Exploring mitochondrial evolution and metabolism organization principles by comparative analysis of metabolic networks

The endosymbiotic theory proposed that mitochondrial genomes are derived from an alpha-proteobacterium-like endosymbiont, which was concluded from sequence analysis. We rebuilt the metabolic networks of mitochondria and 22 relative species, and studied the evolution of mitochondrial metabolism at the level of enzyme content and network topology. Our phylogenetic results based on network alignment and motif identification supported the endosymbiotic theory from the point of view of systems biology for the first time. It was found that the mitochondrial metabolic network were much more compact than the relative species, probably related to the higher efficiency of oxidative phosphorylation of the specialized organelle, and the network is highly clustered around the TCA cycle. Moreover, the mitochondrial metabolic network exhibited high functional specificity to the modules. This work provided insight to the understanding of mitochondria evolution, and the organization principle of mitochondrial metabolic network at the network level.     

Bacillus Anthracis Endospores Regulate Ornithine Decarboxylase and Inducible Nitric Oxide Synthase Through ERK1/2 and p38 Mitogen-Activated Protein Kinases

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (^?NO) from inducible nitric oxide synthase (NOS2) by competing for l-arginine, producing l-ornithine at the expense of ^?NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of l-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores’ germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.     

Style curvature and its adaptive significance in the Malvaceae

Curvature of un-pollinated styles towards the anthers in the Malvaceae has been interpreted as different adaptive mechanisms, presenting a challenging problem for evolutionary biologists. We have surveyed different species of this family to determine in which style curvature occurs, and to determine its possible functional roles. Two distinct types of style curvature occur in 18 species distributed in eight genera of four tribes: Type I, before pollen shedding, occurs in species of the Malvavisceae, Ureneae, and Hibisceae tribes, which have highly receptive stigmas and viable pollen when curvature brings stigmas into contact with anthers; and Type II, after pollen shedding, only appears in the protandrous species of the Malveae tribe. Curvature is often associated with annual and perennial herbs. We conclude that the adaptive significance of style curvature in the Malvaceae includes delayed selfing, promotion of outcrossing, and reduction in intrafloral male–female interference, even if the frequency of two or three of these occurs in one species only. This diversity provides model plants and the opportunity for determining the evolution, relative importance, and harmony mechanism of different adaptive significance, especially when two or three functions occur simultaneously in one floral movement of one species (e.g., Kosteletzkya virginica).     

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13, 14, 18, 20, 29, 49, 50, 51, 52, 53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11, 19, 46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm.nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells of multicellular organisms and unicellular microorganisms (17, 25, 26). Transport across the plasma membrane is the first step in the metabolism of these substrates, which may affect many aspects of the organism, including regulation of energy metabolism (9, 34) and acid-base equilibrium status (10).JEN1p has been identified in several fungal species, e.g., Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces lactis (9, 35, 45), which is a lactate/pyruvate symporter (1, 9, 34). The enzyme imports lactate or some short-chain monocarboxylates across the plasma membrane into cells. Then, the lactate is stereo-specifically oxidized to pyruvate. This reaction is performed by ferricytochrome c oxidoreductase in mitochondria (23, 33) and is tightly connected to the respiratory chain (34). JEN1 was induced by lactic, pyruvic, acetic, and propionic acids and repressed by glucose (2, 9, 35, 45). Nevertheless, for entomopathogenic fungi, the characterization of JEN1p has not been investigated, and its role in infection is still a mystery.For this paper, we studied the functions of a putative carboxylate transport gene, JEN1, in B. bassiana (BbJEN1). Our results demonstrated that BbJEN1 is involved in conidiation of B. bassiana and that the gene is a new factor influencing virulence in entomopathogenic fungi.     

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.     

Co-treating with arecoline and 4-nitroquinoline 1-oxide to establish a mouse model mimicking oral tumorigenesis

The aim of this study was to establish an effective mouse model of oral cancer and to use this model to identify potential markers of oral tumor progression. C57BL/6JNarl mice were treated with arecoline, 4-nitroquinoline 1-oxide (4-NQO), or both arecoline and 4-NQO in high and low doses for 8 weeks to induce oral tumor. The induced oral lesions were observed for 20 weeks to assess the efficiency of cancer induction and survival rate of the mice. In addition, two target proteins that are frequently overexpressed during tongue cancer tumorigenesis, αB-crystallin and Hsp27, were examined by immunohistochemical analysis. In mice exposed to 4-NQO (200 μg/mL) and arecoline (500 μg/mL), the tongue lesions showed evidence of hyperplasia, papilloma, dysplasia, and carcinoma, and the lesions were pathologically similar to those lesions in human oral cancer. The tongue tumor incidence rate was 100% in mice exposed to concomitant 4-NQO (200 μg/mL) and arecoline (500 μg/mL) treatment, 57% in mice exposed to 4-NQO only, and 0% in mice exposed to arecoline only. Immunohistochemical analysis demonstrated that, consistent with human studies, αB-crystallin and Hsp27 were upregulated in murine oral tumors. In conclusion, we have established a powerful animal model that enables the study of the promoting effects of arecoline on tongue tumorigenesis. Data subsequently attained from this mouse model support a role for αB-crystallin and Hsp27 as clinical markers for tumor progression.     

Studies on the structure–activity relationship of 1,3,3,4-tetra-substituted pyrrolidine embodied CCR5 receptor antagonists. Part 2: Discovery of highly potent anti-HIV agents

Modification of 1,3,3,4-tetra-substituted pyrrolidine embodied CCR5 receptor antagonists revealed that introducing a fluoro group at the 3-position of the 3-phenyl group to reduce metabolism did not adversely affect the high potency against HIV infection, and that replacing the piperidine ring with a tropane ring could deliver the most potent anti-HIV agents. Stereochemistry of the substituted tropane ring is essential for maintaining the potent anti-HIV activity because only exo-isomers displayed subnanomolar whole cell activity.     

Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

Upregulation of structurally homologous oncoproteins Hdm2 and Hdmx has been linked to the depletion or inactivation of their common regulation target the tumor suppressor p53 protein leading to the progression of cancer. The restoration of the p53 function, rendered suppressed or dormant by these negative regulators, establishes, therefore, a unique opportunity for a targeted induction of apoptosis in cancers that retain wild-type p53. While several small molecules have been reported to rescue the tumor suppressor by antagonizing the Hdm2–p53 interaction, these agents displayed limited application scope by being ineffective in tumors enriched with active Hdmx. Here, we describe the use of a genetic selection system and encoded library of conformationally pre-organized peptides to perform functional profiling of each regulator revealing specific recognition features that guide the antagonism of Hdm2–p53 and Hdmx–p53 interactions. Structure–activity relationship analysis of the most effective leads identified functional and structural elements mediating selective recognition of the two structurally related regulators, while providing convenient starting points for further activity optimization.