study examining anti-prion mechanism of a positively charged poly-(amino acid), poly-L-Lys, indicated that a negatively charged surface of the Helix 2-Helix 3 region of PrP C (residues 169-230) is the specific site for poly-L-Lys binding [8]. In fact, the Helix 2-Helix 3 region has been suggested as an initiation site for the structural conversion of PrP toward PrP Sc [23][24][25][26] and is a major target for many anti-prion compounds [14,[27][28][29][30].…”

“…These studies led us to hypothesize that poly-(amino acid)s, charged molecules with repeating units, is a potential molecule for inhibiting prion propagation. In fact, several preceding studies have reported anti-prion activity in a positively charged poly-(amino acid)s, such as poly-L-Lys [8][9][10] and poly-L-Arg [11].…”

Effect of poly-L-arginine in inhibiting scrapie prion protein of cultured cells

“…Because previous studies [32] reported that anti-prion activity of PLK correlates with its direct binding to PrP C , investigation on the possibility of PLR interaction with PrP C and its relevance to anti-prion activity of PLR is of interest. If this turns out to be the case, together with our findings demonstrated in this study, it is feasible to speculate that there exist multiple molecular events involved in PLR action to suppress prions.…”