Biochemistry and Molecular Biophysics Faculty Research and Publications

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  • ItemOpen Access
    Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpBfrom Escherichia coli
    (2018-01) Ranaweera, Chathurange B.; Glaza, Przemyslaw; Zolkiewski, Michal
  • ItemOpen Access
    Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB–DnaK bichaperone system
    (2021-01-04) Glaza, Przemyslaw; Ranaweera, Chathurange B.; Shiva, Sunitha; Roy, Anuradha; Geisbrecht, Brian V.; Schoenen, Frank J.; Zolkiewski, Michal
    The ClpB–DnaK bichaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an antitumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd∼60 μM and inhibits the casein-activated, but not the basal, ATPase activity of ClpB with an IC50∼5 μM. The remaining p97 ligands, which displayed a higher affinity toward p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd∼100 μM and did not affect the DnaK ATPase but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB–DnaK bichaperone system in vitro with an IC50∼5 μM and suppressed the growth of cultured Escherichia coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ toward ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.
  • ItemOpen Access
    Synthesis and Characterization of Multifunctional Branched Amphiphilic Peptide Bilayer Conjugated Gold Nanoparticles
    (2018) Natarajan, Pavithra; Sukthankar, Pinakin; Changstrom, Jessica; Holland, Christopher S.; Barry, Shannon; Hunter, Wayne B.; Sorensen, Christopher M.; Tomich, John M.
    We provide strong chemical and biophysical evidence that documents that branched amphiphilic peptides, BAPs, known to assemble into spherical nanoassemblies in solution, do assemble as peptide-bilayer-delimited capsules. These nanoassemblies are termed branched amphiphilic peptide capsules (BAPCs). BAPCs are taken up by cells and accumulate in the perinuclear region to persist there without apparent degradation. BAPCs also entrap small proteins and solutes and stably encapsulate α-particle-emitting radionuclides. We have devised a method utilizing thiol chemistry to conjugate these peptide sequences onto gold nanoparticles (≤5 nm) with the objective of demonstrating the assembly of these peptides into a bilayer. The peptides are initially assembled as a monolayer on the gold surface via interaction with cysteine residues on the peptide C-terminus in an organic solvent. The subsequent transition of these peptide-monolayer-protected gold nanoparticles to an aqueous solution in the presence of excess peptides led to the formation of the peptide bilayer on the gold surface. The approach was exploited further to produce bilayer-coated magnetic nanoparticles. The innovation described in this study provides a stable metallic nanoparticle–peptide conjugate system that will help to determine interactions of BAPs in a biological system, with relative ease, important for developing future applications such as simultaneous delivery and imaging of surface-bound molecules of interest.
  • ItemOpen Access
    Intracellular complexes of the early-onset torsion dystonia-associated AAA+ ATPase TorsinA
    (2014-12-16) Li, Hui; Wu, Hui-Chuan; Liu, Zhonghua; Zacchi, Lucia F.; Brodsky, Jeffery L.; Zolkiewski, Michal
    A single GAG codon deletion in the gene encoding torsinA is linked to most cases of early-onset torsion dystonia. TorsinA is an ER-localized membrane-associated ATPase from the AAA+ superfamily with an unknown biological function. We investigated the formation of oligomeric complexes of torsinA in cultured mammalian cells and found that wild type torsinA associates into a complex with a molecular weight consistent with that of a homohexamer. Interestingly, the dystonia-linked variant torsinAΔE displayed a reduced propensity to form the oligomers compared to the wild type protein. We also discovered that the deletion of the N-terminal membrane-associating region of torsinA abolished oligomer formation. Our results demonstrate that the dystonia-linked mutation in the torsinA gene produces a protein variant that is deficient in maintaining its oligomeric state and suggest that ER membrane association is required to stabilize the torsinA complex.
  • ItemOpen Access
    CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains
    (2017-09-20) Roggenkamp, Emily; Giersch, Rachael M.; Wedeman, Emily; Eaton, Muriel; Turnquist, Emily; Schrock, Madison N.; Alkotami, Linah; Jirakittisonthon, Thitikan; Schluter-Pascua, Samantha E.; Bayne, Gareth H.; Wasko, Cory; Halloran, Megan; Finnigan, Gregory C.; gfinnigan; Roggenkamp, Emily; Giersch, Rachael M.; Wedeman, Emily; Eaton, Muriel; Turnquist, Emily; Schrock, Madison N.; Alkotami, Linah; Jirakittisonthon, Thitikan; Schluter-Pascua, Samantha E.; Bayne, Gareth H.; Wasko, Cory; Halloran, Megan; Finnigan, Gregory C.
    Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.
  • ItemOpen Access
    Allosteric Regulation of Fibronectin/α5 β1 Interaction by Fibronectin-Binding MSCRAMMs
    (2016-07-19) Liang, X.; Garcia, Brandon L.; Visai, L.; Prabhakaran, S.; Meenan, N. A. G.; Potts, J. R.; Humphries, M. J.; Höök, M.; garciab; Garcia, Brandon L.
    Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5 β1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5 β1 integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5 β1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5 β1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5 β1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5 β1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs. © 2016 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
  • ItemOpen Access
    Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose
    Tran, T. N. T.; Breuer, R. J.; Narasimhan, R. A.; Parreiras, L. S.; Zhang, Y. P.; Sato, T. K.; Durrett, Timothy P.; tdurrett; Durrett, Timothy
    Background: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. Results: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media. Conclusion: Our results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.
  • ItemOpen Access
    Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer
    (2017-02-01) Duhachek-Muggy, Sara; Qi, Yue; Wise, Randi; Alyahya, Linda; Li, Hui; Hodge, Jacob; Zolkiewska, Anna; zolkiea; Zolkiewska, Anna
    Background: ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells. Methods: ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown. Results: We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR(+) and CD44(hi)/CD24(-/lo) CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12-and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44(hi)/CD24(-/lo) cell population by ADAM12 knockdown. Conclusions: These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.
  • ItemOpen Access
    In vitro and in vivo studies on the structural organization of Chs3 from Saccharomyces cerevisiae
    (2017-03-25) Gohlke, S.; Muthukrishnan, Subbaratnam; Merzendorfer, H.; smk; Muthukrishnan, Subbaratnam
    Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between. In particular, we identified an amphipathic, crescent-shaped α-helix attached to the inner side of the membrane that presumably controls the channel entrance and a finger helix pushing the polymer into the channel. Evidence has accumulated in the past years that chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. However, the functional significance for living yeast cells has remained elusive. To test Chs3 oligomerization in vivo, we used bimolecular fluorescence complementation. We detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles, and analyzed their transport route using various trafficking mutants. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.
  • ItemOpen Access
    Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII
    (2017-03-22) Wei, W.; Zheng, C. L.; Zhu, M.; Zhu, X. F.; Yang, R. C.; Misra, Saurav; Zhang, B.; misras; Misra, Saurav
    Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 3(10)-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 3(10)-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 3(10)-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.
  • ItemOpen Access
    Symmetry breaking during homodimeric assembly activates an E3 ubiquitin ligase
    Ye, Z.; Needham, P. G.; Estabrooks, S. K.; Whitaker, Susan K.; Garcia, Brandon L.; Misra, Saurav; Brodsky, J. L.; Camacho, C. J.; misras; garciab; Misra, Saurav; Garcia, Brandon L.
    C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase.Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain.In contrast to nearly all homodimeric proteins, CHIP is asymmetric.To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly.We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box.We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization.This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues.Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion.The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites.Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function.This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly. © 2017 The Author(s).
  • ItemOpen Access
    Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase
    (2016-11-08) Bansal, Sunil; Durrett, Timothy P.; tdurrett; Durrett, Timothy P.
    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.
  • ItemOpen Access
    Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS)
    (2016-09-26) Perkins, S. J.; Wright, D. W.; Zhang, H. L.; Brookes, E. H.; Chen, Jianhan H.; Irving, T. C.; Krueger, S.; Barlow, D. J.; Edler, K. J.; Scott, D. J.; Terrill, N. J.; King, S. M.; Butler, P. D.; Curtis, J. E.; jianhanc; Chen, Jianhan H.
    The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration (http://www.ccpsas.org/) is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-art molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in which GenApp provides the deployment infrastructure for running applications on both standard and high-performance computing hardware, and SASSIE provides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data. GenApp produces the accessible web-based front end termed SASSIE-web, and GenApp and SASSIE also make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two-to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic 'bottlebrush' polymers.
  • ItemOpen Access
    Confined Mobility of TonB and FepA in Escherichia coli Membranes
    (2016-12-06) Lill, Y.; Jordan, L. D.; Smallwood, C. R.; Newton, Salete M.; Lill, M. A.; Klebba, Phillip E.; Ritchie, K.; sallyn; peklebba; Newton, Salete; Klebba, Phillip
    The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E. coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region 0.180(-0.007)(+0.006) mu m in radius in the outer membrane and TonB confined to a region 0.266(0.009)(+0.007) mu m in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be 21(-5)(+9) mu m(2)/s and 5.4(-0.8)(+1.5) mu m(2)/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.
  • ItemOpen Access
    Immunoreactivity of the AAA plus chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals
    (2016-07-16) Krajewska, J.; Arent, Z.; Wieckowski, D.; Zolkiewski, Michal; Kedzierska-Mieszkowska, S.; michalz; Zolkiewski, Michal
    Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpB(Li)) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpB(Li) as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. Results: ClpB(Li) consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpB(Li) and E. coli ClpB is 52 %. The coding sequence of the clpB(Li) gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpB(Li) protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpB(Li) activates the host immune system, as evidenced by an increased level of antibodies against ClpB(Li) in the sera from infected animals, as compared to the control group. Additionally, ClpB(Li) was found in kidney tissues of Leptospira-infected hamsters. Conclusions: ClpB(Li) is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpB(Li) point to its potential value as a diagnostic antigen for the detection of leptospirosis.
  • ItemUnknown
    Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum
    Tabunoki, Hiroko; Gorman, Maureen J.; Dittmer, Neal Thomas; Kanost, Michael R.; mgorman; ndittmer; kanost; Gorman, Maureen J.; Dittmer, Neal Thomas; Kanost, Michael R.; Tabunoki, Hiroko
    Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails.
  • ItemUnknown
    Allosteric Regulation of Fibronectin/alpha(5)beta(1) Interaction by Fibronectin-Binding MSCRAMMs
    Liang, X. W.; Garcia, Brandon L.; Visai, L.; Prabhakaran, S.; Meenan, N. A. G.; Potts, J. R.; Humphries, M. J.; Hook, M.; garciab; Garcia, Brandon L.
    Adherence ofmicrobes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with alpha(5)beta(1) integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/alpha(5)beta(1) integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/alpha(5)beta(1) integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/alpha(5)beta(1) on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic alpha(5)beta(1) interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/alpha(5)beta(1) affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs.
  • ItemUnknown
    Immunoreactivity of the AAA+ chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals
    Krajewska, J.; Arent, Z.; Wieckowski, D.; Zolkiewski, Michal; Kedzierska-Mieszkowska, S.; michalz; Zolkiewski, Michal
    Background: Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpB(Li)) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpB(Li) as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. Results: ClpB(Li) consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpB(Li) and E. coli ClpB is 52 %. The coding sequence of the clpB(Li) gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpB(Li) protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpB(Li) activates the host immune system, as evidenced by an increased level of antibodies against ClpB(Li) in the sera from infected animals, as compared to the control group. Additionally, ClpB(Li) was found in kidney tissues of Leptospira-infected hamsters. Conclusions: ClpB(Li) is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpB(Li) point to its potential value as a diagnostic antigen for the detection of leptospirosis.
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    Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex
    (2016-01-25) Garcia, Brandon L.; Zhi, H.; Wager, B.; Hook, M.; Skare, J. T.; garciab
    Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.