Prion‐like Doppel gene polymorphisms and scrapie susceptibility in portuguese sheep breeds

The establishment of an association between prion protein gene (PRNP) polymorphisms and scrapie susceptibility in sheep has enabled the development of breeding programmes to increase scrapie resistance in the European Union. Intense selection for PRNP genotype may lead to correlated selection for genes linked to PRNP. We intended to investigate if any association exists between genetic variation in prion‐like protein Doppel gene (PRND) and scrapie susceptibility, determined through PRNP genotyping. Sampling included 460 sheep from eight Portuguese breeds and the PRND gene coding region was analysed by multiple restriction fragment‐single strand conformation polymorphism (MRF‐SSCP), whereas PRNP genotyping was carried out by primer extension. A synonymous substitution (c.78G>A) was detected in codon 26 of the PRND gene, in all breeds except Churra Mondegueira. Linkage disequilibrium was found between the PRND and PRNP loci (P = 0.000). Specifically, PRND was monomorphic in the 45 animals with the more resistant ARR/ARR PRNP genotype (P = 0.003), whereas a higher frequency of PRND heterozygotes (GA) was associated with ARQ/AHQ (P = 0.029). These results constitute preliminary evidence of an association between a polymorphism in the PRND gene and scrapie susceptibility, and indicate that the possibility of undesirable consequences from widespread selection for PRNP genotype on genetic diversity and reproduction traits needs to be further investigated.     

BCL-2 counteracts Doppel-induced apoptosis of prion-protein-deficient Purkinje cells in the Ngsk Prnp(0/0) mouse

The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.     

Doppel-induced cytotoxicity in human neuronal SH-SY5Y cells is antagonized by the prion protein

Doppel （Dpl） is a prion （PrP）-like protein due to the structural and biochemical similarities; however, the natural functions of Dpl and PrP remain unclear. In this study, a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes. Furl-length and various truncated human Dpl and PrP proteins were expressed and purified from Escherichia coil Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity, and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids （aa） 81-122]. Interestingly, DpMnduced cytotoxicity was antagonized by the presence of full- length wild-type PrP. Analysis on fragments of PrP mutants showed that the N-terminal fragment （aa 23- 90） of PrP was responsible for the protective activity. A truncated PrP （PrPA32-121） with similar secondary structure as Dpl induced DpMike cytotoxicity on SH- SY5Y cells. Furthermore, binding of copper ion could enhance the antagonizing effect of PrP on Dpi-induced cytotoxicity. Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism. These results suggested that the function of Dpl is antagonistic to PrP rather than synergistic.     

Copper(II) complexes with peptide fragments encompassing the sequence 122-130 of human doppel protein

Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH₂ species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.     

重组人Doppel蛋白的表达及其细胞毒性作用的研究

Doppel(Dpl)蛋白是新近发现的PrP相关蛋白.根据GenBank公布的人PRND cDNA序列设计合成特异性引物,用PCR方法从人外周血白细胞总DNA中扩增获得531 bp的人PRND完整基因序列,测序正确后克隆至表达载体,转化大肠杆菌JM109,IPTG诱导表达重组人Doppel(rhDpl)蛋白.目的蛋白以包涵体形式表达,表达量占菌体总蛋白的60%以上.表达产物用Ni2 亲和层析柱纯化及羟胺化学裂解法去除标签蛋白,SDS-PAGE检测证实,纯化的rhDpl蛋白相对分子量约为15 kD.Trypan Blue及MTT实验显示,在50μg/mL及以上剂量时,rhDpl蛋白具有抑制人神经母细胞瘤SH-SY5Y细胞生长的作用,在100μg/mL及以上剂量时,rhDpl具有杀伤HeLa细胞的活性,呈现明显的剂量和时间依赖性.Hoechst33342荧光染色观察显示经rhDpl作用的人神经母细胞瘤细胞SH-SY5Y呈现出细胞凋亡现象.这些结果提示Dpl蛋白具有体外细胞毒作用,并呈现明显的组织类型特异性.以上工作为进一步了解人Dpl蛋白体内外生物学作用奠定了基础.     

Doppel-induced Purkinje cell death is stoichiometrically abrogated by prion protein

Activin A can induce erythroid differentiation, whereas basic fibroblast growth factor (bFGF) can maintain the undifferentiated status of erythroid progenitors. How these two factors together can affect the regulation of erythroid differentiation in hematopoietic cells has not been elucidated. This study demonstrates that bFGF antagonizes activin A-mediated growth inhibition and hemoglobin (Hb) synthesis in K562 cells. Analyses of mitogen-activated protein kinases revealed that activin A-induced p38 phosphorylation and inhibited ERK1/2 phosphorylation. In contrast, bFGF worked antagonistically to induce ERK1/2 phosphorylation and inhibited p38 phosphorylation in K562 cells. Furthermore, co-treatment of cells with activin A and bFGF decreased p38 phosphorylation and increased ERK1/2 phosphorylation. SB203580 inhibition of p38 activity eliminated activin A-mediated growth inhibition and Hb synthesis, whereas U0126 inhibition of ERK1/2 activity augmented the effects of activin A on K562 cells. These results suggest that bFGF can negatively modulate p38 and positively modulate ERK1/2 to antagonize activin A-mediated growth inhibition and Hb synthesis in K562 cells.     

Interaction of Doppel with the full-length laminin receptor precursor protein

Doppel (Dpl) is a homolog of normal cellular prion protein (PrPc) with unknown functions. Ectopic expression of Dpl in the central nervous system (CNS) causes neurotoxicity and this effect is rescued by the expression of PrPc. However, the molecular basis for the protective effect of PrPc remains unclear. Using a yeast two-hybrid system, we showed that Dpl binds the full-length 37-kDa laminin receptor precursor protein (LRP), one of the receptors of PrPc. The interaction was also validated by immunoprecipitation and immunoblotting using transfected cell lines and in vivo derived tissues. Further mapping experiments showed that although the middle fragment containing residues 100-220 of LRP was able to interact with Dpl, deletion of the N-terminal domain of the full-length LRP abolished its interaction with Dpl. These results suggest that while both PrPc and Dpl interact with LRP, the domains that are involved in the binding are not the same. Our results may have implications for the molecular mechanisms of Dpl-PrPc antagonism and physiological roles of Dpl.     

The doppel gene biology: a scientific journey from brain to testis, and return

Doppel is a newly recognized prion-like molecule encoded by a novel gene locus, PRND, located on the same chromosomal region of the prion (PRNP) coding gene. Doppel was considered a paralogue and the first member of the prion-gene family, possibly originated through an ancestral gene duplication event. Prion and doppel have different expression patterns, suggesting that the gene products exhibit different biological functions. Actually, doppel is not involved in the aetiology of the Transmissible Spongiform Encephalopathies (TSEs) or “prion diseases” and is highly expressed only within the testicular tissue, suggesting an important physiological role in the process of spermatogenesis. The restricted spatial and temporal expression profile of doppel has suggested its investigation within particular pathological contexts, such as cancers, showing that it might represent a novel and attractive diagnostic molecular marker and that might provide insights into the regulatory pathways of tumor-cell transformation.     

Tissue- and cell type-specific modification of prion protein (PrP)-like protein Doppel, which affects PrP endoproteolysis

A prion protein (PrP)-like protein, Doppel (Dpl) is a homologue of cellular PrP (PrP^C). Immunoblotting revealed heterogeneous glycosylation patterns of Dpl and PrP^C in several cell lines and tissues, including brain and testis. To investigate whether the glycosylation and modification of Dpl and PrP^C could influence each other, PrP gene (Prnp)-deficient neuronal cells, transfected with Prnp and/or the Dpl gene (Prnd), were analyzed by deglycosylation with peptide N-glycosidase F. The modification of Dpl was not influenced by PrP^C, whereas an N-terminally truncated fragment of PrP^C was reduced by Dpl expression. These results indicated that Dpl was glycosylated in a cell type- and tissue-specific manner regardless of PrP^C, while PrP^C endoproteolysis was modulated by Dpl expression.     

Doppel蛋白的最新研究进展

Dpl是第一个经过鉴定的类朊蛋白。称为叠朊蛋白。对其分子结构、分布情况,生物学功能,已经有所了解。Dpl蛋白在Prp基因敲除小鼠(Prnp~(0/0)或Prnp~(-/-))表现出的神经毒性。以及在Prnd基因敲除小鼠表现出对雄性小鼠的可育性的重要性,使其具有很大的研究价值。本综述对Dpl的最新研究进展进行了总结,便于进一步研究工作的进行。