AbstractThe Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

AbstractType IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus (‘wide’ and ‘narrow’), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

. Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell–cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different
. Sulfolobus
. strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.
.

The differentiation of mouse embryonic stem cells (ESCs) is controlled by the interaction of multiple signaling pathways, typically mediated by post-translational protein modifications. The addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O-GlcNAcylation), whose function in ESCs is only now beginning to be elucidated. Here, we demonstrate that the specific inhibition of O-GlcNAc hydrolase (Oga) causes increased levels of protein O-GlcNAcylation and impairs differentiation of mouse ESCs both in serum-free monolayer and in embryoid bodies (EBs). Use of reporter cell lines demonstrates that Oga inhibition leads to a reduction in the number of Sox1-expressing neural progenitors generated following induction of neural differentiation as well as maintained expression of the ESC marker Oct4 (Pou5f1). In EBs, expression of mesodermal and endodermal markers is also delayed. However, the transition of naïve cells to primed pluripotency indicated by Rex1 (Zfp42), Nanog, Esrrb, and Dppa3 downregulation and Fgf5 upregulation remains unchanged. Finally, we demonstrate that increased O-GlcNAcylation results in upregulation of genes normally epigenetically silenced in ESCs, supporting the emerging role for this protein modification in the regulation of histone modifications and DNA methylation.

The adverse effects of melphalan and cyclophosphamide on hematopoietic stem cells are well-known; however, the effects on the mesenchymal stem cells (MSCs) residing in the bone marrow are less well characterised. Examining the effects of chemotherapeutic agents on patient MSCs in vivo is difficult due to variability in patients and differences in the drug combinations used, both of which could have implications on MSC function. As drugs are not commonly used as single agents during high-dose chemotherapy (HDC) regimens, there is a lack of data comparing the short- or long-term effects these drugs have on patients post treatment. To help address these problems, the effects of the alkylating chemotherapeutic agents cyclophosphamide and melphalan on human bone marrow MSCs were evaluated in vitro. Within this study, the exposure of MSCs to the chemotherapeutic agents cyclophosphamide or melphalan had strong negative effects on MSC expansion and CD44 expression. In addition, changes were seen in the ability of MSCs to support hematopoietic cell migration and repopulation. These observations therefore highlight potential disadvantages in the use of autologous MSCs in chemotherapeutically pre-treated patients for future therapeutic strategies. Furthermore, this study suggests that if the damage caused by chemotherapeutic agents to marrow MSCs is substantial, it would be logical to use cultured allogeneic MSCs therapeutically to assist or repair the marrow microenvironment after HDC.

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.

AIMS/HYPOTHESIS: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out. METHODS: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells. RESULTS: TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death. CONCLUSIONS/INTERPRETATION: Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.

BACKGROUND: Protein 4.1R is an important component of the red cell membrane skeleton. It imparts structural integrity and has transmembrane signaling roles by direct interactions with transmembrane proteins and other membrane skeletal components, notably p55 and calmodulin. DESIGN AND METHODS: Spontaneous and ligation-induced phosphatidylserine exposure on erythrocytes from two patients with 4.1R deficiency were studied, using CD47 glycoprotein and glycophorin C as ligands. We also looked for protein abnormalities in the 4.1R-based multiprotein complex. RESULTS: Phosphatidylserine exposure was significantly increased in 4.1R-deficient erythrocytes obtained from the two different individuals when ligands to CD47 glycoprotein were bound. Spontaneous phosphatidylserine exposure was normal. 4.1R, glycophorin C and p55 were missing or sharply reduced. Furthermore there was an alteration or deficiency of CD47 glycoprotein and a lack of CD44 glycoprotein. Based on a recent study in 4.1R-deficient mice, we found that there are clear functional differences between interactions of human red cell 4.1R and its murine counterpart. CONCLUSIONS: Glycophorin C is known to bind 4.1R, and we have defined previously that it also binds CD47. From our evidence, we suggest that 4.1R plays a role in the phosphatidylserine exposure signaling pathway that is of fundamental importance in red cell turnover. The linkage of CD44 to 4.1R may be relevant to this process.

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting 'top' and 'bottom' strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.

DNA vaccinations effectively induce both humoral and cellular immune responses to immunogens from diverse infectious agents. However, DNA vaccines expressing the HIV-1 envelope glycoprotein (Env) are poorly immunogenic when expressed from wild-type (wt) DNA sequences. Two recent approaches used to enhance the immunogenicity of Env expressed from a DNA vaccine are the fusion of the molecular adjuvant, C3d, to a soluble form of Env and the use of codon-optimized (co) env gene inserts. Independently, each approach enhances antibody titer and cellular responses against Env expressed from gene inserts. The goal of this study was to examine if both codon-optimization of env gene inserts and C3d conjugation to Env could function in a synergistic manner to enhance immunogenicity. Mice (BALB/c) were inoculated with decreasing doses (2.0 microg, 0.2 microg or 0.02 microg) of co DNA expressing Env alone or fused to three copies of murine C3d (mC3d3) gene. Mice vaccinated with the highest dose (2.0 microg) of DNA had high anti-Env specific antibody titers regardless of the addition of mC3d3. At lower doses (0.2 microg and 0.02 microg) of DNA, mice vaccinated with Env-mC3d3 had enhanced immune responses compared to mice vaccinated with DNA expressing Env only. In addition, mice vaccinated with Env-mC3d3 at the highest doses of DNA had enhanced interleukin-4 secreting cells, while mice vaccinated with the lowest dose of DNA had enhanced interferon-gamma secreting cells. Therefore, both codon-optimization of env sequences and C3d conjugation to Env appear to enhance anti-Env antibodies in an independent and additive manner.