Professional Education

  • Doctor of Philosophy, Princeton University (2012)

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  • Sirtuins Are Evolutionarily Conserved Viral Restriction Factors MBIO Koyuncu, E., Budayeva, H. G., Miteva, Y. V., Ricci, D. P., Silhavy, T. J., Shenk, T., Cristea, I. M. 2014; 5 (6)


    The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD(+)-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity.We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.

    View details for DOI 10.1128/mBio.02249-14

    View details for Web of Science ID 000347073600058

    View details for PubMedID 25516616

  • Predicting Functionally Informative Mutations in Escherichia coli BamA Using Evolutionary Covariance Analysis GENETICS Dwyer, R. S., Ricci, D. P., Colwell, L. J., Silhavy, T. J., Wingreen, N. S. 2013; 195 (2): 443-?


    The essential outer membrane β-barrel protein BamA forms a complex with four lipoprotein partners BamBCDE that assembles β-barrel proteins into the outer membrane of Escherichia coli. Detailed genetic studies have shown that BamA cycles through multiple conformations during substrate assembly, suggesting that a complex network of residues may be involved in coordinating conformational changes and lipoprotein partner function. While genetic analysis of BamA has been informative, it has also been slow in the absence of a straightforward selection for mutants. Here we take a bioinformatic approach to identify candidate residues for mutagenesis using direct coupling analysis. Starting with the BamA paralog FhaC, we show that direct coupling analysis works well for large β-barrel proteins, identifying pairs of residues in close proximity in tertiary structure with a true positive rate of 0.64 over the top 50 predictions. To reduce the effects of noise, we designed and incorporated a novel structured prior into the empirical correlation matrix, dramatically increasing the FhaC true positive rate from 0.64 to 0.88 over the top 50 predictions. Our direct coupling analysis of BamA implicates residues R661 and D740 in a functional interaction. We find that the substitutions R661G and D740G each confer OM permeability defects and destabilize the BamA β-barrel. We also identify synthetic phenotypes and cross-suppressors that suggest R661 and D740 function in a similar process and may interact directly. We expect that the direct coupling analysis approach to informed mutagenesis will be particularly useful in systems lacking adequate selections and for dynamic proteins with multiple conformations.

    View details for DOI 10.1534/genetics.113.155861

    View details for Web of Science ID 000325286200014

    View details for PubMedID 23934888

  • Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for beta-barrel assembly in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rigel, N. W., Ricci, D. P., Silhavy, T. J. 2013; 110 (13): 5151-5156


    In gram-negative bacteria, integral outer membrane ?-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam) complex. The essential components of this complex are the OMP BamA [which contains a carboxyl-terminal ?-barrel and an amino-terminal periplasmic module composed of five polypeptide transport associated (POTRA) domains] and the lipoprotein BamD. In Escherichia coli, the Bam complex also contains three nonessential lipoproteins (BamBCE), all of which require the barrel-proximal POTRA domain (P5) for stable interactions with BamA. We have previously reported that the BamA ?-barrel assumes two different conformations. A method for conformation-specific labeling of BamA described here reveals that these conformers reflect the degree of surface exposure of the conserved sixth extracellular loop (L6). L6 is surface accessible in one conformation but not in the other, likely because it occupies the lumen of the BamA ?-barrel in the latter case. A gain-of-function mutation that promotes Bam activity (bamDR197L) and a loss-of-function mutation that decreases the activity of Bam (?bamE) both favor surface exposure of BamA L6, suggesting that BamD and BamE normally act to control L6 exposure through opposing functions. These results, along with the synthetic lethality of the bamDR197L ?bamE double mutant, imply a cyclic mechanism in which the Bam lipoproteins regulate the conformation of BamA during the OMP assembly reaction. Our results further suggest that BamDE controls L6 exposure via conformational signals transmitted through P5 to L6.

    View details for DOI 10.1073/pnas.1302662110

    View details for Web of Science ID 000318031900064

    View details for PubMedID 23479609

  • The Activity and Specificity of the Outer Membrane Protein Chaperone SurA Are Modulated by a Proline Isomerase Domain. mBio Ricci, D. P., Schwalm, J., Gonzales-Cope, M., Silhavy, T. J. 2013; 4 (4)


    ABSTRACT SurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) in Escherichia coli. Although SurA contains two well-conserved proline isomerase (PPIase) domains, the contribution of these domains to SurA function is unclear. In the present work, we show that defects in OMP assembly caused by mutation of the β-barrel assembly factors BamA or BamB can be corrected by gain-of-function mutations in SurA that map to the first PPIase domain. These mutations apparently bypass the requirement for a stable interaction between SurA and the Bam complex and enhance SurA chaperone activity in vivo despite destabilization of the protein in vitro. Our findings suggest an autoinhibitory mechanism for regulation of SurA chaperone activity through interdomain interactions involving a PPIase domain. We propose a model in which SurA activity is modulated by an interaction between SurA and the Bam complex that alters the substrate specificity of the chaperone. IMPORTANCE The dominant surA mutations described here alter amino acid residues that are highly conserved in eukaryotic homologs of SurA, including Pin1, the human proline isomerase (PPIase) implicated in Alzheimer's disease and certain cancers. Consequently, a mechanistic description of SurA function may enhance our understanding of clinically important PPIases and their role(s) in disease. In addition, the virulence of Gram-negative bacterial pathogens, such as Salmonella, Shigella, and Escherichia coli O157:H7, is largely dependent on SurA, making this PPIase/chaperone an attractive antibiotic target. Investigating the function of SurA in outer membrane (OM) biogenesis will be useful in the development of novel therapeutic strategies for the disruption of the OM or the processes that are essential for its assembly.

    View details for DOI 10.1128/mBio.00540-13

    View details for PubMedID 23943764

  • The Bam machine: A molecular cooper BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Ricci, D. P., Silhavy, T. J. 2012; 1818 (4): 1067-1084


    The bacterial outer membrane (OM) is an exceptional biological structure with a unique composition that contributes significantly to the resiliency of Gram-negative bacteria. Since all OM components are synthesized in the cytosol, the cell must efficiently transport OM-specific lipids and proteins across the cell envelope and stably integrate them into a growing membrane. In this review, we discuss the challenges associated with these processes and detail the elegant solutions that cells have evolved to address the topological problem of OM biogenesis. Special attention will be paid to the Bam machine, a highly conserved multiprotein complex that facilitates OM ?-barrel folding. This article is part of a Special Issue entitled: Protein Folding in Membranes.

    View details for DOI 10.1016/j.bbamem.2011.08.020

    View details for Web of Science ID 000301761500017

    View details for PubMedID 21893027

  • BamE Modulates the Escherichia coli Beta-Barrel Assembly Machine Component BamA JOURNAL OF BACTERIOLOGY Rigel, N. W., Schwalm, J., Ricci, D. P., Silhavy, T. J. 2012; 194 (5): 1002-1008


    Biogenesis of the outer membrane (OM) is an essential process in gram-negative bacteria. One of the key steps of OM biogenesis is the assembly of integral outer membrane beta-barrel proteins (OMPs) by a protein machine called the Bam complex. In Escherichia coli, the Bam complex is composed of the essential proteins BamA and BamD and three nonessential lipoproteins, BamB, BamC, and BamE. Both BamC and BamE are important for stabilizing the interaction between BamA and BamD. We used comprehensive genetic analysis to clarify the interplay between BamA and the BamCDE subcomplex. Combining a ?bamE allele with mutations in genes that encode other OMP assembly factors leads to severe synthetic phenotypes, suggesting a critical function for BamE. These synthetic phenotypes are not nearly as severe in a ?bamC background, suggesting that the functions of BamC and BamE are not completely overlapping. This unique function of BamE is related to the conformational state of BamA. In wild-type cells, BamA is sensitive to externally added proteinase K. Strikingly, when ?bamE mutant cells are treated with proteinase K, BamA is degraded beyond detection. Taken together, our findings suggest that BamE modulates the conformation of BamA, likely through its interactions with BamD.

    View details for DOI 10.1128/JB.06426-11

    View details for Web of Science ID 000300530800011

    View details for PubMedID 22178970

  • Activation of the Escherichia coli beta-barrel assembly machine (Bam) is required for essential components to interact properly with substrate PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ricci, D. P., Hagan, C. L., Kahne, D., Silhavy, T. J. 2012; 109 (9): 3487-3491


    The outer membrane (OM) of gram-negative bacteria such as Escherichia coli contains lipoproteins and integral ?-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of ?-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.

    View details for DOI 10.1073/pnas.1201362109

    View details for Web of Science ID 000300828200060

    View details for PubMedID 22331884

  • Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci HUMAN MUTATION Kim, U., Wooding, S., Ricci, D., Jorde, L. B., Drayna, D. 2005; 26 (3): 199-204


    Bitter taste perception in humans is mediated by receptors encoded by 25 genes that together comprise the TAS2R (or T2R) gene family. The ability to identify the ligand(s) for each of these receptors is dependent on understanding allelic variation in TAS2R genes, which may have a significant effect on ligand recognition. To investigate the extent of coding variation among TAS2R alleles, we performed a comprehensive evaluation of sequence and haplotype variation in the human bitter taste receptor gene repertoire. We found that these genes exhibit substantial coding sequence diversity. In a worldwide population sample of 55 individuals, we found an average of 4.2 variant amino acid positions per gene. In aggregate, the 24 genes analyzed here, along with the phenylthiocarbamide (PTC) receptor gene analyzed previously, specify 151 different protein coding haplotypes. Analyses of the ratio of synonymous and nonsynonymous nucleotide substitutions using the Ka/Ks ratio revealed an excess of amino acid substitutions relative to most other genes examined to date (Ka/Ks = 0.94). In addition, comparisons with more than 1,500 other genes revealed that levels of diversity in the TAS2R genes were significantly greater than expected (pi = 0.11%; p < 0.01), as were levels of differentiation among continental populations (FST = 0.22; p < 0.05). These diversity patterns indicate that unusually high levels of allelic variation are found within TAS2R loci and that human populations differ appreciably with respect to TAS2R allele frequencies. Diversity in the TAS2R genes may be accounted for by natural selection, which may have favored alleles responsive to toxic, bitter compounds found in plants. These findings are consistent with the view that different alleles of the TAS2R genes encode receptors that recognize different ligands, and suggest that the haplotypes we have identified will be important in studies of receptor-ligand recognition.

    View details for DOI 10.1002/humu.20203

    View details for Web of Science ID 000231631200005

    View details for PubMedID 16086309

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