Critical roles of interactions among switch I-preceding residues and between switch II and its neighboring alpha-helix in conformational dynamics of the GTP-bound Ras family small GTPases

GTP-bound forms of Ras family small GTPases exhibit dynamic equilibrium between two interconverting conformations, "inactive" state 1 and "active" state 2. A great variation exists in their state distribution; H-Ras mainly adopts state 2, whereas M-Ras predominantly adopts state 1. Our previous studies based on comparison of crystal structures representing state 1 and state 2 revealed the importance of the hydrogen-bonding interactions of two flexible effector-interacting regions, switch I and switch II, with the γ-phosphate of GTP in establishing state 2 conformation. However, failure to obtain both state structures from a single protein hampered further analysis of state transition mechanisms. Here, we succeed in solving two crystal structures corresponding to state 1 and state 2 from a single Ras polypeptide, M-RasD41E, carrying an H-Ras-type substitution in residue 41, immediately preceding switch I, in complex with guanosine 5'-(β,γ-imido)triphosphate. Comparison among the two structures and other state 1 and state 2 structures of H-Ras/M-Ras reveal two new structural features playing critical roles in state dynamics; interaction of residues 31/41 (H-Ras/M-Ras) with residues 29/39 and 30/40, which induces a conformational change of switch I favoring its interaction with the γ-phosphate, and the hydrogen-bonding interaction of switch II with its neighboring α-helix, α3-helix, which induces a conformational change of switch II favoring its interaction with the γ-phosphate. The importance of the latter interaction is proved by mutational analyses of the residues involved in hydrogen bonding. These results define the two novel functional regions playing critical roles during state transition.     

Characterization of early EDEM1 protein maturation events and their functional implications

The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.     

Home Range Size of Sympatric Squirrel Species Inhabiting a Lowland Dipterocarp Forest in Malaysia1

Home range sizes and spatial overlap of four sympatric squirrel species were investigated in a lowland dipterocarp forest in Malaysia using a radio‐tracking method. The population density of Callosciurus caniceps was highest and C. notatus was next highest, while C. nigrovittatus and Lariscus insignis were scarce. C. caniceps was larger than C. nigrovittatus and C. notatus while L. insignis was extremely small. For females, home range size was smaller in L. insignis than Callosciurus spp., which may support the body weight hypothesis: larger species have larger home ranges. Among the three Callosciurus species, female C. caniceps had the smallest home range. These differences were accounted for by habitat characteristics rather than by density or body weight; C. caniceps was dominant in bushy areas and used crowded small trees while C. notatus and C. nigrovittatus used large trees in the forest. In this study, home range size did not change seasonally; this differs from studies in temperate regions, possibly because food availability is much less variable among seasons in tropical rain forest. Home range overlap among heterospecific individuals was common but different species seemed to partition space by using different vertical levels of the forest. Consequently, the home range size and spatial overlap of sympatric squirrel species may be affected by habitat diversity in tropical rain forest.     

Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein

Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A(1), which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.     

A perennial ryegrass CBF gene cluster is located in a region predicted by conserved synteny between Poaceae species

CBF/DREB1 proteins are the most important regulators of the cold temperature signaling pathway in many plants. CBF genes are candidates for low-temperature tolerance QTL in wheat and barley. Ten novel putative CBF cDNAs of perennial ryegrass (Lolium perenne L.) have been isolated from cold-treated leaf tissue. Their primary structures contain some conserved motifs, characteristic of the gene class. Phylogenetic analysis revealed that LpCBF genes were attributable to the HvCBF3-, and HvCBF4-subgroups following the previously proposed classification of barley CBF genes. RT-PCR analysis revealed that the expression of LpCBF genes was rapidly induced in response to low temperature and that the expression pattern under the low-temperature conditions for a long period was different between the various LpCBF genes. Five of the ten LpCBF genes were assigned to the genetic linkage map using the p150/112 reference mapping population. LpCBFIb, LpCBFII, LpCBFIIIb and LpCBFIIIc were mapped on LG5 forming a cluster within 2.2 cM, while LpCBFVb was located on LG1. Based on comparative genetic studies, conserved synteny for CBF gene family was observed between the Triticeae cereals and perennial ryegrass. Information on the perennial ryegrass CBF genes at both the molecular and genetic level obtained in this study would be useful for the further study on the role of CBF genes and low-temperature tolerance in grasses.     

Reduction of Aluminum Toxicity by 2-Isopropylmalic Acid in the Budding Yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae secretes 2-isopropylmalic acid (2-iPMA), an intermediate in leucine biosynthesis. Because 2-iPMA binds Al(III) in the culture medium, it is thought to reduce toxicity by Al(III). The effects of 2-iPMA and malic acid (MA) on Al toxicity were investigated in a medium with a low pH and low concentrations of phosphates and magnesium. The reduction in the growth of S. cerevisiae observed in the presence of 100 μM Al(III) ions was relieved more by the addition of 1.0 mM 2-iPMA than by 1.0 mM MA, indicating that 2-iPMA possesses superior Al(III)-ion detoxification ability. Investigations using the wild type and the Δleu4 and Δleu9 mutant strains indicated that secretion of a sufficient level of 2-iPMA was required to enhance the Al tolerance. It is thought that 2-iPMA secreted from the yeast cells chelates Al ions and prevents them from entering the cells, resulting in Al tolerance. Suzuki and Tamura contributed equally to this work.     

The effect of calcium ion on the biodegradation of octylphenol polyethoxylates,and the antiandrogenic activity of their biodegradates

Because limes have been used as important fertilizers to neutralize acidified farmland in Japan, our interest in this study was focused on the effect of calcium ion on the biodegradation of octylphenol polyethoxylates (OPEOn) by a pure culture of Pseudomonas putida S5 isolated from a rice paddy field in Japan. In the presence of calcium ion, P. putida S5 accelerated the formation of octylphenol oligoethoxy carboxylates (OPECn) rather than that of octylphenol oligoethoxylates under an aerobic condition, indicating that more soluble biodegradates with terminal carboxyl group may liquate out easily to surface and ground water rather than more hydrophobic biodegradates with shorter ethylene oxide residues. Therefore, the androgen receptor (AR) activity of their degradation products was characterized using an in vitro reporter gene assay. As ethylene oxide chain length decreased, the biodegradates, OPEOn (n < 3), increased their AR antagonist activity. However, OPECn (n < 3) were unable to determine their AR activity because of their cytotoxicity in our reporter gene assay system.     

Transformation of an alpha-helix peptide into a beta-hairpin induced by addition of a fragment results in creation of a coexisting state

Intrinsic rules of determining the tertiary structure of a protein have been unknown partly because physicochemical factors that contribute to stabilization of a protein structure cannot be represented as a linear combination of local interactions. To clarify the rules on the nonlinear term caused by nonlocal interaction in a protein, we tried to transform a peptide that has a fully helical structure (Target Peptide or TP) into a peptide that has a beta-hairpin structure (Designed Peptide or DP) by adding seven residues to the C terminus of TP. According to analyses of nuclear magnetic resonance measurements, while the beta-hairpin structure is stabilized in some DPs, it is evident that the helical structure observed in TP is also persistent and even extended throughout the length of the molecule. As a result, we have produced a peptide molecule that contains both the alpha-helix and beta-hairpin conformation at an almost equally populated level. The helical structures contained in these DPs were more stable than the helix in TP, suggesting that stabilizing one conformation does not result in destabilizing the other conformation. These DPs can thus be regarded as an isolated peptide version of the chameleon sequence, which has the capability of changing the secondary structure depending on the context of the surrounding environment in a protein structure. The fact that the transformation of one secondary structure caused stabilization of both the original and the induced structure would shed light on the mechanism of protein folding.