Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.     

Species cohesion of an extremophyte (Carex angustisquama,Cyperaceae) in solfatara fields maintained under interspecific natural hybridization

Background and AimsHybridization is the main driver of plant diversification, and gene flow via hybridization has multifaceted effects on plant evolution. Carex angustisquama is an extremophyte that grows on soils heavily acidified by volcanism. Despite its habitat distinct from that of other species, this species is known to form interspecific hybrids, implying interspecific gene flow. It is crucial to verify the extent and direction of interspecific gene flow between C. angustisquama and closely related species to understand the evolutionary process of an extremophyte in solfatara fields.MethodsIn this study, expressed sequence tag–simple sequence repeat markers were utilized to infer the extent and direction of interspecific gene flow between C. angustisquama and closely related species.Key ResultsBayesian clustering and simulation analyses revealed that all individuals of the three hybrid species were classified into the first hybrid generation or first backcross to C. angustisquama; therefore, current interspecific gene flow is limited. Moreover, in the Bayesian inference of historical gene flow based on multispecies samples, the model that assumed no interspecific gene flow was the most strongly supported across all species pairs, including phylogenetically close but ecologically distinctive species pairs.ConclusionsOur results revealed that interspecific gene flow between C. angustisquama and its related species has been limited both currently and historically. Moreover, our results of Bayesian inference of historical gene flow indicated that extrinsic, rather than intrinsic, factors probably act as isolating barriers between Carex species, with hybrid breakdown via microhabitat segregation being the probable potential barrier. Overall, our findings provide insights into the evolutionary process of an extremophyte in solfatara fields and offer an important example of the mechanisms of diversification of the speciose genus Carex.     

Intraspecific karyotypic polymorphism is highly concordant with allozyme variation in Lysimachia mauritiana (Primulaceae: Myrsinoideae) in Taiwan: implications for the colonization history and dispersal patterns of coastal plants

Background and Aims

Investigating intraspecific karyotypic and genetic variations jointly can provide unique insights into how historical, ecological and cytogenetic factors influence microevolution. A coastal herb, Lysimachia mauritiana, exhibits extensive karyotypic polymorphism and displays a complex cytogeographic pattern across the Ryukyus. To explore whether a similar degree of chromosomal variation exists south of the Ryukyus, and in an attempt to ascertain the mechanisms that may have generated the patterns, comprehensive sampling was conducted in Taiwan.

Methods

Karyotypes were analysed at mitotic metaphase for 550 individuals from 42 populations throughout Taiwan Proper and its adjacent islands. In addition, genetic variation was estimated using 12 allozymes (21 loci) of 314 individuals sampled from 12 localities.

Key Results

Four chromosome numbers and eight cytotypes, including four endemic cytotypes, were detected. Cytotype distributions were highly structured geographically, with single cytotypes present in most populations and four major cytotypes dominating the north, east and south of Taiwan and the Penghu Archipelago. Allozyme variation was very low and F-statistics indicated an extremely high level of population differentiation, implying limited gene flow among populations. Cluster analysis of allozyme variation uncovered four geographic groups, each corresponding perfectly to the four dominant cytotypes. The geographic structure of cytotype distribution and allozyme variation probably resulted from severe genetic drift triggered by genetic bottlenecks, suggesting that Taiwanese populations were likely to be derived from four independent founder events. In the few localities with multiple cytotypes, cytogeographic patterns and inferences of chromosomal evolution revealed a trend of northward dispersal, consistent with the course of the Kuroshio Current that has been influential in shaping the coastal biota of the region.

Conclusions

The data elucidate the patterns of colonization and the effects of the Kuroshio Current on the distribution of L. mauritiana in Taiwan. These inferences are highly relevant to other coastal plant species in the region and will stimulate further studies.     

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4)P recognition site. An extensive interaction study using NMR and other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.     

Complete genome sequence of Mycoplasma pneumoniae type 2a strain 309, isolated in Japan

Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain of this bacterium, has variations in the P1 protein, which is responsible for attachment of the bacterium to host cells. Here, we report the complete genome sequence of M. pneumoniae strain 309 isolated from a pneumonia patient in Japan.     

Proinsulin C-peptide activates α-enolase: implications for C-peptide--cell membrane interaction

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.     

The use of mammalian cultured cells loaded with a fluorescent dye shows specific membrane penetration of undissociated acetic acid

Acetic acid induces unique physiological responses in mammalian cells. Our previous study found that fura-2-loaded human embryonic kidney (HEK) 293T cells showed a robust intracellular fluorescence response immediately after stimulation with acetic acid, and no such response in the case of citric acid. In the present study, we aimed to identify the unique characteristics of acetic acid responsible for this phenomenon. We found that one such feature is its hydrophobicity. We also discovered that acetic acid induces cell responses by intracellular acidification. Of the components of acetic acid in solution (protons, acetate ions, and undissociated acetic acid), undissociated acetic acid might be the functional unit that penetrates the lipid bilayer of cell membranes to acidify the intracellular environment, thereby inducing cell responses. The method used in this study might be convenient in evaluating the intracellular acidification of cultured cells by acids in the external environment.     

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC₅₀ = 7.7–11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.