CgDN24: A gene involved in hyphal development in the fungal phytopathogen Colletotrichum gloeosporioides

A cDNA corresponding to a transcript induced in culture by N starvation, was identified in Colletotrichum gloeosporioides by a differential hybridisation strategy. The cDNA comprised 905 bp and predicted a 215 aa protein; the gene encoding the cDNA was termed CgDN24. No function for CgDN24 could be predicted by database homology searches using the cDNA sequence and no homologues were found in the sequenced fungal genomes. Transcripts of CgDN24 were detected in infected leaves of Stylosanthes guianensis at stages of infection that corresponded with symptom development. The CgDN24 gene was disrupted by homologous recombination and this led to reduced radial growth rates and the production of hyphae with a hyperbranching phenotype. Normal sporutation was observed, and following conidia inoculation of S. guianensis, normal disease development was obtained. These results demonstrate that CgDN24 is necessary for normal hyphal development in axenic culture but dispensable for phytopathogenicity. (c) 2005 Elsevier GmbH. Alt rights reserved.

Defenses against oxidative stress in Neisseria gonorrhoeae: A system tailored for a challenging environment

Neisseria gonorrhoeae is a host-adapted pathogen that colonizes primarily the human genitourinary tract. This bacterium encounters reactive oxygen and reactive nitrogen species as a consequence of localized inflammatory responses in the urethra of males and endocervix of females and also of the activity of commensal lactobacilli in the vaginal flora. This review describes recent advances in the understanding of defense systems against oxidative stress in N. gonorrhoeae and shows that while some of its defenses have similarities to the paradigm established with Escherichia coli, there are also some key differences. These differences include the presence of a defense system against superoxide based on manganese ions and a glutathione-dependent system for defense against nitric oxide which is under the control of a novel MerR-like transcriptional regulator. An understanding of the defenses against oxidative stress in N. gonorrhoeae and their regulation may provide new insights into the ways in which this bacterium survives challenges from polymorphonuclear leukocytes and urogenital epithelial cells.

Patched1 functions as a gatekeeper by promoting cell cycle progression

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G(1)-S phase and G(2)-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a gatekeeper. In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

The 2.1 angstrom crystal structure of copGFP, a representative member of the copepod clade within the green fluorescent protein superfamily

The green fluorescent protein (avGFP), its variants, and the closely related GFP-like proteins are characterized structurally by a cyclic tri-peptide chromophore located centrally within a conserved beta-can fold. Traditionally, these GFP family members have been isolated from the Cnidaria although recently, distantly related GFP-like proteins from the Bilateria, a sister group of the Cnidaria have been described, although no representative structure from this phylum has been reported to date. We have determined to 2.1 angstrom resolution the crystal structure of copGFP, a representative GFP-like protein from a copepod, a member of the Bilateria. The structure of copGFP revealed that, despite sharing only 19% sequence identity with GFP, the tri-peptide chromophore (Gly57-Tyr58-Gly59) of copGFP adopted a cis coplanar conformation within the conserved beta-can fold. However, the immediate environment surrounding the chromophore of copGFP was markedly atypical when compared to other members of the GFP-superfamily, with a large network of bulky residues observed to surround the chromophore. Arg87 and Glu222 (GFP numbering 96 and 222), the only two residues conserved between copGFP, GFP and GFP-like proteins are involved in autocatalytic genesis of the chromophore. Accordingly, the copGFP structure provides an alternative platform for the development of a new suite of fluorescent protein tools. Moreover, the structure suggests that the autocatalytic genesis of the chromophore is remarkably tolerant to a high degree of sequence and structural variation within the beta-can fold of the GFP superfamily. (c) 2006 Elsevier Ltd . All rights reserved.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. it operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

The PhD Finger Protein VRN5 Functions in the Epigenetic Silencing of Arabidopsis FLC

Vernalization, the acceleration of flowering by the prolonged cold of winter, ensures that plants flower in favorable spring conditions. During vernalization in Arabidopsis, cold temperatures repress FLOWERING LOCUS C (FLC) expression [1,2] in a mechanism involving VERNALIZATION INSENSITIVE 3 (VIN3) [3], and this repression is epigenetically maintained by a Polycomb-like chromatin regulation involving VERNALIZATION 2 (VRN2), a Su(z)12 homolog, VERNALIZATION 1 (VRN1), and LIKE-HETEROCHROMATIN PROTEIN 1 [4,5,6,7,8]. In order to further elaborate how cold repression triggers epigenetic silencing, we have targeted mutations that result in FLC misexpression both at the end of the prolonged cold and after subsequent development. This identified VERNALIZATION 5 (VRN5), a PHD finger protein and homolog of VIN3. Our results suggest that during the prolonged cold, VRN5 and VIN3 forma heterodimer necessary for establishing the vernalization-induced chromatin modifications, histone deacetylation, and H3 lysine 27 trimethylation required for the epigenetic silencing of FLC. Double mutant and FLC misexpression analyses reveal additional VRN5 functions, both FLC-dependent and -independent, and indicate a spatial complexity to FLC epigenetic silencing with VRN5 acting as a common component in multiple pathways.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Small Ubiquitin-related Modifier (SUMO)-1 promotes glycolysis in hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.

Uncovering changes in the redox protein interactome of PTEN

Phosphatase and tensin homolog (PTEN) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating a number of cellular processes including metabolism, apoptosis, cell proliferation and survival. It acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of a redox regulation of PTEN downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on the PTEN interactome is still poorly defined. To investigate this, PTEN-GST fusion protein was prepared in its reduced form and an H2O2-oxidized form that was reversible by DTT treatment, and these were immobilized on a glutathione-sepharose-based support. The immobilized protein was incubated with cell lysate to capture interacting proteins. Captured proteins were eluted from the beads, analyzed by LC-MSMS and comparatively quantified using label-free methods. After subtraction of interactors that were also present in the resin and GST controls, 97 individual protein interactors were identified, including several that are novel. Fourteen interactors that varied significantly with the redox status of PTEN were identified, including thioredoxin and peroxiredoxin-1. Except for one interactor, their binding was higher for oxidized PTEN. Using western blotting, altered binding to PTEN was confirmed for 3 selected interactors (Prdx1, Trx, and Anxa2) and DDB1 was validated as a novel interactor with unaltered binding. Our results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome which is important for the cellular function of PTEN. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Structural flexibility and oxygen diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.

Understanding Ten-Eleven Translocation-2 in Hematological and Nervous Systems

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Mechanisms of Arsenic Detoxification and Resistance

Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-, dimethyl- and trimethyl products. Synthetic purified CrArsM was crystallized in an unliganded form. Biochemical and biophysical studies conducted on CrArsM sheds new light on the pathways of biomethylation. While in microbes ArsM detoxifies arsenic, the human homolog, hAS3MT, converts inorganic arsenic into more toxic and carcinogenic forms. An understanding of the enzymatic mechanism of ArsM will be critical in deciphering its parallel roles in arsenic detoxification and carcinogenesis.