Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

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.

Engineered receptors for human cytomegalovirus that are orthogonal to normal human biology.

A trimeric glycoprotein complex on the surface of human cytomegalovirus (HCMV) binds to platelet-derived growth factor (PDGF) receptor α (PDGFRα) to mediate host cell recognition and fusion of the viral and cellular membranes. Soluble PDGFRα potently neutralizes HCMV in tissue culture, and its potential use as an antiviral therapeutic has the benefit that any escape mutants will likely be attenuated. However, PDGFRα binds multiple PDGF ligands in the human body as part of developmental programs in embryogenesis and continuing through adulthood. Any therapies with soluble receptor therefore come with serious efficacy and safety concerns, especially for the treatment of congenital HCMV. Soluble virus receptors that are orthogonal to human biology might resolve these concerns. This engineering problem is solved by deep mutational scanning on the D2-D3 domains of PDGFRα to identify variants that maintain interactions with the HCMV glycoprotein trimer in the presence of competing PDGF ligands. Competition by PDGFs is conformation-dependent, whereas HCMV trimer binding is independent of proper D2-D3 conformation, and many mutations at the receptor-PDGF interface are suitable for functionally separating trimer from PDGF interactions. Purified soluble PDGFRα carrying a targeted mutation succeeded in displaying wild type affinity for HCMV trimer with a simultaneous loss of PDGF binding, and neutralizes trimer-only and trimer/pentamer-expressing HCMV strains infecting fibroblasts or epithelial cells. Overall, this work makes important progress in the realization of soluble HCMV receptors for clinical application.

Role of PDGF receptor-α during human cytomegalovirus entry into fibroblasts.

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 genome-wide 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.

Cellular responses to human cytomegalovirus infection: Induction of a mesenchymal-to-epithelial transition (MET) phenotype.

Human cytomegalovirus (HCMV) is the prototypical human ß-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.

Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome.

Replication of human cytomegalovirus (HCMV) is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of HCMV by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high-resolution MS. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication.

Subconfluent ARPE-19 Cells Display Mesenchymal Cell-State Characteristics and Behave like Fibroblasts, Rather Than Epithelial Cells, in Experimental HCMV Infection Studies.

Human cytomegalovirus (HCMV) has a broad cellular tropism and epithelial cells are important physiological targets during infection. The retinal pigment epithelial cell line ARPE-19 has been used to model HCMV infection in epithelial cells for decades and remains a commonly used cell type for studying viral entry, replication, and the cellular response to infection. We previously found that ARPE-19 cells, despite being derived from an epithelial cell explant, express extremely low levels of canonical epithelial proteins, such as E-cadherin and EpCAM. Here, we perform comparative studies of ARPE-19 and additional epithelial cell lines with strong epithelial characteristics. We find that ARPE-19 cells cultured under subconfluent conditions resemble mesenchymal fibroblasts, rather than epithelial cells; this is consistent with previous studies showing that ARPE-19 cultures require extended periods of high confluency culture to maintain epithelial characteristics. By reanalyzing public gene expression data and using machine learning, we find evidence that ARPE-19 cultures maintained across many labs exhibit mesenchymal characteristics and that the majority of studies employing ARPE-19 use them in a mesenchymal state. Lastly, by performing experimental HCMV infections across mesenchymal and epithelial cell lines, we find that ARPE-19 cells behave like mesenchymal fibroblasts, producing logarithmic yields of cell-free infectious progeny, while cell lines with strong epithelial character exhibit an atypical infectious cycle and naturally restrict the production of cell-free progeny. Our work highlights important characteristics of the ARPE-19 cell line and suggests that subconfluent ARPE-19 cells may not be optimal for modeling epithelial infection with HCMV or other human viruses. It also suggests that HCMV biosynthesis and/or spread may occur quite differently in epithelial cells compared to mesenchymal cells. These differences could contribute to viral persistence or pathogenesis in epithelial tissues.

Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry.

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.

A tumor-specific endogenous repetitive element is induced by herpesviruses.

Tandem satellite repeats account for 3% of the human genome. One of them, Human Satellite II (HSATII), is highly expressed in several epithelial cancers and cancer cell lines. Here we report an acute induction of HSATII RNA in human cells infected with two herpes viruses. We show that human cytomegalovirus (HCMV) IE1 and IE2 proteins cooperate to induce HSATII RNA affecting several aspects of the HCMV replication cycle, viral titers and infected-cell processes. HSATII RNA expression in tissue from two chronic HCMV colitis patients correlates with the strength of CMV antigen staining. Thus, endogenous HSATII RNA synthesis after herpesvirus infections appears to have functionally important consequences for viral replication and may provide a novel insight into viral pathogenesis. The HSATII induction seen in both infected and cancer cells suggests possible convergence upon common HSATII-based regulatory mechanisms in these seemingly disparate diseases.

Crystal structure of the Bcl-XL-Beclin 1 peptide complex: Beclin 1 is a novel BH3-only protein.

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 2.5A 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.

Site-specific transgenesis by Cre-mediated recombination in Drosophila.

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 role of binding site cluster strength in Bicoid-dependent patterning in Drosophila.

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.

Groucho-dependent repression by sloppy-paired 1 differentially positions anterior pair-rule stripes in the Drosophila embryo.

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.

Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms.

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers.