Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase

The Sec6/8 complex, also known as the exocyst complex, is an octameric protein complex that has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. Two subunits of the Sec6/8 complex, Exo84 and Sec5, have recently been shown to be effector targets for active Ral GTPases. However, the mechanism by which Ral proteins regulate the Sec6/8 activities remains unclear. Here, we present the crystal structure of the Ral-binding domain of Exo84 in complex with active RalA. The structure reveals that the Exo84 Ral-binding domain adopts a pleckstrin homology domain fold, and that RalA interacts with Exo84 via an extended interface that includes both switch regions. Key residues of Exo84 and RalA were found that determine the specificity of the complex interactions; these interactions were confirmed by mutagenesis binding studies. Structural and biochemical data show that Exo84 and Sec5 competitively bind to active RalA. Taken together, these results further strengthen the proposed role of RalA-regulated assembly of the Sec6/8 complex.     

Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.     

hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.     

Nuclear translocation of the heterotrimeric CCAAT binding factor of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> is dependent on two redundant localising signals in a single subunit

The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a ‘piggy back mechanism’ in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.     

In vitro degradation of the antimicrobial human peptide HEM-gamma 130-146 in plasma analyzed by a validated quantitative LC-MS/MS procedure

In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-gamma 130-146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative muHPLC-MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5-15%), accuracy (-3 to 15%), and ruggedness with a lower limit of quantification at 0.29 microg/ml plasma (0.15 microM). Because of good linearity (r2>0.999), the recovery (84+/-3%) and ion suppression (86+/-4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.     

Structural basis of FFAT motif-mediated ER targeting

The FFAT motif is a targeting signal responsible for localizing a number of proteins to the cytosolic surface of the endoplasmic reticulum (ER) and to the nuclear membrane. FFAT motifs bind to members of the highly conserved VAP protein family, which are tethered to the cytoplasmic face of the ER by a C-terminal transmembrane domain. We have solved crystal structures of the rat VAP-A MSP homology domain alone and in complex with an FFAT motif. The co-crystal structure was used to design a VAP mutant that disrupts rat and yeast VAP-FFAT interactions in vitro. The FFAT binding-defective mutant also blocked function of the VAP homolog Scs2p in yeast. Finally, overexpression of the FFAT binding-defective VAP in COS7 cells dramatically altered ER morphology. Our data establish the structural basis of FFAT-mediated ER targeting and suggest that FFAT-targeted proteins play an important role in determining ER morphology.     

Structure of the cytochrome complex SoxXA of Paracoccus pantotrophus, a heme enzyme initiating chemotrophic sulfur oxidation

The sulfur-oxidizing enzyme system (Sox) of the chemotroph Paracoccus pantotrophus is composed of several proteins, which together oxidize hydrogen sulfide, sulfur, thiosulfate or sulfite and transfers the gained electrons to the respiratory chain. The hetero-dimeric cytochrome c complex SoxXA functions as heme enzyme and links covalently the sulfur substrate to the thiol of the cysteine-138 residue of the SoxY protein of the SoxYZ complex. Here, we report the crystal structure of the c-type cytochrome complex SoxXA. The structure could be solved by molecular replacement and refined to a resolution of 1.9A identifying the axial heme-iron coordination involving an unusual Cys-251 thiolate of heme2. Distance measurements between the three heme groups provide deeper insight into the electron transport inside SoxXA and merge in a better understanding of the initial step of the aerobic sulfur oxidation process in chemotrophic bacteria.     

A previously unrecognized radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences

In sub-Saharan Africa, amphibians are represented by a large number of endemic frog genera and species of incompletely clarified phylogenetic relationships. This applies especially to African frogs of the family Ranidae. We provide a molecular phylogenetic hypothesis for ranids, including 11 of the 12 African endemic genera. Analysis of nuclear (rag-1, rag-2, and rhodopsin genes) and mitochondrial markers (12S and 16S ribosomal RNA genes) provide evidence for an endemic clade of African genera of high morphological and ecological diversity thus far assigned to up to five different subfamilies: Afrana, Cacosternum, Natalobatrachus, Pyxicephalus, Strongylopus, and Tomopterna. This clade has its highest species diversity in southern Africa, suggesting a possible biogeographic connection with the Cape Floral Region. Bayesian estimates of divergence times place the initial diversification of the southern African ranid clade at approximately 62-85 million years ago, concurrent with the onset of the radiation of Afrotherian mammals. These and other African ranids (Conraua, Petropedetes, Phrynobatrachus, and Ptychadena) are placed basally within the Ranoidae with respect to the Eurasian groups, which suggests an African origin for this whole epifamily.     

Cation fluxes cause plasma membrane depolarization involved in beta-glucan elicitor-signaling in soybean roots

Inducible and specific ion fluxes on plasma membranes represent very early events during elicitation of plant cells. The hierarchy of such ion fluxes involved is still unknown. The effect of Phytophthora sojae-derived beta-glucan elicitors on the plasma membrane potential as well as on surface K+, Ca2+, and H+ fluxes has been investigated on soybean roots using ion-selective microelectrodes. Beta-Glucans with different degrees of polymerization transiently depolarized the plasma membrane. The elicitor concentration necessary for half-maximal depolarization closely resembled the corresponding binding affinities of soybean root membranes toward the respective beta-glucans. Upon repeated elicitor treatment, the root cells responded partially refractory, suggesting a complex responsiveness of the system. Within the root hair space, characteristic decreasing K(+)- and Ca(2+)-free concentrations were induced by the elicitors, probably causing depolarization through the influx of positive charges. Whereas K+ fluxes were inverted after passing the K+ equilibrium (Nernst-) potential, Ca2+ influx continued. No anion fluxes sufficient to account for charge compensation were observed under the same experimental conditions. K+ and Ca2+ fluxes as well as depolarization were inhibited by 100 microM or less of the Ca2+ antagonist La3+. Contrasting other systems, in soybean the main cause for elicitor-induced plasma membrane depolarization is the activation of cation instead of anion fluxes.