Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement.

The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

Activation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP-ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide-induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis-inducing factor could not be observed, but cells could be saved by methyl pyruvate and alpha-ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis-necrosis switch in severely damaged cells.

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230.

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence (916)NFNHIHKRIRRVADKYLSG(934) comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin alpha/beta mechanism employing importins alpha1 and alpha3 but not alpha5. This transport was inhibited by wheat germ agglutinin and GTPgammaS. The sequence (1414)SKKTNRGSQLHKYYMKRRTL(1433), a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin alpha1/beta or alpha3/beta complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

BACKGROUND: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals. METHODS: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230. RESULTS: PI4K230 gives a prominent signal in the nucleolus of ethanol fixed rat brain cryosections and of several cell types in addition to its presence in the nucleus and cytoplasm. The PI4K230 immunoreactivity of the nucleolus is masked in PFA fixed cells, but it can be restored by treatment of PFA fixed cells with hot wet citrate buffer or by washing the cryosections with PBS prior to PFA fixation. Nucleolar PI4K230 occurs in a Triton X-100 resistant complex. Treatment of COS-7 cells with siRNA targeting PI4K230 and permeabilized B50 cells with DNase or RNase results in the loss of PI4K230 signal from the nucleolus. CONCLUSION: These experiments suggest the participation of PI4K230 in a DNase and RNase sensitive complex with a unique localization and function in the nucleolus.

Mammalian phosphatidylinositol 4-kinases.

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.