Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membraneThis study identifies FtsW as the flippase that translocates lipid-linked peptidoglycan precursors across the cell membrane during bacterial cell wall synthesis.

a b s t r a c tThe bacterial cell wall is mainly composed of peptidoglycan, which is a three-dimensional network of long aminosugar strands located on the exterior of the cytoplasmic membrane. These strands consist of alternating MurNAc and GlcNAc units and are interlinked to each other via peptide moieties that are attached to the MurNAc residues. Peptidoglycan subunits are assembled on the cytoplasmic side of the bacterial membrane on a polyisoprenoid anchor and one of the key components in the synthesis of peptidoglycan is Lipid II. Being essential for bacterial cell survival, it forms an attractive target for antibacterial compounds such as vancomycin and several lantibiotics. Lipid II consists of one GlcNAcMurNAc-pentapeptide subunit linked to a polyiosoprenoid anchor 11 subunits long via a pyrophosphate linker. This review focuses on this special molecule and addresses three questions. First, why are special lipid carriers as polyprenols used in the assembly of peptidoglycan? Secondly, how is Lipid II translocated across the bacterial cytoplasmic membrane? And finally, how is Lipid II used as a receptor for lantibiotics to kill bacteria?

Neuroblastoma (NB) is a frequently lethal tumor of childhood. MYCN amplification accounts for the aggressive phenotype in a subset while the majority have no consistently identified molecular aberration but frequently express MYC at high levels. We hypothesized that activated Wnt/b-catenin (CTNNB1) signaling might account for this as MYC is a b-catenin transcriptional target and multiple embryonal and neural crest malignancies have oncogenic alterations in this pathway. NB cell lines without MYCN amplification express higher levels of MYC and b-catenin (with aberrant nuclear localization) than MYCNamplified cell lines. Evidence for aberrant b-catenin-TCF transcriptional activity was demonstrated using expression profiles from 73 primary NBs. Findings included increased WNT ligands (WNT1, WNT6, WNT7A, WNT10B), DVL1 and TCF7 expression in high-risk NBs without MYCN amplification, consistent with canonical b-catenin signaling. More directly, Patterns of Gene Expression and Gene Set Enrichment Analyses demonstrated b-catenin target genes (for example, MYC, PPARD, NRCAM, CD44, TCF7) as coordinately upregulated in high-risk NBs without MYCN amplification in comparison to high-risk MYCN-amplified or intermediate-risk NBs, supporting pathway activation in this subset. Thus, high-risk NBs without MYCN amplification may deregulate MYC and other oncogenic genes via altered b-catenin signaling providing a potential candidate pathway for therapeutic inhibition.

SummaryTranslocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.

The major impediment to cure for many malignancies is the development of therapy resistance with resultant tumor progression. Genetic alterations leading to subversion of inherent apoptosis pathways are common themes in therapy resistance. Bcl-2 family proteins play a critical role in regulating mitochondrial apoptosis that governs chemotherapeutic effects, and defective engagement of these pathways contributes to treatment failure. We have studied the efficacy of BH3 peptidomimetics consisting of the minimal death, or BH3, domains of the proapoptotic BH3-only proteins Bid and Bad to induce apoptosis using neuroblastoma (NB) as a model system. We demonstrate that BH3 peptides, modified with an arginine homopolymer for membrane transduction (called r8-BidBH3 and r8-BadBH3, respectively), potently induce apoptosis in NB cells, including those with MYCN amplification. Cell death is caspase 9 dependent, consistent with a requirement for the intrinsic mitochondrial pathway. Substitutions at highly conserved residues within the r8-BidBH3 peptide abolish apoptotic efficacy supporting activity through specific BH domain interactions. Concomitant exposure to r8-BadBH3 and r8-BidBH3 at sublethal monotherapy doses revealed potent synergy consistent with a competitive displacement model, whereby BH3 peptides displace sequestered BH3 proteins to induce cell death. Further, BH3 peptides demonstrate antitumor efficacy in a xenograft model of NB in the absence of additional genotoxic or trophic stressors. These data provide proof of principle that targeted reengagement of apoptosis pathways may be of therapeutic utility, and BH3-like compounds are attractive lead agents to re-establish therapy-induced apoptosis in refractory malignancies.