Naomi S. Hachiya

A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.

Rat liver cytosol contained an activity that stimulated the import of wheat germ lysate‐synthesized precursor proteins into mitochondria. The activity was purified 10,000‐fold from the cytosol as a homogeneous heterodimeric protein. This protein (termed mitochondrial import stimulation factor or MSF) stimulated the binding and import of mitochondrial precursor proteins. MSF was also found to recognize the presequence portion of mitochondrial precursors and catalyze the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP‐dependent manner; in this connection, MSF exhibited ATPase activity depending on the important‐incompetent mitochondrial precursor protein. The mitochondrial binding and import‐stimulating activities were strongly inhibited by the pretreatment of MSF with NEM, whereas the ATP‐dependent depolymerization activity was insensitive to the NEM treatment, suggesting that the process subsequent to the unfolding was inhibited with the NEM treatment. We conclude that MSF is a multifunctional cytoplasmic chaperone specific for mitochondrial protein import.

MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria.

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N‐ethylmaleimide (NEM)‐treated MSF was not inhibited by OM. In this context, the MSF‐precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor‐targeting function.

14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

Huntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.

Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC.

Recent studies suggest that the disease isoform of prion protein (PrPSc) is non-neurotoxic in the absence of cellular isoform of prion protein (PrPC), indicating that PrPC may participate directly in the neurodegenerative damage by itself. Meanwhile, transgenic mice harboring a high-copy-number of wild-type mouse (Mo) PrPC develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of PrPSc and thus, investigations of these aged transgenic mice may lead to the understanding how PrPC participate in the neurotoxic property of PrP. Here we demonstrate mitochondria-mediated neuronal apoptosis in aged transgenic mice overexpressing wild-type MoPrPC (Tg(MoPrP)4053/FVB). The aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasomal activity, while younger littermates did not. Such aberrant mitochondrial localization was accompanied by decreased mitochondrial manganese superoxide dismutase (Mn-SOD) activity, cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal neuronal cells. Following cell culture studies confirmed that decrease in the proteasomal activity is fundamental for the PrPC-related, mitochondria-mediated apoptosis. Hence, the neurotoxic property of PrPC could be explained by the mitochondria-mediated neuronal apoptosis, at least in part.

A screening method for DNA aptamers that bind to␣a␣specific, unidentified protein in tissue samples

Aptamers are oligonucleotide ligands with a high affinity to, and specificity for, various target molecules and they are expected to be powerful tools for proteomic analysis. To select aptamers that bind to a specific unidentified protein in tissues for protein analysis, a screening method was developed using chicken skeletal muscle as a model. Target proteins in the target mixture were separated by electrophoresis and transferred to a membrane, and a DNA library was added onto it. The aptamers that bound to the target protein were visualized by chemiluminescence and collected by cutting out the visualized band. The specific aptamers to the target protein were selected by only one round of selection using this screening, suggesting this screening method might be useful for selecting aptamers for proteome analysis.

14-3-3 proteins sequester a pool of soluble TRIM32 ubiquitin ligase to repress autoubiquitylation and cytoplasmic body formation

Summary Deregulated expression of tripartite motif-containing protein 32 (TRIM32, an E3 ubiquitin-protein ligase) contributes to various diseases. Here we report, using quantitative proteomics and biochemistry, that 14-3-3 proteins bind to phosphorylated TRIM32 and prevent TRIM32 autoubiquitylation and the formation of TRIM32-containing cytoplasmic bodies, which are potential autoregulatory mechanisms that can reduce the concentration of soluble free TRIM32. The 14-3-3–TRIM32 interaction is dependent on protein-kinase-A-catalyzed phosphorylation of TRIM32 at Ser651. We found that the inhibitory effect of 14-3-3 is, in part, a consequence of disrupting the propensity of TRIM32 to undergo higher-order self-association without affecting its dimerization. Consequently, dimerized TRIM32 bound to 14-3-3 was sequestered in a distinct cytoplasmic pool away from the microtubule network, whereas a TRIM32 mutant that cannot bind 14-3-3 underwent multimerization and was unavailable to facilitate cell growth. Our results reveal a novel connection between ubiquitylation and phosphorylation pathways, which could modulate a variety of cell events by stimulating the formation of the 14-3-3–TRIM32 signaling complex.

Temporospatial patterns of COX-2 expression and pyramidal cell degeneration in the rat hippocampus after trimethyltin administration

The temporospatial profile of cyclooxygenase-2 (COX-2) expression and neuronal degeneration following trimethyltin (TMT) administration was investigated in the rat hippocampus region. In the CA1 region, significant COX-2 expression was detected on day 3 after TMT administration but pyramidal cell degeneration was detected only on day 5 and thereafter. In the CA3 region, on the other hand, the constitutive COX-2 expression remained unchanged, and more severe pyramidal cell degeneration started on day 3. Concomitant with these observations, we observed that the coadministration of a COX-2 inhibitor prevented such neuronal degeneration only in the CA1 region and not in the CA3 region. In addition, COX-2 inhibition did not affect the increase in the plasma corticosterone concentration after TMT administration. Furthermore, the COX-2 inhibitor did not alleviate TMT-induced locomotor hyperactivity in rats, for which inhibitors of corticosterone synthesis are known to be effective. These data suggest that the COX-2-dependent pathway appears to assist TMT-induced degeneration of CA1 pyramidal cells but not CA3 pyramidal cells in a corticosterone-independent manner.

Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro

Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 μM, 99% effective concentration (EC99) = 0.86 μM vs. EC50 = 0.45 μM, EC99 = 1.5 μM. Besides, no apparent cell damage was observed at the concentration of up to 4.3 μM (100 μg/ml). In combination treatment with 0.43 μM (10 μg/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 μM to 0.5 μM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.

Three-repeat Tau 69 is a major tau isoform in laser-microdissected Pick bodies

By utilizing a novel combinatorial method of a Laser Microdissection System and Western blot analysis, we demonstrate that a distinct isoform of abnormally phosphorylated tau (69 kDa, Tau 69) predominantly aggregated in laser-microdissected Pick bodies (PBs) in sporadic Picks disease. By contrast, tau migrated as two major bands of 60 and 64 kDa (Tau 60 and 64) in total brain homogenates as previously reported. Comparative immunohistochemical analysis with anti–4-repeat antibody revealed that a major component of the abnormally phosphorylated tau in these PBs was 3-repeat tau (3R-tau). Whether 29 amino acid repeat encoded by exons 2 and 3 in the Tau 69 might accelerate the formation of PBs remains to be further investigated. Such a combination of morphological and biochemical techniques significantly complements the existing histopathological methods.

Intracerebroventricular delivery of dominant negative prion protein in a mouse model of iatrogenic Creutzfeldt-Jakob disease after dura graft transplantation

We have developed a novel procedure in which a small collagen sheet (3 mm x 3 mm) absorbing prion-infected brain homogenates was transplanted onto the brain surface of highly prion-susceptible transgenic mice (Tg(MoPrP)4053/FVB), as an animal model of iatrogenic Creutzfeldt-Jakob disease (iCJD) caused by prion-contaminated cadaveric dura graft transplantation. Using the iCJD model, we further investigated the in vivo efficacy of dominant negative recombinant prion protein with lysine substitution at mouse codon 218 (rPrP-Q218K), which is known to inhibit prion replication in vitro (H. Kishida, Y. Sakasegawa, K. Watanabe, Y. Yamakawa, M. Nishijima, Y. Kuroiwa, N.S. Hachiya, K. Kaneko, Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro, Amyloid, vol. 11, 2004, pp. 14-20.). Following 7-day intracerebroventricular administration of the rPrP-Q218K via an indwelling catheter connected to the implanted osmotic pump, the median incubation period of Tg(MoPrP)4053/FVB was prolonged considerably from 117 days to 131 days (p=0.016, log-rank test) in the rPrP-Q218K-treated group, even after a lengthy latency period of as long as 30 days by starting the rPrP-Q218K injection. Whether wild-type rPrP, other mutant rPrPs, or the combination of rPrP-Q218K with other anti-prion compounds might extend the survival period in that condition must be further investigated.