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Abstract Background Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood. Results In a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-ΔC in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-ΔC in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease. Conclusions We identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders.

Abstract Background A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. Results HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. Conclusion HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

The studies on the molecular targets of a photoreceptor G protein transducin (T[beta][gamma]) analogue that was generated to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of T[gamma] were performed to explore the farnesyl-mediated molecular interactions. The results shed light on the active role of the modifying group T[gamma] in protein-protein and protein-membrane interactions and also on the efficient formation of the signaling ternary complex between transducin and rhodopsin supported by the farnesyl-PE/PS phosphatidylethanolamine/phosphatidylserine) interaction.

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L-type-like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy. [ABSTRACT FROM AUTHOR]

G protein γ-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Tα/Tβγ). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tβγ analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tγ. POG- modified Tβγ was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tβγ. Photoaffinity labeling experiment with the photoreactive Tβγ analogue in its free form indicated that the POG moiety of Tγ interacted with Tβ. In the trimeric Tα/Tβγ complex, the POG moiety was cross-linked with Tα in addition to concurrent affinity labeling of Tβ. When photoreactive Tβγ was reconstituted with Tα and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Tα and Tβ but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tγ plays an active role in protein-protein and protein-membrane interactions and suggest that the farnesyl-PEIPS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*. [ABSTRACT FROM AUTHOR]

Farnesylation of the γ-subunit of the retinal G-protein, transducin (T&alfa;/Tβγ), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tβγ analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or T&alfa;-GDP, UV irradiation of POG-modified Tβγ (POG-Tβγ) invariably yielded a cross-linked product Tγ-Tβ, reflecting a constitutive interaction of the Tγ C-terminal lipid with Tβ. In addition to the Tγ-Tβ adduct, a Tγ-T&alfa; cross-link was detected in the aqueous fraction. Reconstitution of POG-Tβγ with T&alfa; and light- activated rhodopsin (Rh*) in photoreceptor membranes resulted in cross-linking of Tγ with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tγphospholipid cross-link was observed only in the presence of both T&alfa;-GDP and Rh*, and was abolished by the addition of GTPγS or by replacing Rh* with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh* ternary complex. On the other hand, UV irradiation of POG-Tβγ in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tγ---phosducin adduct in addition to the Tγ-Tβ cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process. [ABSTRACT FROM AUTHOR]

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrPSc; PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrPC) to PrPSc and the subsequent conversion of PrPC to PrPSc. We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrPC and PrPSc. Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrPSc state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrPSc and interfered with the conversion of endogenous MoPrPC. The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrPSc. Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrPC reduced the accumulation of PrPSc after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrPC plays a key role in conversion after binding to MoPrPSc [ABSTRACT FROM AUTHOR]

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).

Transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative disorders. In aetiological viewpoint, human prion diseases are classified into 1) sporadic Creutzfeldt-Jakob disease (CJD) which comprises 80-90% of the total population of human prion disaeses, 2) inherited forms, and 3) acquired types by prion-contaminated surgical instruments, biopharmaceuticals or foodstuffs. The diseases cause an accumulation of the disease-associated form(s) of prion protein (PrP(Sc)) in the central nervous system. PrP(Sc) is regarded as the entity of prion agents and generally exerts infectivity, irrespective of its origin being from the sporadic cases or the inherited cases. Variant CJD (vCJD), first identified in the United Kingdom (UK) in 1996, is an acquired type of human CJD by oral intake of BSE prion. Cumulative numbers of 215 patients in the world have been reported for definite or probable vCJD cases according to the UK National Creutzfeldt-Jakob Disease Surveillance Unit by September, 2009. Different from sporadic CJD cases, vCJD patients show an accumulation of PrP(Sc) in spleen and tonsils. Such distribution of PrP(Sc) in lymphoid tissues raised clinical concern about the potential infectivity in the blood or blood components used for blood transfusion. To date, five instances of probable transfusion-mediated transmission of vCJD prion have been found in UK. Here we review the past and the present issues about the acquired human prion diseases.