Protein and Peptide Letters - Volume 16, Issue 3, 2009

Recently, several detection methods for prion and/or prion protein (PrP) (including abnormal and cellular PrP) have been developed. An important method is protein misfolding cyclic amplification (PMCA), which enables the amplification of abnormal PrP in vitro. Prospects for therapy or preventative vaccine have also been explored. Accumulated knowledge on the role of cellular PrP would provide useful information for exploring strategies for the treatment of prion diseases. To discuss this hot topic, scientists using various approaches to study the pathogenesis, diagnosis, and prevention of prion diseases were invited. I am glad to have the honor to organize a Hot Topic issue for Protein and Peptide Letters and to experience working with eminent scientists to review research on prion diseases. This issue covers the broad topic of prion biology and includes articles on the following topics: fundamental knowledge and possible pathogenesis of prion diseases written by myself and Prof. Kazuyoshi Ikuta, recent studies on a neurocytotoxicity model of a PrP-derived peptide written by Dr. Neville Vassallo, the role of cellular PrP in functions of macrophage and dendritic cells written by Prof. Takashi Onodera and colleagues, uptake of abnormal PrP in the intestines written by Prof. Hiroyuki Nakayama and colleagues, abnormal PrP amplification by PMCA written by Dr. Ryuichiro Atarashi, prospects for preventative vaccine against prion diseases written by Prof. Suehiro Sakaguchi, and yeast prions written by Dr. Yuji Inoue. Hopefully, readers will enjoy this issue, obtain useful information for their own research, and be inspired with new ideas for future research on prion biology.

Prion diseases are fatal degenerative disorders whose features include the accumulation of abnormal isoform of prion protein (PrPSc), vacuolation, and astrocytosis in the brain. After a prion infection, the cellular isoform of prion protein (PrPC) is converted into PrPSc, resulting in PrPC deficiency and PrPSc accumulation in the brain. These changes are major etiological events, thought to be closely related to the pathogenesis of prion diseases, and used as an index for diagnosis. Studies using recently developed research tools such as transgenic and knockout mice and cell lines have shown that the accumulation of PrPSc is not the sole factor contributing to the clinical onset of prion diseases and that loss-of function of PrPC by depletion leads to neuronal cell loss. Notably, PrPC plays an important role in anti-oxidative defense and its deficiency increases susceptibility to oxidative stress. Furthermore, there is a possible interrelationship between Alzheimer's disease and prion disease through loss-of-function of PrPC, which leads to the production of amyloid β. In this review, we introduce research and diagnostic tools for prion diseases and the involvement of loss-of-function of PrPC in the pathogenicity of prion diseases.

Prion diseases are neurodegenerative disorders characterized by a hallmark event involving the posttranslational misfolding of the normal cellular prion protein (PrPC) into an infectious and toxic protease-resistant conformation (PrPSc). Studies on identification of the pathological prion species and on the mechanisms involved in triggering neuronal death have been hampered by the heterogeneous nature of PrPSc aggregates. The use of synthetic PrP-derived peptides has made possible exploration of the relationship between amino acid sequence, biophysical structure and biological effect. Indeed, most PrP-derived peptides replicate the fundamental aspects of full-length PrPSc, including: a β- sheet-rich structure; destabilization of lipid membranes; intracellular calcium dysregulation; increased oxidative stress; activation of pro-apoptotic signaling pathways; and, more contentiously, neurotoxicity dependent upon endogenous PrPC expression. Crucially, in vivo toxicity of the important PrP-peptides, e.g. PrP(106-126) and PrP(118-135), has additionally been established. Therefore, the use of prion-derived peptides facilitates the development of therapeutic strategies based on small-molecule inhibitors of aggregation and other pharmacological agents that protect against the lethal effect of these peptides in vivo.

The cellular isoform of prion proteins (PrPC) is expressed in hematopoietic stem cells, granulocytes, T and B lymphocyte natural killer cells, platelets, monocytes, dendritic cells, and follicular dendritic cells, which may act as carrier cells for the spread of its abnormal isoform (PrPSc) before manifesting transmissible spongiform encephalopathies (TSEs). In particular, macrophages and dendritic cells seem to be involved in the replication of PrPSc after ingestion. In addition, information on the role of PrPC during phagocytotic activity in these cells has been obtained. A recent study showed that resident macrophages from ZrchI PrP gene (Prnp)-deficient (Prnp-/-) mice show augmented phagocytotic activity compared to Prnp+/+ counterparts. In contrast, our study suggests that Rikn Prnp-/- peritoneal macrophages show pseudopodium extension arrest and up-regulation of phagocytotic activity compared to Prnp+/+ cells. Although reports regarding phagocytotic activity in resident and peritoneal macrophages are inconsistent between ZrchI and Rikn Prnp-/- mice, it seems plausible that PrPC in macrophages could contribute to maintain the immunological environment. This review will introduce the recent progress in understanding the functions of PrPC in macrophages and dendritic cells under physiological conditions and its involvement in the pathogenesis of prion diseases.

Transmissible spongiform encephalopathies (TSEs) are characterized by the accumulation of a proteaseresistant abnormal isoform of the prion protein (PrPSc), which is converted from the cellular isoform of the prion protein (PrPC). In the oral transmission of prion protein, PrPSc can invade a host body through the intestinal tract. There is only limited information available on how the infectious agent passes through one or several biological barriers before it can finally reach the brain. After oral administration, PrPSc withstands the digestive process and may be incorporated by microfold (M) cells or villous columnar epithelial cells in the intestine. After entry into the intestinal epithelium, PrPSc accumulates and is amplified in follicular dendritic cells (FDCs) within Peyer's patches and other isolated lymphoid follicles possibly by an interaction with dendritic cells or macrophages. Following accumulation in gut-associated lymphoid tissues, PrPSc is thought to move to the enteric nervous systems (ENS) by an interaction with FDCs or dendritic cells. As a result of neuroinvasion into the ENS, PrPSc spreads to the central nervous system. In addition, an epidemiological study suggested that most bovine spongiform encephalopathy cases had been exposed to the agent in the first 6 months of life. Developments of the intestinal defense and immune system may be involved in the susceptibility to infection.

The development of amplification technology for abnormal forms of prion protein in vitro has had a great impact on the field of prion research. This novel technology has generated new possibilities for understanding the molecular basis of prions and for developing an early diagnostic test for prion diseases. This review provides an overview of recent progress in cell-free PrPSc amplification techniques.

Emergence of variant type of Creutzfeldt-Jakob disease (vCJD) in humans due to infection from bovine spongiform encephalopathy contaminated beef and recent reports of human-to-human transmission of vCJD via blood transfusion have raised great concern about an epidemic of vCJD. The disease is currently difficult to diagnose during preclinical stages and requires a very long incubation period for neurological symptoms to be evident. This therefore suggests that the disease is already latently spreading and that opportunity for infection is thus growing among human populations. Interestingly, passive immunization with antibodies against prion protein (PrP), a major component of the prion infectious agents, was shown to protect mice from infection, indicating the possibility of prion vaccines. However, PrP is a host protein therefore immune tolerance to PrP has hampered development of them. Here, the so far reported attempts to overcome the tolerance to elicit protective immunity to prions are briefly reviewed.

Currently, prion phenomena have been detected in various organisms, in addition to mammals affected by transmissible spongiform encephalopathies. In the budding yeast Saccharomyces cerevisiae, various proteins have prion properties and adopt atypical phenotypes as genetic elements, such as the Sup35 and Ure2 proteins, corresponding to the [PSI+] and [URE3] phenotypes, respectively. Each yeast prion protein has a prion-forming region rich in glutamines and/or asparagines, and can form amyloid fibrils in its prion conformation. Studies on yeast prions have revealed that the amyloid fibrils play critical roles in the life cycle of the yeast prion. First, the amyloid fibril binds the normal prion protein and catalyzes a structural conversion into the abnormal form, the key event of the prion phenomenon. Second, the amyloid fibril is related to the strain differences of the prion phenotypes, by its substructural differences. Third, the number of prion elements multiplies by the fragmentation of amyloid fibrils, which is mediated by a chaperone system in which Hsp104 plays a central role, and the prion elements are distributed to the daughter cells during cell division. Moreover, heterologous prion-prion communications may occur, probably by cross-seeding of amyloid fibrils among different prion proteins in the same yeast cell. Findings achieved by yeast prion studies are making great contributions toward understanding the characteristics of amyloid fibrils and prions.

Three trypsin-chymotrypsin inhibitors were isolated from seeds of the black gram (Vigna mungo) with a procedure that entailed cation exchange chromatography on SP-Sepharose, anion exchange chromatography on Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q and Mono S, and gel filtration by FPLC on Superdex 75. Two of the trypsin-chymotrypsin inhibitors were adsorbed on the first four types of chromatographic media. All three inhibitors have a molecular mass of 16 kDa as judged by gel filtration and sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The trypsin inhibitory activity of the inhibitors was attenuated in the presence of the reducing agent dithiothreitol. The remaining inhibitor was unadsorbed on SP-Sepharose but adsorbed on Q-Sepharose, Mono Q and Mono S. The protease inhibitors did not exert any inhibitory effect on hepatoma (Hep G2) and breast cancer (MCF 7) cells or antifungal action toward Botrytis cinerea, Fusarium oxysporum and Mycosphaerella arachidicola. Two of the inhibitors slightly inhibited the activity of HIV-1 reverse transcriptase, with an IC50 in the millimolar range.

NPTX-594 is a neurotoxic acylpolyamine isolated from a Madagascar Joro spider and acts as a specific blocker of glutamate receptors (GluRs). The present study reports the creation of novel NPTX-594 analogs with thiol-containing fluorophores for use as visualization probes for the analysis of the binding mode between spider toxins and GluRs, together with the result of their cricket bioassay.

The mutation trend and pattern in protein are currently studied directly using amino acid sequence, however, it would be more efficient if the amino acid sequence is transferred into other domains through quantification because sophisticated mathematical tools can be applied to a numeric sequence. In this study, we apply the amino-acid pair predictability to quantifying the human androgen receptor with its 215 missense point mutations to analyze which amino-acid pairs are sensitive to mutations. The results show that 94.88% mutations occurred at the unpredictable amino-acid pairs, 79% mutations targeted at one or two original amino-acid pairs whose actual frequency is larger than predicted frequency and 63.26% mutations lead to one or two mutated amino-acid pairs with their actual frequency smaller than predicted one.

Stereochemical preference for homochiral or heterochiral couplings was investigated in the reactions of Nprotected L-amino acid 1-hydroxybenzotriazole esters with racemic amino acid derivatives. It was found to be dependent on the combination of amino acid residues as the carboxyl and amino components and the protecting groups of the amino acid residues, especially the N-protecting groups. Very high diastereomeric ratios were observed when t-leucine was employed as the carboxyl component and/or proline as the amino component and when the N-protecting group was the phthaloyl group.

We screened SMG7-binding peptides with phage display and docking simulation analysis. Although a consensus motif was absent in the phage display-derived candidates, we succeeded to find a peptide CDDRPPKSC, which can bind specifically to SMG7. We conclude that docking simulation helps to find high-affinity peptides efficiently, even if the phage display-screened candidates lack a consensus region.

The coiled-coil neck domain of pulmonary surfactant protein D (SP-D) is required for trimeric association and the subsequent assembly of functional dodecamers of SP-D. It is also necessary and sufficient for trimerization of a heterologous collagen sequence. To investigate whether it is capable of driving trimerization of heterologous noncollagenous proteins, we expressed and purified a fusion of a heterologous non-collagenous sequence (thioredoxin) to the coiled-coil neck domain of human SP-D here. While western blot analysis detected a small population of stable trimers of the fusion protein, chemical cross-linking and SEC-HPLC indicated that the fusion protein was predominantly a trimer. In contrast, purified thioredoxin without the fusion was found only as monomers and dimers. We also measured the thermal stabilities (with circular dichroism) and degradation rates of these two proteins. Our data showed that the fusion protein had a melting temperature that was 13 K higher than that of thioredoxin and a longer degradation half life than thioredoxin. Our findings indicate that the coiled-coil neck domain of SP-D enables the trimerization and stabilization of the heterologous non-collagenous thioredoxin. It may provide new clues for further study on the application of this human original coiled-coil domain in protein engineering to construst trimeric functional fusion proteins.

Cyclosporin O (CyO), an immunosuppressent cyclic undecapeptide, was synthesized by convergent approach employing Bsmoc-Nmethyl amino acid fluorides and Potassium Salt of 7-Aza-1-hydroxybenzotriazole (KOAt) in solution by stepwise assembly. The couplings were found to be epimerisation free. The difficulty in the coupling of four consecutive N-methyl amino acids at position 8, 9, 10 and 11 was overcome by repeating the coupling thrice at these critical positions. All the ten protected peptide fragments of CyO starting from the dipeptide to the undecapeptide and final protected as well as CyO were isolated and fully characterized.

A novel grass carp growth hormone receptor (gcGHR) cDNA was cloned by RT-PCR method. CHO-K1 cells were transfected with eukaryotic expression vector pcDNA3.0 containing gcGHR entire coding region, resulting in a stable CHO cell line expressing gcGHR (CHO-gcGHR). Bioactivity assay revealed that silver carp growth hormone (scGH) bound gcGHR with dissociation constant (Kd) value of 1.96 nM and stimulated CHO-gcGHR cells proliferation with the sensitivity of 20 ng/ml. The cells proliferation assay was also provided to measure fish growth hormone levels of pituitary and refolded protein.

Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9), a subunit of an Elongin Ccullin- SOCS box (ECS) E3 ubiquitin ligase complex, is believed to be involved in specific substrate-recognition for ubiquitination and degradation. In fact, this specific substrate-recognition is determined by the ankyrin repeats of hASB9 protein. Here, we have cloned and overexpressed the hASB9-2, the splice variant of hASB9 with only one ankyrin repeat domain, as a 6His-tagged recombinant protein in Escherichia coli. The purified hASB9-2 protein was crystallized by the hanging-drop vapor-diffusion technique and diffracted to 2.2Å resolution. The data showed that the cubic hASB9-2 crystal belongs to space group P4332 with unit-cell parameters (a=b=c=129.25Å, α=β=γ=90°). An asymmetric unit in the crystal was assumed to contain one protein molecule giving the Matthews Coefficient factor of 2.81 and the solvent content of 56.3%.

A novel human Glyco_18 domain-containing protein, SI-CLP, was detected recently in human bronchoalveolar lavage of patients with chronic inflammatory disorders of the respiratory tract and peripheral-blood leukocytes. The expression of SI-CLP is up-regulated by dexamethasone or IL-4 and involved in the Th2 cell pathway. To further investigate its structure and function will provide new insights into human immunity and related disorders. Here we provide a preliminary crystal image of SI-CLP using the hanging-drop vapor diffusion method. The crystals of SI-CLP diffracted Xrays to a resolution of 2.7 Å. The crystals belong to the space group P3221 with unit cell parameters a = b =99.79 Å, c =250.53 Å, α=β=90°, γ=120°. There are two molecules per asymmetry unit.

Human calcyphosine was cloned into the pET-28a vector and highly expressed in Escherichia coli BL21 (DE3) cells. The protein was purified and crystallized. The crystal diffracted to 2.8 Å and belonged to space group P21212, with the unit cell parameters a=70.39 Å, b=132.02 Å, c=46.20 Å.