Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Tertiary and quaternary structures of photoreactive Fe-type nitrile hydratase from Rhodococcus sp. N-771: roles of hydration water molecules in stabilizing the structures and the structural origin of the substrate specificity of the enzyme.

The crystal structure analysis of the Fe-type nitrile hydratase from Rhodococcus sp. N-771 revealed the unique structure of the enzyme composed of the alpha- and beta-subunits and the unprecedented structure of the non-heme iron active center [Nagashima, S., et al. (1998) Nat. Struct. Biol. 5, 347-351]. A number of hydration water molecules were identified both in the interior and at the exterior of the enzyme. The study presented here investigated the roles of the hydration water molecules in stabilizing the tertiary and the quaternary structures of the enzyme, based on the crystal structure and the results from a laser light scattering experiment for the enzyme in solution. Seventy-six hydration water molecules between the two subunits significantly contribute to the alphabeta heterodimer formation by making up the surface shape, forming extensive networks of hydrogen bonds, and moderating the surface charge of the beta-subunit. In particular, 20 hydration water molecules form the extensive networks of hydrogen bonds stabilizing the unique structure of the active center. The amino acid residues hydrogen-bonded to those hydration water molecules are highly conserved among all known nitrile hydratases and even in the homologous enzyme, thiocyanate hydrolase, suggesting the structural conservation of the water molecules in the NHase family. The crystallographic asymmetric unit contained two heterodimers connected by 50 hydration water molecules. The heterotetramer formation in crystallization was clearly explained by the concentration-dependent aggregation state of NHase found in the light scattering measurement. The measurement proved that the dimer-tetramer equilibrium shifted toward the heterotetramer dominant state in the concentration range of 10(-2)-1.0 mg/mL. In the tetramer dominant state, 50 water molecules likely glue the two heterodimers together as observed in the crystal structure. Because NHase exhibits a high abundance in bacterial cells, the result suggests that the heterotetramer is physiologically relevant. In addition, it was revealed that the substrate specificity of this enzyme, recognizing small aliphatic substrates rather than aromatic ones, came from the narrowness of the entrance channel from the bulk solvent to the active center. This finding may give a clue for changing the substrate specificity of the enzyme. Under the crystallization condition described here, one 1,4-dioxane molecule plugged the channel. Through spectroscopic and crystallographic experiments, we found that the molecule prevented the dissociation of the endogenous NO molecule from the active center even when the crystal was exposed to light.     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Polyamine oxidase from water hyacinth: purification and properties

Polyamine oxidase was purified to homogeneity from leaves of water hyacinth by the criterion of sodium dodecyl sulfate gel electrophoresis (SDS disc PAGE). The enzyme showed a high specificity for spermidine and spermine (K_m values 28 micromolar and 20 micromolar, respectively). The optimal pH of the enzyme for both spermidine and spermine was 6.5. The molecular weight of the enzyme estimated by Sephadex G-200 gel filtration was 87,000, while SDS disc PAGE gave a single band at the molecular weight of 60,000. Octamethylenediamine and quinacrine were strong inhibitors of the enzyme, but p-chloromercuribenzoate was without effect. A prosthetic group in the enzyme was identified as flavin adenine dinucleotide.     

Schistosoma mansoni: migration potential of normal and radiation attenuated parasites in naive guinea pigs

Compressed tissue autoradiography using [75Se]selenomethionine labelled parasites has been used to investigate the migration potential of normal and radiation attenuated cercariae of Schistosoma mansoni in naive guinea pigs. By Day 14 after infection. 44% of normal parasites were detected as reduced silver foci in the liver; this value corresponded well with the number of liver parasites recovered by retrograde perfusion of the hepatic portal system on Day 42 (42% of the challenge). In contrast, cercariae subjected to 50 krad of gamma irradiation failed to migrate out of the skin. The migration capacity of 20 krad irradiated parasites was less severely affected in that about half of the challenge parasites reached the lungs, but virtually none moved to the liver. These data are discussed in relation to the kinetics of immunity induced in guinea pigs by infection or vaccination with normal or radiation attenuated parasites.     

Stimulation of in vitro motility of Chlamydomonas axonemes by inhibition of cAMP-dependent phosphorylation

When demembranated axonemes of Chlamydomonas were reactivated with Mg-ATP, the proportion of motile axonemes was significantly increased by the presence of either phosphodiesterase (PDE) or protein inhibitor of cAMP-dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10(-5) M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP-dependent regulation of motility was observed with the axonemes from which detergent-soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation studies have implicated two polypeptides as candidates for the target protein of cAMP-dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.     

ADP-ribosylation by type C1 and D botulinum neurotoxins: stimulation by guanine nucleotides and inhibition by guanidino-containing compounds

We recently reported that type D botulinum neurotoxin ADP-ribosylates a specific protein of Mr 21,000 in membrane fractions of various tissues (Ohashi, Y. and Narumiya, S. (1987) J. Biol. Chem. in press). We examined similar enzyme activities in other types (types A, B, C1 and E) of botulinum neurotoxins. Of these, only type C1 toxin showed the activity similar to type D toxin and ADP-ribosylated the same Mr 21,000 protein in membranes of mouse brain. No enzyme activities were detected in type A, B and E toxins under the present experimental conditions. GTP stimulated ADP-ribosylation by the two toxins in a concentration dependent manner from 10 nM to 100 microM. The maximum stimulation was about 6 fold. GDP was 10 times less potent than GTP and achieved similar maximum at 1 mM, while GMP, ADP and ATP had little effect. Several guanidino-containing compounds dose-dependently inhibited the activities of both toxins. The IC50 values were 8.5, 14.5 and 45 mM for agmatine, L-arginine methyl ester and guanidine, respectively.     

Modification of expression of the malignant phenotype in radiation-initiated cells

Carcinogenesis is a multistage process consisting minimally of initiation and promotion/progression stages. Radiation and many environmental xenobiotics are potent initiating agents. We have shown that initiation of carcinogenesis in vivo by these agents is a common cellular event. In the irradiated thyroid (5 Gy) at least one in 20 cells is initiated. Initiation by both radiation and chemicals has also been shown to be a common cellular event in the mammary gland. Initiation therefore is most likely not the sole rate-limiting event in the carcinogenic process. The propensity of the initiated cell to express the malignant phenotype is modulated by many factors, including environmental chemicals and physiological and genetic factors. Scopal and abscopal physiological factors can either enhance or suppress the progression of initiated cells to a frank tumour. For example, prolactin enhances the rate of progression of radiation and chemically initiated mammary tumours while glucocorticoids suppress this progression. TSH enhances the progression of radiation-initiated thyroid tumours while a scopal factor associated with unirradiated thyroid cells suppresses progression of this tumour type.