MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis

DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.     

Acetylation/Deacetylation Modulates the Stability of DNA Replication Licensing Factor Cdt1

Proper expression of the replication licensing factor Cdt1 is primarily regulated post-translationally by ubiquitylation and proteasome degradation. In a screen to identify novel non-histone targets of histone deacetylases (HDACs), we found Cdt1 as a binding partner for HDAC11. Cdt1 associates specifically and directly with HDAC11. We show that Cdt1 undergoes acetylation and is reversibly deacetylated by HDAC11. In vitro, Cdt1 can be acetylated at its N terminus by the lysine acetyltransferases KAT2B and KAT3B. Acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. These results extend the list of non-histone acetylated proteins to include a critical DNA replication factor and provide an additional level of complexity to the regulation of Cdt1.To maintain genomic integrity, DNA replication must be tightly controlled to ensure that each portion of the genome replicates once and only once per cell cycle (reviewed in Ref. 1). Replication licensing begins by the formation of the prereplication complex at multiple potential origins of replication. This is established sequentially, with the origin recognition complex (ORC)² proteins binding first, followed by the recruitment of Cdc6 and Cdt1, which in turn recruit the MCM2–7 proteins. MCM proteins act as the replicative helicase. The licensed replication origins are activated by cyclin-dependent kinases at the start of S phase. Licensing occurs throughout the cell cycle once S phase is complete.Cdt1 levels fluctuate throughout the cell cycle. It is destabilized at G₁/S transition, and then levels begin to climb again upon S phase completion. To prevent licensing at inappropriate times, two separate processes regulate the inactivation or destruction of Cdt1. First, geminin negatively regulates Cdt1 function by prevention of the association of Cdt1 with MCM2–7 via steric hindrance (2). Interestingly, geminin also positively regulates Cdt1 by preventing its ubiquitylation, perhaps by prevention of its interaction with an E3 ligase. This allows Cdt1 to accumulate in G₂ and M phases, to ensure adequate pools of Cdt1 to license the next cycle of replication (3). The ratio of geminin to Cdt1 likely determines whether geminin positively or negatively regulates Cdt1 (4). Second, Cdt1 is targeted for proteolysis by two distinct ubiquitin E3 ligases: the SCF-Skp2 complex and the DDB1-Cul4 complex (5). Phosphorylation by cyclin A/Cdk2 promotes interaction of Cdt1 with Skp2, leading to Cdt1 degradation during S phase (6–8). In addition, DDB1-Cul4 utilizes proliferating cell nuclear antigen as a binding platform to contact Cdt1, targeting the destruction of Cdt1 in S phase or following DNA damage (9, 10). Ubiquitylation by either of these E3 ligases promotes degradation of Cdt1 by the proteasome.Ubiquitylation occurs primarily (but not exclusively) on the ε-amino group of lysine residues. Another prominent post-translational modification that occurs on that residue is acetylation. Acetylation and, correspondingly, deacetylation can modulate the function and activity of a variety of proteins (see Ref. 11 for review). Here, we report that Cdt1 physically interacts with HDAC11, a class IV histone deacetylase (12, 13), as well as with several lysine acetyltransferases (KATs). We show that Cdt1 is an acetylated protein and further show that acetylation protects Cdt1 from ubiquitylation and subsequent proteasomal degradation. This study uncovers yet another layer of complexity to the regulation of the critical licensing factor Cdt1.     

Bmovo-1 Regulates Ovary Size in the Silkworm,Bombyx mori

The regulation of antagonistic OVO isoforms is critical for germline formation and differentiation in Drosophila. However, little is known about genes related to ovary development. In this study, we cloned the Bombyx mori ovo gene and investigated its four alternatively spliced isoforms. BmOVO-1, BmOVO-2 and BmOVO-3 all had four C2H2 type zinc fingers, but differed at the N-terminal ends, while BmOVO-4 had a single zinc finger. Bmovo-1, Bmovo-2 and Bmovo-4 showed the highest levels of mRNA in ovaries, while Bmovo-3 was primarily expressed in testes. The mRNA expression pattern suggested that Bmovo expression was related to ovary development. RNAi and transgenic techniques were used to analyze the biological function of Bmovo. The results showed that when the Bmovo gene was downregulated, oviposition number decreased. Upregulation of Bmovo-1 in the gonads of transgenic silkworms increased oviposition number and elevated the trehalose contents of hemolymph and ovaries. We concluded that Bmovo-1 was involved in protein synthesis, contributing to the development of ovaries and oviposition number in silkworms.     

Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis

Here we report the presence and expression levels of the vanC ₁ and vanC _2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC ₁ and vanC _2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC ₁ gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.     

Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine: A NOVEL CATEGORY OF BIOACTIVE 1-DEOXYSPHINGOID BASES AND 1-DEOXYDIHYDROCERAMIDES BIOSYNTHESIZED BY MAMMALIAN CELL LINES AND ANIMALS*

Fumonisin B₁ (FB₁) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB₁ increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C₁₈H₄₀NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH₃CH=NH ⁺₂) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-¹³C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK₁ cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-¹³C]palmitate into 1-[¹³C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[¹³C]deoxySa. 1-DeoxySa was elevated in FB₁-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK₁ and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB₁, substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and “ceramides” that might play important roles in cell regulation.Fumonisins (FB)² cause diseases of horses, swine, and other farm animals and are regarded to be potential risk factors for human esophageal cancer (1) and, more recently, birth defects (2). Studies of this family of mycotoxins, and particularly of the highly prevalent subspecies fumonisin B₁ (FB₁) (reviewed in Refs. 1 and 2), have established that FB_1, is both toxic and carcinogenic for laboratory animals, with the liver and kidney being the most sensitive target organs (3, 4). Other FB are also toxic, but their carcinogenicity is unknown.FB are potent inhibitors of ceramide synthase(s) (CerS) (5), the enzymes responsible for acylation of sphingoid bases using fatty acyl-CoA for sphingolipid biosynthesis de novo and recycling pathways (6). As a consequence of this inhibition, the substrates sphinganine (Sa) and, usually to a lesser extent, sphingosine (So), accumulate and are often diverted to sphinganine 1-phosphate (Sa1P) and sphingosine 1-phosphate (S1P), respectively (7), while the product N-acylsphinganines (dihydroceramides), N-acylsphingosines (ceramides, Cer), and more complex sphingolipids decrease (5, 7). This disruption of sphingolipid metabolism has been proposed to be responsible for the toxicity, and possibly carcinogenicity, of FB, based on mechanistic studies with cells in culture (5, 7–9). This has been borne out by a number of animal feeding studies that have correlated the elevation of Sa in blood, urine, liver, and kidney with liver and kidney toxicity (4, 7, 10, 11).Most of the mechanistic studies have focused on the accumulation of free Sa and other sphingoid bases, because these compounds are highly cytotoxic, although the large number of bioactive metabolites in this pathway make it likely that multiple mediators may participate (7, 9). Nonetheless, inhibition of serine palmitoyltransferase (SPT), the initial enzyme of de novo sphingolipid biosynthesis, reverses the increased apoptosis and altered cell growth induced by FB₁ treatment (12–19). Therefore, it is likely that these effects of FB₁ are due to the accumulation of cytotoxic intermediate(s) rather than depletion of downstream metabolites, because the latter also occurs when SPT is inhibited.In studies of the effects of FB₁ on the renal cell line LLC-PK₁ (20),³ we have noted that in addition to the elevation of Sa and So, there is a large increase in an unidentified species that appears to be a sphingoid base, because it is extracted by organic solvents, derivatized with ortho-phthalaldehyde (OPA), and eluted from reverse-phase liquid chromatography (LC) in the sphingoid base region. Herein we report: (i) the isolation and characterization of this novel sphingoid base as 1-deoxysphinganine (1-deoxySa); (ii) that its origin is the utilization of alanine instead of serine by SPT as well as that the N-acyl-derivatives of 1-deoxySa (1-deoxydihydroceramides (1-deoxyDHCer)) are normally found in mammalian cells; (iii) that 1-deoxySa has cytotoxicity comparable to other sphingoid bases elevated by FB₁; and (iv) that 1-deoxySa is not only elevated in cells in culture but also in tissues of animals exposed to dietary FB and, therefore, might contribute to diseases caused by these mycotoxins.     

Nonindependent K+ Movement through the Pore in IRK1 Potassium Channels

We measured unidirectional K⁺ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K⁺ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K⁺ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K⁺ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K⁺ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K⁺ concentration, and may differ in the number of K⁺ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.