Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-X L . However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-X L . We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-X L at 2.5 Å resolution. Reminiscent of previously determined Bcl-X L -BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-X L . These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.Programmed cell death (1) is a critical process in metazoans responsible for basic tissue homeostasis, selective elimination of damaged cells, and structural reorganization during development. Apoptosis, the most clearly defined form of programmed cell death, is initiated by an array of intracellular and extracellular signals (e.g. DNA damage, cytokine removal, anoikis, etc.) and is characterized by a canonical morphology exhibiting chromatin condensation, nuclear fragmentation, and membrane blebbing (2, 3). The molecular mechanisms responsible for this morphology involve the release of cytochrome c and other apoptogenic factors from the mitochondria, and the subsequent activation of caspases, a family of cysteine proteases that dismantle the cell in a precise and systematic manner (4 -6). The Bcl-2 family of proteins integrate apoptotic signal transduction upstream of and at the mitochondrial membrane (7,8). Three classes of Bcl-2 family proteins have been extensively described in the literature: anti-apoptotic Bcl-2-like proteins (Bcl-2, Bcl-X L , Bcl-W, and Mcl-1, A1) containing three or four Bcl-2 homology (BH) 2 domains; pro-apoptotic Bax-like proteins (Bax, Bak, and Bok) containing BH1, -2, and -3 domains; and pro-apoptotic BH3-only proteins (e.g. Bim, Bad, Bid, PUMA, and Noxa) containing only the BH3 domain. Upon receipt of an apoptotic stimulus, BH3-only proteins are then "activated," repress anti-apoptotic Bcl-2 family members, and activate pro-apoptotic Bax-like proteins via direct protein-protein interaction (9 -11). This leads to oligomerization of Bax and Bak at the mitochondrial membrane, release of cytochrome c, and subsequent activation of caspases. Thus, BH3-only proteins form a family of critical cell death ligands, which initiate a canonical death pathway and the resultant apoptotic morphology. Autophagy, a highly conserved stress response, may function as an alternative, non-apoptotic death mechanism under certain conditions, for which it is sometimes referred to as type II programmed cell death (...

Summary Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism.

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix alpha4 approximately 10 A below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix alpha5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix alpha5.

The maternal morphogen Bicoid (Bcd) is distributed in an embryonic gradient that is critical for patterning the anterior-posterior (AP) body plan in Drosophila. Previous work identified several target genes that respond directly to Bcd-dependent activation. Positioning of these targets along the AP axis is thought to be controlled by cis-regulatory modules (CRMs) that contain clusters of Bcd-binding sites of different ''strengths.'' Here we use a combination of Bcd-site cluster analysis and evolutionary conservation to predict Bcd-dependent CRMs. We tested 14 predicted CRMs by in vivo reporter gene assays; 11 show Bcd-dependent activation, which brings the total number of known Bcd target elements to 21. Some CRMs drive expression patterns that are restricted to the most anterior part of the embryo, whereas others extend into middle and posterior regions. However, we do not detect a strong correlation between AP position of target gene expression and the strength of Bcd site clusters alone. Rather, we find that binding sites for other activators, including Hunchback and Caudal correlate with CRM expression in middle and posterior body regions. Also, many Bcd-dependent CRMs contain clusters of sites for the gap protein Kruppel, which may limit the posterior extent of activation by the Bcd gradient. We propose that the key design principle in AP patterning is the differential integration of positive and negative transcriptional information at the level of individual CRMs for each target gene.morphogen ͉ network ͉ transcription G radients of two transcription factors, Bicoid (Bcd) and Dorsal (Dl), are critical for patterning the major body plan axes in the Drosophila embryo. This studied focused on Bcd, a maternal effect gene whose mRNA is initially localized at the anterior pole of the oocyte (Fig. 1A) (1). Upon egg deposition, the trapped bcd RNA is translated, and a nuclear gradient forms, with highest concentrations near the anterior pole and progressively lower concentrations in more posterior regions (Fig. 1B) (2). Bcd protein distribution matches its biological activity, with high levels required for the positioning of the most anterior structures, intermediate levels for head structures, and low levels for the thoracic and anterior abdominal segments. Varying the shape of the Bcd gradient has a direct effect on positional identity in the blastoderm (3). Increasing the maternal input of bcd can shift morphological landmarks posteriorly, whereas decreasing it shifts them anteriorly. These experiments suggest that the concentration of Bcd protein present at each position along the length of the embryo (EL) determines the destiny of cells that will occupy that region.Bcd protein contains a homeodomain and functions as a morphogen by activating the transcription of multiple target genes in different positions along the anterior-posterior (AP) axis (reviewed in refs. 4 and 5). Driever, Thoma, and Nüsslein-Volhard (6) first proposed that differential positioning could occur by means of gene-specific regulatory ele...

Human CMV (HCMV) exhibits a broad cell tropism that depends on two virion glycoprotein complexes: a trimeric complex (gH/gL/gO) that facilitates viral infection primarily in fibroblasts and a pentameric complex (gH/gL/pUL128-pUL130-pUL131A) that mediates infection in epithelial and endothelial cells. We performed genomewide CRISPR screens in which the PDGF receptor-α (PDGFRα) was identified as the most significant cellular gene product essential for infection by HCMV virions containing only trimeric complex (trimer-only virus). Trimer-only virus did not enter PDGFRα knockout fibroblasts. By using knockout fibroblasts, the extracellular domain of PDGFRα required for virus entry was mapped, and the intracellular tyrosine kinase domain was shown to be nonessential. In addition, direct cell-to-cell spread of virus from knockout cells transfected with trimer-only viral DNA was blocked, despite the production of infectious virus in the transfected cells. In contrast to trimer-only virus, wild-type HCMV virions containing both trimeric and pentameric complexes entered PDGFRα knockout cells, reinforcing the view that fibroblasts contain a second, independent receptor for the pentameric complex. Importantly, however, wild-type virus entered the knockout fibroblasts at reduced efficiency compared with parental fibroblasts, arguing that the cellular receptor for the virion pentameric complex is limiting or that virions are produced containing different relative amounts of the two glycoprotein complexes. Finally, ectopic expression of PDGFRα in ARPE-19 epithelial cells and THP-1 monocytic cells, which have little to no endogenous PDGFRα expression, markedly enhanced their susceptibility to trimer-only virions. In sum, our data clarify several key determinants of HCMV tropism. herpesvirus | tropism | cellular receptor

Human cytomegalovirus (HCMV) is the prototypical human β-herpes virus. Here we perform a systems analysis of the HCMV host-cell transcriptome, using gene set enrichment analysis (GSEA) as an engine to globally map the host-pathogen interaction across two cell types. Our analysis identified several previously unknown signatures of infection, such as induction of potassium channels and amino acid transporters, derepression of genes marked with histone H3 lysine 27 trimethylation (H3K27me3), and inhibition of genes related to epithelial-to-mesenchymal transition (EMT). The repression of EMT genes was dependent on early viral gene expression and correlated with induction E-cadherin (CDH1) and mesenchymal-to-epithelial transition (MET) genes. Infection of transformed breast carcinoma and glioma stem cells similarly inhibited EMT and induced MET, arguing that HCMV induces an epithelium-like cellular environment during infection.

Transposons such as P elements are routinely used to stably transfer exogenous DNA (transgenes) into the Drosophila genome. Transgene insertion events, however, are essentially random and are subject to 'position effects' from nearby endogenous regulatory elements. Here we describe a microinjection-based system that uses Cre-mediated recombination to insert transgenes into precise genomic 'landing sites'. The system is simple and efficient, and will permit precise comparisons between multiple transgenic constructs.

The Drosophila body plan is composed of a linear array of cephalic, thoracic, and abdominal segments along the anterior posterior axis. The number and positions of individual segments are established by a transcriptional network comprised of maternal effect, gap, pair-rule, and segment polarity genes. The sloppy-paired (slp) locus contains two genes (slp1 and slp2) that are expressed in overlapping striped patterns in the presumptive thorax and abdomen. Previous studies suggest that these genes function at the pair-rule and segment polarity levels to establish the spacing and polarity of thoracic and abdominal segments. One of these genes (slp1) is also expressed in a broad anterior domain that appears before the striped patterns. There are severe cephalic defects in slp1 mutants, including the complete loss of the mandibular segment, but the molecular roles played by Slp1 in anterior patterning are not clear. Here, we present evidence that the anterior Slp1 domain acts as a gradient to differentially repress the anteriormost stripes of several different pair-rule genes. This repressive gradient contributes to the precise spatial arrangement of anterior pair-rule stripe borders required for expression of the first engrailed stripe and the formation of the mandibular segment. These results suggest that Slp1 functions as a gap gene-like repressor, in addition to its roles at the pair-rule and segment polarity levels of the hierarchy. The Slp1 protein contains a protein motif (EH1) which mediates binding to the transcriptional corepressor Groucho (Gro). We show that this domain is required for Slp1-mediated repression in vivo.