Life Sciences Commons^™

The Interdomain Interface In Bifunctional Enzyme Protein 3/4a (Ns3/4a) Regulates Protease And Helicase Activities, Cihan Aydin, Sourav Mukherjee, Alicia Hanson, David Frick, Celia Schiffer

Celia A. Schiffer

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that …

Stomatin-Like Protein 2 Deficiency In T Cells Is Associated With Altered Mitochondrial Respiration And Defective Cd4+ T Cell Responses., Darah A Christie, Panagiotis Mitsopoulos, Julianna Blagih, Stanley D Dunn, Julie St-Pierre, Russell G Jones, Grant M Hatch, Joaquín Madrenas

Stanley D Dunn

Stomatin-like protein 2 (SLP-2) is a mostly mitochondrial protein that regulates mitochondrial biogenesis and function and modulates T cell activation. To determine the mechanism of action of SLP-2, we generated T cell-specific SLP-2-deficient mice. These mice had normal numbers of thymocytes and T cells in the periphery. However, conventional SLP-2-deficient T cells had a posttranscriptional defect in IL-2 production in response to TCR ligation, and this translated into reduced CD4(+) T cell responses. SLP-2 deficiency was associated with impaired cardiolipin compartmentalization in mitochondrial membranes, decreased levels of the NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, NADH dehydrogenase (ubiquinone) 1β subcomplex subunit …

Paracrine Action Of Transforming Growth Factor-Alpha In Rectal Crypt Epithelium Of Humans, Ivan Cameron, W. Hardman

Elaine Hardman Ph.D.

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor α(TGF-α) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-α was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-α stained cells that exhibited intense positive staining in a continuous column from the mouth down …

Inwardly Rectifying Potassium Channels In Drosophila, Zhuo Luan, Hong-Sheng Li

zhuo luan

Inwardly rectifying potassium channels (Kir) are a special subset of potassium selective ion channels which pass potassium more easily into rather than out of the cell. These channels mediate a variety of cellular functions, including control of membrane resting potential, maintenance of potassium homeostasis and regulation of cellular metabolism. Given the existence of fifteen Kir genes in mammals, current genetic studies using mutant animals that lack a single channel may have missed many important physiological functions of these channels due to gene redundancy. This issue can be circumvented by using a simple model organism like Drosophila, whose genome encodes only …

Cooperative Effects Of Drug-Resistance Mutations In The Flap Region Of Hiv-1 Protease, Jennifer Foulkes-Murzycki, Christina Rosi, Nese Yilmaz, Robert Shafer, Celia Schiffer

Celia A. Schiffer

Understanding the interdependence of multiple mutations in conferring drug resistance is crucial to the development of novel and robust inhibitors. As HIV-1 protease continues to adapt and evade inhibitors while still maintaining the ability to specifically recognize and efficiently cleave its substrates, the problem of drug resistance has become more complicated. Under the selective pressure of therapy, correlated mutations accumulate throughout the enzyme to compromise inhibitor binding, but characterizing their energetic interdependency is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A) displays extreme entropy-enthalpy compensation relative to wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does not. Individual mutations of sites …

Protein Translocation Across The Rough Endoplasmic Reticulum, Elisabet Mandon, Steven Trueman, Reid Gilmore

Elisabet Mandon

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.

Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven Trueman, Elisabet Mandon, Reid Gilmore

Elisabet Mandon

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in …

Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, …

Crystal Structure Of The Dna Cytosine Deaminase Apobec3f: The Catalytically Active And Hiv-1 Vif-Binding Domain, Markus-Frederik Bohn, Shivender Shandilya, John Albin, Takahide Kouno, Brett Anderson, Rebecca Mcdougle, Michael Carpenter, Anurag Rathore, Leah Evans, Ahkillah Davis, Jingying Zhang, Yongjian Lu, Mohan Somasundaran, Hiroshi Matsuo, Reuben Harris, Celia Schiffer

Celia A. Schiffer

Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this …

A High Omega-3 Fatty Acid Diet Has Different Effects On Early And Late Stage Myeloid Progenitors, Melinda Varney, James Buchanan, Yulia Dementieva, W. Hardman, Vincent Sollars

Yulia Dementieva

The effects of the polyunsaturated omega-3 (n-3) and omega-6 (n-6) fatty acids (FA) on hematopoiesis are complex in that both FA forms are processed into leukotrienes, eicosanoids, and prostaglandins, which can have independent effects. These FA have antagonistic effects in that n-6 FA prostaglandins tend to be pro-proliferative and pro-inflammatory, while the effects of n-3 FA prostaglandins are the opposite. We have previously shown that diets high in n-3 FA reduce the size of the middle to later stage myeloid progenitor compartment in FVB X sv129 F1hybrid mice. To assay the effects of high n-3 FA diets on earlier stages …

Human Monoclonal Antibody Mbl-Hcv1 Delays Hcv Viral Rebound Following Liver Transplantation: A Randomized Controlled Study, R. Chung, F. Gordon, M. Curry, T. Schiano, S. Emre, K. Corey, J. Markmann, M. Hertl, J. Pomposelli, E. Pomfret, S. Florman, M. Schilsky, Teresa Broering, Robert Finberg, Gyongyi Szabo, Phillip Zamore, U. Khettry, Gregory Babcock, Donna Ambrosino, Brett Leav, Mark Leney, H. Smith, Deborah Molrine

Gyongyi Szabo

Rapid allograft infection complicates liver transplantation (LT) in patients with hepatitis C virus (HCV). Pegylated interferon-alpha and ribavirin therapy after LT has significant toxicity and limited efficacy. The effect of a human monoclonal antibody targeting the HCV E2 glycoprotein (MBL-HCV1) on viral clearance was examined in a randomized, double-blind, placebo-controlled pilot study in patients infected with HCV genotype 1a undergoing LT. Subjects received 11 infusions of 50 mg/kg MBL-HCV1 (n=6) or placebo (n=5) intravenously with three infusions on day of transplant, a single infusion on days 1 through 7 and one infusion on day 14 after LT. MBL-HCV1 was well-tolerated …

Tca Cycle Inactivation In Staphylococcus Aureus Alters Nitric Oxide Production In Raw 264.7 Cells, Chandirasegaran Massilamany, Arunakumar Gangaplara, Donald Gardner, James Musser, David Steffen, Greg Somerville, Jay Reddy

Greg A. Somerville

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts …

Acute Toxicity Of Copper Sulfate And Potassium Dichromate On Stygobiont Proasellus: General Aspects Of Groundwater Ecotoxicology And Future Perspectives, Ana Reboleira, Nelson Abrantes, Pedro Oromí, Fernando Gonçalves

Ana Sofia P.S. Reboleira

Karst systems harbor large groundwater resources for human consumption and represent an important habitat for rare and unprotected specialized animals, the so-called stygofauna. Due to the highly adapted features towards underground life, together with the geographic isolation provided by the subterranean aquifers, groundwater-dwelling animals may lose the ability to face sudden changes on their ecosystems, and therefore the risk of extinction is remarkably high. A little is known about their sensitiveness, especially linked to contamination pressure in urbanized karst areas. Understanding the impact of contaminants on stygofauna is important for setting groundwater environmental quality and management of karst systems. We …

The Intermediate Monoclinic Phase Of Phosphatidylcholines, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared …

Lateral Phase Separations In Binary Mixtures Of Phospholipids Having Different Charges And Different Crystalline Structures, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Synthetic dipalmitoyl phosphatidylserine exhibits a sharp chain-melting transition temperature at 51 degrees C as judged by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Phase diagrams representing lateral phase separations in binary mixtures of dipalmitoyl phosphatidylserine with dipalmitoyl phosphatidylcholine as well as with dimyristoyl phosphatidylcholine are derived from paramagnetic resonance determinations of 2,2,6,6,-tetramethylpiperidine-1-oxyl partitioning, freeze-fracture electron microscopic studies and theoretical arguments that limit the general form of acceptable phase diagrams. The reported phase diagrams are the first to describe binary mixtures in which one lipid is charged and the second lipid uncharged. These phase diagrams also are the first to include the …

Multiple Phase Equilibria In Binary Mixtures Of Phospholipids, Elizabeth Luna, Harden Mcconnell

Elizabeth J. Luna

Approximate phse diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidycholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data. These phase diagrams represent an improvement over previous studies in that both solid phses (Pbeta' and Lbeta') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two Pbeta' phases that do not form a continuous range of solid solutions.

Transmembrane Domain Three Contributes To The Ion Conductance Pathway Of Channelrhodopsin-2, Robert Dempski, Olga Gaiko

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a light-activated nonselective cation channel that is found in the eyespot of the unicellular green alga Chlamydomonas reinhardtii. Despite the wide employment of this protein to control the membrane potential of excitable membranes, the molecular determinants that define the unique ion conductance properties of this protein are not well understood. To elucidate the cation permeability pathway of ion conductance, we performed cysteine scanning mutagenesis of transmembrane domain three followed by labeling with methanethiosulfonate derivatives. An analysis of our experimental results as modeled onto the crystal structure of the C1C2 chimera demonstrate that the ion permeation pathway includes …

An N-Terminal, 830 Residues Intrinsically Disordered Region Of The Cytoskeleton-Regulatory Protein Supervillin Contains Myosin Ii- And F-Actin-Binding Sites, Stanislav Fedechkin, Jacob Brockerman, Elizabeth Luna, Michail Lobanov, Oxana Galzitskaya, Serge Smirnov

Elizabeth J. Luna

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1-830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1-830) represents an IDR, which …

Transmembrane Serine Mutations Reduce The Minimum Pore Diameter Of Channelrhodopsin-2, Robert Dempski, Ryan Richards

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin that, together with channelrhodopsin-1, mediates phototactic behavior in the green algae Chlamydomonas reinhardtii. Like all other microbial-type rhodopsins, ChR2 has seven transmembrane domains with the chromophore all-trans retinal bound to a single lysine residue. However, unlike other microbial-type rhodopsins, ChR2 functions as a non-selective cation channel and not an ion pump. A sequence alignment of ChR2 with the proton pump bacteriorhodopsin (bR) reveals that ChR2 lacks specific motifs within the transmembrane domains that facilitate non-covalent interactions and contribute to protein stability.

Periplasmic Response Upon Disruption Of Transmembrane Cu Transport In Pseudomonas Aeruginosa., José Argüello, Daniel Raimunda, Teresita Padilla-Benavides, Stefan Vogt, Sylvain Boutigny, Kaleigh Tomkinson, Lydia Finney

José M. Argüello

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the …

Mechanisms Of Copper Homeostasis In Bacteria, José Argüello, Daniel Raimunda, Teresita Padilla-Benavides

José M. Argüello

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu(+) transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of …

Sinorhizobium Meliloti Nia Is A P(1b-5)-Atpase Expressed In The Nodule During Plant Symbiosis And Is Involved In Ni And Fe Transport., José Argüello, Eliza Zielazinski, Manuel Gonzalez-Guerrero, Poorna Subramanian, Timothy Stemmler, Amy Rosenzweig

José M. Argüello

The P1B-ATPases are a ubiquitous family of metal transporters. These transporters are classified into subfamilies on the basis of substrate specificity, which is conferred by conserved amino acids in the last three transmembrane domains. Five subfamilies have been identified to date, and representative members of four (P1B-1 to P1B-4) have been studied. The fifth family (P1B-5), of which some members contain a C-terminal hemerythrin (Hr) domain, is less well characterized. The S. meliloti Sma1163 gene encodes for a P1B-5-ATPase, denoted Nia (Nickel-iron ATPase), that is induced by exogenous Fe(2+) and Ni(2+). The nia mutant accumulates nickel and iron, suggesting a …

Structure And Dynamics Of A Primordial Catalytic Fold Generated By In Vitro Evolution, Fa-An Chao, Aleardo Morelli, John C. Haugner Iii, Lewis Churchfield, Lei Shi, Larry R. Masterson, Ritimukta Sarangi, Gianluigi Veglia, Burckhard Seelig

Larry Masterson