scholarly journals Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

2008 ◽  
Vol 19 (11) ◽  
pp. 4707-4716 ◽  
Author(s):  
Rogier W. Sanders ◽  
Shang-Te D. Hsu ◽  
Eelco van Anken ◽  
I. Marije Liscaljet ◽  
Martijn Dankers ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Side Chain ◽  
Viral Glycoprotein ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Β Sheet

The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues.

scholarly journals “Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

2015 ◽  
Vol 1 (7) ◽  
pp. e1500263 ◽  
Author(s):  
Akihiko Nakamura ◽  
Takuya Ishida ◽  
Katsuhiro Kusaka ◽  
Taro Yamada ◽  
Shinya Fushinobu ◽  
...  
Keyword(s):  
Glycoside Hydrolases ◽  
Side Chain ◽  
Enzymatic Reactions ◽  
X Ray Diffraction ◽  
Peptide Bonds ◽  
Proton Relay ◽  
Newton’S Cradle ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

scholarly journals Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

1998 ◽  
Vol 72 (6) ◽  
pp. 5121-5127 ◽  
Author(s):  
Prasad S. Koka ◽  
John K. Fraser ◽  
Yvonne Bryson ◽  
Gregory C. Bristol ◽  
Grace M. Aldrovandi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Erythroid Differentiation ◽  
Inhibitory Effect ◽  
Immunodeficiency Virus ◽  
The Many ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4+ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.

scholarly journals Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon

2015 ◽  
Vol 112 (32) ◽  
pp. 10038-10043 ◽  
Author(s):  
Noortje Ijssennagger ◽  
Clara Belzer ◽  
Guido J. Hooiveld ◽  
Jan Dekker ◽  
Saskia W. C. van Mil ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Tumor Suppressors ◽  
Disulfide Bonds ◽  
Red Meat ◽  
Cell Turnover ◽  
Degrading Bacteria ◽  
Mucus Barrier

Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.

scholarly journals Enabling microbial syringol conversion through structure-guided protein engineering

2019 ◽  
Vol 116 (28) ◽  
pp. 13970-13976 ◽  
Author(s):  
Melodie M. Machovina ◽  
Sam J. B. Mallinson ◽  
Brandon C. Knott ◽  
Alexander W. Meyers ◽  
Marc Garcia-Borràs ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Protein Engineering ◽  
Aromatic Compounds ◽  
Side Chain ◽  
Microbial Conversion ◽  
Sinapyl Alcohol ◽  
Analogous Compound ◽  
Dynamics Simulations ◽  
Rate Limiting

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism isO-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringolO-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover inPseudomonas putidaKT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromaticO-demethylases in the biological conversion of lignin-derived aromatic compounds.

The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

2020 ◽  
Vol 73 (4) ◽  
pp. 312
Author(s):  
Balasubramanyam Chittoor ◽  
Bankala Krishnarjuna ◽  
Rodrigo A. V. Morales ◽  
Raymond S. Norton
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Conformational Stability ◽  
Disulfide Bridge ◽  
Solution Nmr ◽  
Peptide Epitope ◽  
The Core ◽  
Native Fold

Disulfide bonds play a key role in the oxidative folding, conformational stability, and functional activity of many peptides. A few disulfide-rich peptides with privileged architecture such as the inhibitor cystine knot motif have garnered attention as templates in drug design. The single disulfide-directed β-hairpin (SDH), a novel fold identified more recently in contryphan-Vc1, has been shown to possess remarkable thermal, conformational, and chemical stability and can accept a short bioactive epitope without compromising the core structure of the peptide. In this study, we demonstrated that the single disulfide bond is critical in maintaining the native fold by replacing both cysteine residues with serine. We also designed an analogue with an additional, non-native disulfide bridge by replacing Gln1 and Tyr9 with Cys. Contryphan-Vc11–22[Q1C, Y9C] was synthesised utilising orthogonal cysteine protection and its solution structure determined using solution NMR spectroscopy. This analogue maintained the overall fold of native contryphan-Vc1. Previous studies had shown that the β-hairpin core of contryphan-Vc1 was resistant to proteolysis by trypsin and α-chymotrypsin but susceptible to cleavage by pepsin. Contryphan-Vc11–22[Q1C, Y9C] proved to be completely resistant to pepsin, thus confirming our design strategy. These results highlight the role of the disulfide bond in maintaining the SDH fold and provide a basis for the design of more stable analogues for peptide epitope grafting.

scholarly journals Increased Immunogenicity of Human Immunodeficiency Virus gp120 Engineered To Express Galα1-3Galβ1-4GlcNAc-R Epitopes

2006 ◽  
Vol 80 (14) ◽  
pp. 6943-6951 ◽  
Author(s):  
Ussama Abdel-Motal ◽  
Shixia Wang ◽  
Shan Lu ◽  
Kim Wigglesworth ◽  
Uri Galili
Keyword(s):  
Human Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
T Cell Response ◽  
Immunogenic Peptides ◽  
Immunodeficiency Virus ◽  
Glycoprotein Gp120 ◽  
Antigen Presenting ◽  
Hiv Envelope ◽  
Carbohydrate Chains

ABSTRACT The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galα1-3Galβ1-4GlcNAc-R (α-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting ∼1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing α-gal epitopes (gp120αgal) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcγ receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in α-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120αgal produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120αgal than in mice immunized with gp120. It is suggested that gp120αgal can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120αgal, thereby creating effective prophylactic vaccines.

scholarly journals Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins

2015 ◽  
Vol 90 (6) ◽  
pp. 2884-2894 ◽  
Author(s):  
Eden P. Go ◽  
Albert Cupo ◽  
Rajesh Ringe ◽  
Pavel Pugach ◽  
John P. Moore ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Protein A ◽  
Native Conformation ◽  
Purification Method ◽  
Immunodeficiency Virus ◽  
Monomeric Gp120 ◽  
Env Protein ◽  
The Impact ◽  
Hiv 1

ABSTRACTWe investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 proteins and native-like SOSIP.664 trimers based on the BG505envgene. Our results show that uncleaved gp140 proteins were not homogeneous, in that substantial subpopulations (20 to 80%) contained aberrant disulfide bonds. In contrast, the gp120 subunits of the native-like SOSIP.664 trimer almost exclusively retained the canonical disulfide bond pattern. We also observed that the purification method could influence the proportion of an Env protein population that contained aberrant disulfide bonds. We infer that gp140 proteins may always contain a variable but substantial proportion of aberrant disulfide bonds but that the impact of this problem can be minimized via design and/or purification strategies that yield native-like trimers. The same factors may also be relevant to the production and purification of monomeric gp120 proteins that are free of aberrant disulfide bonds.IMPORTANCEIt is widely thought that a successful HIV-1 vaccine will include a recombinant form of the Env protein, a trimer located on the virion surface. To increase yield and simplify purification, Env proteins are often made in truncated, soluble forms. A consequence, however, can be the loss of the native conformation concomitant with the virion-associated trimer. Moreover, some soluble recombinant Env proteins contain aberrant disulfide bonds that are not expected to be present in the native trimer. To assess whether these observations are linked, to determine the extent of disulfide bond scrambling, and to understand why scrambling occurs, we determined the disulfide bond profiles of two soluble Env proteins with different designs that are being assessed as vaccine candidates. We found that uncleaved gp140 forms heterogeneous mixtures in which aberrant disulfide bonds abound. In contrast, BG505 SOSIP.664 trimers are more homogeneous, native-like entities that contain predominantly the native disulfide bond profile.

The role of disulfide bonds and N-terminus in the structural properties of hepcidins: Insights from molecular dynamics simulations

Biopolymers ◽  
2010 ◽  
Vol 93 (10) ◽  
pp. 917-926 ◽  
Author(s):  
Massimiliano Aschi ◽  
Argante Bozzi ◽  
Renato Di Bartolomeo ◽  
Raffaele Petruzzelli
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Structural Properties ◽  
Disulfide Bonds ◽  
N Terminus ◽  
Dynamics Simulations

scholarly journals Identification of a Subset of Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus Strains Able To Exploit an Alternative Coreceptor on Untransformed Human Brain and Lymphoid Cells

2003 ◽  
Vol 77 (11) ◽  
pp. 6138-6152 ◽  
Author(s):  
Samantha J. Willey ◽  
Jacqueline D. Reeves ◽  
Richard Hudson ◽  
Koichi Miyake ◽  
Nathalie Dejucq ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Simian Immunodeficiency Virus ◽  
Cell Lines ◽  
Chemokine Receptors ◽  
Adult Brain ◽  
Immunodeficiency Virus ◽  
Siv Infection

ABSTRACT The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4+ transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4− astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Δ32/Δ32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.

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