Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

CD8 T cell expansion and memory differentiation are facilitated by simultaneous and sustained exposure to antigenic and inflammatory milieu

Understanding the factors contributing to the generation of immune memory is important for rational vaccine design. In this study, we addressed the individual and combined roles of Ag and inflammation in sustaining the ability of primed CD8 T cells to clonally expand and differentiate into memory cells. We transferred CD8 T cells that were primed for a brief period into naive mice, mice infected with a pathogen not carrying the specific Ag (inflammation only), mice infected with a pathogen carrying the donor cell-specific Ag (inflammation plus Ag), or into mice exposed to soluble Ag (Ag only). We found that the donor CD8 T cells continued to proliferate in all the four conditions, but their ability to clonally expand and differentiate into memory cells was approximately 1000-fold higher when transferred into mice acutely infected with pathogen carrying the relevant Ag. Memory cells generated under conditions of sustained exposure to inflammation and Ag during the priming phase were superior in their ability to elicit recall responses on a per cell basis. Thus, simultaneous and sustained exposure of donor CD8 T cells to inflammatory and antigenic stimuli, following the initial priming phase, leads to the greatest expansion of CD8 T cells at the peak of the immune response and induces an optimal memory differentiation program. These results suggest that vaccination strategies should attempt to provide sustained exposure to Ag plus inflammation but not either alone following the initial priming.     

An InCytes from MBC Selection: Importin β Regulates the Seeding of Chromatin with Initiation Sites for Nuclear Pore Assembly

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the “seeding” of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin β. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin β is only partially reversed by RanGTP. Importin β forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin β and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.     

Improved visualization of vertebrate nuclear pore complexes by field emission scanning electron microscopy

Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.     

The Drosophila hus1 gene is required for homologous recombination repair during meiosis

The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Previously, we demonstrated that Drosophila hus1 is essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. In this study, we further analyzed the function of hus1 during meiosis and discovered that the synaptonemal complex (SC) disassembles abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, we showed that in hus1, DSBs that form during meiosis are not processed efficiently, and that this defect is not suppressed by a mutation in DmChk2. We found a genetic interaction between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, our results imply that hus1 is required for repair of DSBs during meiotic recombination.     

Loss of IFN-gamma production by invariant NK T cells in advanced cancer

Invariant NK T cells express certain NK cell receptors and an invariant TCRalpha chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-gamma, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (alpha-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-gamma and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-gamma production and a striking decrease in their IFN-gamma:IL-4 ratio. The IFN-gamma deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-gamma and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.     

Peptide-based hydrogel nanoparticles as effective drug delivery agents

Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules.     

Are microbial symbionts involved in the speciation of the gall-inducing aphid, <Emphasis Type="Italic">Slavum wertheimae</Emphasis>?

Microbial symbionts have come to be recognized as agents in the speciation of their eukaryote hosts. In this study, we asked if bacterial symbionts are, or were in the past, involved in the speciation of the gall-inducing aphid Slavum wertheimae (Hemiptera: Aphididae). This aphid is specific to the tree Pistacia atlantica, which has a fragmented distribution among mesic and xeric habitats, leading to corresponding fragmentation of the aphid population. Previous studies revealed genetic differentiation among populations of the gall-inducing aphid, suggesting cryptic allopatric speciation. Pistacia atlantica trees show no such variation. By means of diagnostic PCR, we screened several populations of S. wertheimae from mesic and xeric sites in Israel for the presence of nine known aphid symbionts: Arsenophonus, Hamiltonella, Regiella, Rickettsia, Rickettsiella, Serratia, Spiroplasma, Wolbachia, and X-type, as well as Cardinium, known to be a reproductive manipulator. Only one symbiont, Wolbachia, was detected in S. wertheimae. Wolbachia was found in all the aphids of the mesic populations, compared to 26% in the aphids from the xeric populations. Multilocus Sequence typing of Wolbachia revealed new haplotypes in the fbpA and coxA genes in both the mesic and xeric populations. Phylogenetic analysis showed that Wolbachia of S. wertheimae is closely related to Wolbachia strains from assorted hosts, mostly lepidopterans, but only distantly related to Wolbachia strains from other aphid species. We conclude that the cryptic speciation of mesic and xeric populations of S. wertheimae was likely driven by geographical isolation rather than by Wolbachia.     

A novel role for Dun1 in the regulation of origin firing upon hyper-acetylation of H3K56

During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader.