ppGpp Controls Global Gene Expression in Light and in Darkness in S. elongatus.

The bacterial and plant stringent response involves production of the signaling molecules guanosine tetraphosphate and guanosine pentaphosphate ((p)ppGpp), leading to global reorganization of gene expression. The function of the stringent response has been well characterized in stress conditions, but its regulatory role during unstressed growth is less studied. Here, we demonstrate that (p)ppGpp-deficient strains of S. elongatus have globally deregulated biosynthetic capacity, with increased transcription rate, translation rate, and cell size in unstressed conditions in light and impaired viability in darkness. Synthetic restoration of basal guanosine tetraphosphate (ppGpp) levels is sufficient to recover transcriptional balance and appropriate cell size in light and to rescue viability in light/dark conditions, but it is insufficient to enable efficient dark-induced transcriptional shutdown. Our work underscores the importance of basal ppGpp signaling for regulation of cyanobacterial physiology in the absence of stress and for viability in energy-limiting conditions, highlighting that basal (p)ppGpp level is essential in cyanobacteria in the environmental light/dark cycle.

Systematic production of inactivating and non-inactivating suppressor mutations at the relA locus that compensate the detrimental effects of complete spot loss and affect glycogen content in Escherichia coli.

In Escherichia coli, ppGpp is a major determinant of growth and glycogen accumulation. Levels of this signaling nucleotide are controlled by the balanced activities of the ppGpp RelA synthetase and the dual-function hydrolase/synthetase SpoT. Here we report the construction of spoT null (ΔspoT) mutants obtained by transducing a ΔspoT allele from ΔrelAΔspoT double mutants into relA+ cells. Iodine staining of randomly selected transductants cultured on a rich complex medium revealed differences in glycogen content among them. Sequence and biochemical analyses of 8 ΔspoT clones displaying glycogen-deficient phenotypes revealed different inactivating mutations in relA and no detectable ppGpp when cells were cultured on a rich complex medium. Remarkably, although the co-existence of ΔspoT with relA proficient alleles has generally been considered synthetically lethal, we found that 11 ΔspoT clones displaying high glycogen phenotypes possessed relA mutant alleles with non-inactivating mutations that encoded stable RelA proteins and ppGpp contents reaching 45-85% of those of wild type cells. None of the ΔspoT clones, however, could grow on M9-glucose minimal medium. Both Sanger sequencing of specific genes and high-throughput genome sequencing of the ΔspoT clones revealed that suppressor mutations were restricted to the relA locus. The overall results (a) defined in around 4 nmoles ppGpp/g dry weight the threshold cellular levels that suffice to trigger net glycogen accumulation, (b) showed that mutations in relA, but not necessarily inactivating mutations, can be selected to compensate total SpoT function(s) loss, and (c) provided useful tools for studies of the in vivo regulation of E. coli RelA ppGpp synthetase.

[Roles of hMMS2 gene in reversing the oxaliplatin tolerance of human colon carcinoma cells].

In this study, the roles of hMMS2 (human methyl methanesulfonate sensitive mutant 2) gene encoding the human ubiquitin-conjugating enzyme E2 variant 2 in the drug resistance in human colon carcinoma were investigated by using a well-differentiated human colorectal carcinoma L-OHP-resistant cell line, THC8307/L-OHP. THC8307/L-OHP cells were transfected via liposome along with plasmid pcDNA6.2-GW/EmGFP-miR-MMS2 expressing both miRNA against hMMS2 and GFP, followed by real-time fluorescent quantitative PCR and immunofluorescence to select stable transfectants with significantly reduced hMMS2 expression. Compared with untransfected or pcDNA6.2-GW/EmGFP vector-transfected cells, the hMMS2-depleted cells displayed significantly (P<0.05) reduced half inhibition concentration(IC50) resistance index (RI) and colony-forming efficiency (CFE) upon treatment with oxaliplatin (L-OHP), while its relative reverse efficiency(RRE) was significantly higher (P<0.05) than the control cells, indicating compromised ability of cell proliferation. Indeed, Rho-damine 123 staining and flow cytometry analyses revealed an increased rate of apoptosis in hMMS2-depleted cells while no difference in cell proliferation or apoptosis was observed between the two control cell lines. The above observations collec-tively indicate that suppression of hMMS2 reverses L-OHP tolerance in differentiated human colorectal carcinoma cells by promoting apoptosis.

relA enhances the adherence of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a known causative agent of diarrhea in children. In the process of colonization of the small intestine, EPEC synthesizes two types of adhesins, the bundle-forming pilus (BFP) and intimin. The BFP pilus is an adhesin associated with the initial stages of adherence of EPEC to epithelial cells, while the outer membrane protein intimin carries out the intimate adherence that takes place at the third stage of infection. BFP is encoded by the bfp operon located in plasmid EAF, present only in typical EPEC isolates, while eae, the gene that encodes intimin is situated in the LEE, a chromosomal pathogenicity island. Transcription of bfp and eae is regulated by the products of the perABC operon, also present in plasmid EAF. Here we show that deletion of relA, that encodes a guanosine penta and tetraphosphate synthetase impairs EPEC adherence to epithelial cells in vitro. In the absence of relA, the transcription of the regulatory operon perABC is reduced, resulting in lower levels of BFP and intimin. Bacterial adherence, BFP and intimin synthesis and perABC expression are restored upon complementation with the wild-type relA allele.

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen.

Engineering and visualization of bacteria for targeting infarcted myocardium.

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (ΔppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P(BAD)), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of L-arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ΔppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.

RelA-dependent (p)ppGpp production controls exoenzyme synthesis in Erwinia carotovora subsp. atroseptica.

In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen Erwinia carotovora subsp. atroseptica. A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type E. carotovora subsp. atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule, N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in E. carotovora subsp. atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.

Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro.

BACKGROUND & AIMS: The transcription factor nuclear factor (NF)-kappaB plays a critical role in mediating survival of hepatocytes in response to tumor necrosis factor (TNF)-alpha during development because mice deficient for the NF-kappaB subunit RelA/p65 die in utero because of TNF-induced liver apoptosis. For the adult liver, conflicting concepts exist as to whether soluble TNF can trigger apoptosis when NF-kappaB activation is impaired. By creating a mouse model in which the transactivating NF-kappaB subunit RelA/p65 can be genetically inactivated in hepatocytes using the Cre/lox system, we sought to clarify the role of NF-kappaB in TNF-mediated hepatocyte apoptosis. METHODS: Deletion of RelA/p65 in the liver was achieved using an inducible conditional knockout system (rela(F/F)MxCre mice) or, hepatocyte-specifically, using a developmental conditional knockout system (rela(F/F)AlbCre mice). RESULTS: Disruption of RelA/p65 rendered mice sensitive to lethal liver injury upon TNF administration. Primary RelA/p65-deficient hepatocytes showed no NF-kappaB activation and undergo rapid apoptosis after TNF treatment. In contrast, hepatocytes deficient for I kappa B-kinase beta (IKK beta), displayed residual NF-kappaB activity and consecutively only mild apoptosis in response to TNF. TNF-induced apoptosis in RelA/p65-deficient hepatocytes was accompanied by prolonged activation of c-jun activating kinase (JNK) and rapid, largely proteasome-independent elimination of the long splice form of the antiapoptotic cellular FLICE inhibitor protein (c-FLIP(L)). Gene silencing of caspase-8, caspase-inhibitors, inhibition of JNK, or administration of antioxidants inhibited apoptosis and elimination of c-FLIP(L). CONCLUSIONS: RelA/p65 is essential for TNF-induced NF-kappaB activation in adult hepatocytes. Genetic deletion of a functional RelA/p65 sensitizes these cells to apoptosis in response to soluble TNF in vivo and in vitro.

A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M.

Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

A novel ubiquitin ligase is deficient in Fanconi anemia.

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

Are ubiquitination pathways central to Parkinson's disease?

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The major motor disabilities of PD are associated with the extensive loss of dopaminergic neurons in the substantia nigra pars compacta. The physiological changes and biochemical pathways involved in the selective demise of these neurons are still unclear. Recent studies have demonstrated that alterations or reductions in ubiquitin-mediated proteasome function can be causal of at least some forms of parkinsonism, and multiple lines of evidence suggest that this mechanism of protein degradation may play an important role in the etiology of PD.

[Parkinson's disease: what have we learned from the genes responsible for familial forms?]. / La maladie de Parkinson: que nous apprennent les gènes responsables des formes familiales?

Parkinson's disease is characterized by the progressive and selective loss of the dopaminergic neurons in the substantia nigra and the presence of ubiquitinated protein inclusions termed Lewy bodies. In the past six years, four genes involved in rare inherited forms of Parkinson's disease have been identified: mutations in the alpha-synuclein and ubiquitin carboxyterminal hydrolase L1 genes (UCH-L1) cause autosomal dominant forms, whereas mutations in the Parkin and DJ-1 genes are responsible for autosomal recessive forms of the disease. A toxic gain of function related to the ability of alpha-synuclein to assemble into insoluble amyloid fibrils may underlie neuronal cell death in parkinsonism due to alpha-synuclein gene mutations. In contrast, loss of protein function appears to be the cause of the disease in parkinsonism due to mutations in the genes encoding Parkin and UCH-L1, which are key enzymes of the ubiquitin-proteasome pathway. The presence of alpha-synuclein, Parkin and UCH-L1 in Lewy bodies suggests that dysfunction of pathways involved in protein folding and degradation is not only involved in the pathogenesis of familial Parkinson's disease, but could also play a role in the frequent sporadic form of the disease (idiopathic Parkinson's disease).

Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor.

Loss of the von Hippel-Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (-) cells). The cyclinD1 expression level was also abnormally high in VHL (-) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.

Meningioma showing VHL gene inactivation in a patient with von Hippel-Lindau disease.

The genetic mechanism of the tumorigenesis of meningioma in conjunction with von Hippel-Lindau (VHL) disease is unclear. The authors present a case of VHL disease associated with a posterior fossa meningioma and with multiple cerebellar hemangioblastomas. A germline mutation of the VHL gene and loss of heterozygosity on the VHL gene locus in 3p were detected in the meningioma. Tumorigenesis of a meningioma associated with VHL disease could be caused by inactivation of both alleles of the VHL gene.

von Hippel-Lindau protein-mediated repression of tumor necrosis factor alpha translation revealed through use of cDNA arrays.

Based on evidence that the von Hippel-Lindau (VHL) tumor suppressor protein is associated with polysomes and interacts with translation regulatory factors, we set out to investigate the potential influence of pVHL on protein translation. To this end, renal cell carcinoma (RCC) cells that either lacked pVHL or expressed pVHL through stable transfection were used to prepare RNA from cytosolic (unbound) and polysome-bound fractions. Hybridization of cDNA arrays using RNA from each fraction revealed a subset of transcripts whose abundance in polysomes decreased when pVHL function was restored. The tumor necrosis factor alpha (TNF-alpha) mRNA was identified as one of the transcripts that preferentially associated with polysomes in pVHL-deficient cells. Additional evidence that the TNF-alpha mRNA was a target of translational repression by pVHL was obtained from reporter gene assays, which further revealed that pVHL's inhibitory influence on protein synthesis occurred through the TNF-alpha 3'-untranslated region. Our findings uncover a novel function for the pVHL tumor suppressor protein as regulator of protein translation.

Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth.

Inactivation of the von Hippel-Lindau (VHL) gene is associated with the development of highly vascularized tumors. pVHL targets the alpha subunits of hypoxia inducible factor (HIF) for ubiquitin-mediated degradation in an oxygen-dependent manner. Although pVHL-deficient tumor cell lines demonstrate constitutive stabilization and activation of HIF, it has yet to be shown that loss of murine Vhl alone is sufficient to dysregulate HIF. We utilized a genetic approach to demonstrate that loss of Vhl is sufficient not only to stabilize HIF-alpha subunits under normoxia, but also fully activate HIF-mediated responses. These studies have implications for the hierarchy of signaling events leading to HIF stabilization, nuclear translocation, and target gene expression. We further demonstrate that loss of murine Vhl does not promote teratocarcinoma growth, indicating that other genetic changes must occur to facilitate Vhl-mediated tumorigenesis.

Negative regulation of Lck by Cbl ubiquitin ligase.

The Cbl-family ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent targeting to lysosomes. Cbl associates with the lymphoid-restricted nonreceptor tyrosine kinase Lck, but the functional relevance of this interaction remains unknown. Here, we demonstrate that T cell receptor and CD4 coligation on human T cells results in enhanced association between Cbl and Lck, together with Lck ubiquitination and degradation. A Cbl(-/-) T cell line showed a marked deficiency in Lck ubiquitination and increased levels of kinase-active Lck. Coexpression in 293T cells demonstrated that Lck kinase activity and Cbl ubiquitin ligase activity were essential for Lck ubiquitination and negative regulation of Lck-dependent serum response element-luciferase reporter activity. The Lck SH3 domain was pivotal for Cbl-Lck association and Cbl-mediated Lck degradation, with a smaller role for interactions mediated by the Cbl tyrosine kinase-binding domain. Finally, analysis of a ZAP-70-deficient T cell line revealed that Cbl inhibited Lck-dependent mitogen-activated protein kinase activation, and an intact Cbl RING finger domain was required for this functional effect. Our results demonstrate a direct, ubiquitination-dependent, negative regulatory role of Cbl for Lck in T cells, independent of Cbl-mediated regulation of ZAP-70.

Serial analysis of gene expression in renal carcinoma cells reveals VHL-dependent sensitivity to TNFalpha cytotoxicity.

We have used serial analysis of gene expression (SAGE) to investigate the influence of the von Hippel-Lindau (VHL) gene on global gene expression profiles. SAGE libraries were prepared from renal cell carcinoma (RCC) lines that either lack (parental) or express wild-type VHL (wtVHL). Comparison of these libraries revealed some differentially expressed genes (Glut-1, for example) that were known to be influenced by VHL, but the majority of genes had not previously been reported to be affected by the cell's VHL status. The identification of several genes involved in TNFalpha-mediated events prompted us to compare the sensitivity of cells with different VHL status in TNFalpha cytotoxicity assays. Strikingly, VHL-deficient cells were much more resistant to the toxic influence of TNFalpha. We propose that VHL-dependent sensitization of RCC cells to TNFalpha-mediated killing may contribute to VHL's growth suppressive function.

Genetic ablation of the steroid receptor coactivator-ubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and defects in reproduction.

The E6-associated protein (E6-AP), although originally identified as a ubiquitin ligase, has recently been shown to function as a coactivator of steroid receptor-dependent gene expression in in vitro assays. In order to determine whether E6-AP acts as a coactivator in vivo, physiological parameters associated with male and female sex steroid action were assessed in the E6-AP null mouse. Gonadal size was reduced in E6-AP null male and female mice in comparison to wild-type controls in conjunction with reduced fertility in both genders. Consistent with this observation, defects in sperm production and function, as well as ovulation were observed. In comparison to wild-type controls, induction of prostate gland growth induced by testosterone and uterine growth by estradiol were significantly reduced. In contrast, estrogen and progesterone-stimulated growth of virgin mammary gland was not compromised by E6-AP ablation despite E6-AP expression in this tissue. This latter finding contrasts with the impaired estrogen and progesterone-induced mammary gland development observed previously for steroid receptor coactivator type 1 (SRC-1) and SRC-3 female knockout mice. Taken together, these results are consistent with a role for E6-AP in mediating a subset of steroid hormone actions in vivo. Nevertheless, differences observed between SRC and E6-AP knockout phenotypes indicate that these two families of steroid receptor coactivators are not functionally equivalent and supports the hypothesis that coactivators contribute to tissue-specific steroid hormone action.

Parkin and the molecular pathways of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective demise of specific neuronal populations leading to impairment of motor functions. Recent genetic studies have uncovered several genes involved in inherited forms of the disease. These gene products are implicated in the biochemical pathways underlying the etiology of sporadic PD. Mutations in the parkin gene causal of autosomal recessive juvenile parkinsonism highlight that ubiquitin-mediated proteolysis may play an important role in the pathobiology of PD.