scholarly journals Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase

2021 ◽  
Vol 118 (23) ◽  
pp. e2022447118
Author(s):  
Simon Klaessens ◽  
Vincent Stroobant ◽  
Delia Hoffmann ◽  
Mads Gyrd-Hansen ◽  
Luc Pilotte ◽  
...  
Keyword(s):  
Amino Acid ◽  
Immune Homeostasis ◽  
Hepatic Enzyme ◽  
Rapid Adaptation ◽  
Tight Control ◽  
Immune Activity ◽  
Sensing Mechanism ◽  
Tryptophan Catabolism ◽  
The Stability

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four–amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

scholarly journals Physical and Functional Interaction of the HECT Ubiquitin-protein Ligases E6AP and HERC2

2011 ◽  
Vol 286 (22) ◽  
pp. 19410-19416 ◽  
Author(s):  
Simone Kühnle ◽  
Ulrike Kogel ◽  
Sandra Glockzin ◽  
Andreas Marquardt ◽  
Aaron Ciechanover ◽  
...  
Keyword(s):  
Amino Acid ◽  
Angelman Syndrome ◽  
Amino Acid Residues ◽  
Cervical Carcinogenesis ◽  
Tight Control ◽  
Functional Interaction ◽  
Ubiquitin Protein Ligase ◽  
Protein Ligases ◽  
Translational Level

Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150–200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.

scholarly journals Structure/Function Studies Involving the V3 Region of the HIV-1 Envelope Delineate Multiple Factors That Affect Neutralization Sensitivity

2015 ◽  
Vol 90 (2) ◽  
pp. 636-649 ◽  
Author(s):  
Susan Zolla-Pazner ◽  
Sandra Sharpe Cohen ◽  
David Boyd ◽  
Xiang-Peng Kong ◽  
Michael Seaman ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hiv Infection ◽  
Chemokine Receptors ◽  
Variable Region ◽  
Single Amino Acid ◽  
V3 Loop ◽  
Infection Rates ◽  
Neutralization Sensitivity ◽  
The Stability ◽  
Hiv 1

ABSTRACTAntibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most tier 1 and many tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans and affect the nature of transmitted viruses in infected vaccinees, despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is metastable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The spring-loaded V3 in the envelope trimer is easily released by disruption of the stability of the V3 pocket in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is, in turn, dependent on the conformation of the bridging sheet and on the stability of the V1V2 B-C strand-connecting loop.IMPORTANCEThe levels of antibodies to the third variable region (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded, as it sits in a pocket of the envelope trimer on the surface of virions; however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120 (i) render viruses sensitive to neutralization by V3 antibodies, (ii) result in altered packing of the V3 loop, and (iii) activate an open conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection and why they should be one of the targets of vaccine-induced antibodies.

A Predictive Energy Landscape Model of Metamorphic Protein Conformational Specificity

2021 ◽  
Author(s):  
James O. Wrabl ◽  
Keila Voortman-Sheetz ◽  
Vincent J. Hilser
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Large Scale ◽  
Energy Landscape ◽  
Thermodynamic Approach ◽  
High Identity ◽  
Denatured State ◽  
Metamorphic Protein ◽  
The Stability ◽  
The Impact

'Metamorphic' proteins challenge state-of-the-art structure prediction methods reliant on amino acid similarity. Unfortunately, this obviates a more effective thermodynamic approach necessary to properly evaluate the impact of amino acid changes on the stability of two different folds. A vital capability of such a thermodynamic approach would be the quantification of the free energy differences between 1) the energy landscape minima of each native fold, and 2) each fold and the denatured state. Here we develop an energetic framework for conformational specificity, based on an ensemble description of protein thermodynamics. This energetic framework was able to successfully recapitulate the structures of high-identity engineered sequences experimentally shown to adopt either Streptococcus protein GA or GB folds, demonstrating that this approach indeed reflected the energetic determinants of fold. Residue-level decomposition of the conformational specificity suggested several testable hypotheses, notably among them that fold-switching could be affected by local de-stabilization of the populated fold at positions sensitive to equilibrium perturbation. Since this ensemble-based compatibility framework is applicable to any structure and any sequence, it may be practically useful for the future targeted design, or large-scale proteomic detection, of novel metamorphic proteins.

scholarly journals Synthesis and Evaluation of Cyclic Acetals of Serine Hydroxylamine for Amide-Forming KAHA Ligations

Synthesis ◽  
2019 ◽  
Vol 51 (05) ◽  
pp. 1273-1283 ◽  
Author(s):  
Simon Baldauf ◽  
Jeffrey Bode
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Amino Acid Residues ◽  
High Demand ◽  
Cyclic Acetals ◽  
Ligation Product ◽  
Canonical Amino Acid ◽  
The Stability ◽  
Peptide Segments ◽  
Derivatives Of

The α-ketoacid–hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. The most widely used variant employs a 5-membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. While very effective, monomers that give canonical amino acid residues are in high demand. In order to preserve the stability and reactivity of cyclic hydroxylamines, but form a canonical amino acid residue upon ligation, we sought to prepare cyclic derivatives of serine hydroxylamine. An evaluation of several cyclization strategies led to cyclobutanone ketals as the leading structures. The preparation, stability, and amide-forming ligation of these serine-derived ketals are described.

scholarly journals The composition of peptidochitodextrins from sarcophagid puparial cases

1971 ◽  
Vol 125 (3) ◽  
pp. 703-716 ◽  
Author(s):  
H. Lipke ◽  
T. Geoghegan
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Protein Complex ◽  
Covalent Bonds ◽  
Aromatic Residues ◽  
Similar Molecular Weight ◽  
X Ray ◽  
Molecular Weights ◽  
Physical Interactions ◽  
The Stability

1. N-Bromosuccinimide cleaved proteins and pigments from fly puparia, increasing the chitin:protein ratio from 0.5 to 1.5. The product afforded subfractions (ratio 5:1) of molecular weights of 1200 and 1600 devoid of aromatic residues and N-terminal β-alanine, direct aryl links between polysaccharide chains being discounted. 2. The chitin–protein complex decreased in molecular weight when treated with Pronase, which suggested polypeptide bridges within the native chitin micelle. The limit dextrins generated by chitinase were mixtures of unsubstituted dextrins and peptidylated oligosaccharides, with the former predominating. 3. Peptidochitodextrins of similar molecular weight but markedly different solubility were prepared, which were indistinguishable with respect to amino acid, glucosamine, acetyl, X-ray or infrared characteristics. It is suggested that physical interactions contribute to the stability of the integument in addition to the covalent bonds that form during sclerotization.

Circular dichroism studies of sheep β-lipotropic hormone

1976 ◽  
Vol 54 (11) ◽  
pp. 992-998 ◽  
Author(s):  
Serge St-Pierre ◽  
Claude Gilardeau ◽  
Michel Chrétien
Keyword(s):  
Circular Dichroism ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Conformational Transition ◽  
Acidic Solutions ◽  
Nacl Concentration ◽  
Helical Content ◽  
The Stability ◽  
Far Ultraviolet ◽  
Circular Dichroïsm

The far ultraviolet circular dichroism spectra of sheep β-lipotropic hormone (β-LPH) were recorded under different conditions of pH, temperature, salt concentration, and solvent composition. Results confirm the stability of the hormone in strong basic or acidic solutions; moreover, temperatures up to 50 °C do not seem to affect noticeably the conformation of β-LPH. However, increasing the NaCl concentration or addition of dioxane in the solution brings about a conformational transition of the chain, interpreted as an increase in the helical content. The method of Yang (Chen, Y. H., Yang, J. T. &Martinez, H. M. (1972) Biochemistry 11, 4120–4131) was used to compute the proportion of helical, β, and unordered forms of the hormone chain. The proportions are compared with those obtained from Fasman's predictive method (Chou, P. Y. &Fasman, G. D. (1974) Biochemistry 13, 211–221 and Chou, P. Y. &Fasman, G. D. (1974) Biochemistry 13, 222–245) based on the known amino acid sequence of β-LPH.

scholarly journals Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Tomokazu Tamura ◽  
Takasuke Fukuhara ◽  
Takuro Uchida ◽  
Chikako Ono ◽  
Hiroyuki Mori ◽  
...  
Keyword(s):  
Life Cycle ◽  
Amino Acid ◽  
Viral Life Cycle ◽  
Luciferase Gene ◽  
Content Type ◽  
Recombinant Viruses ◽  
Nanoluc Luciferase ◽  
The Stability ◽  
Split Luciferase

ABSTRACTThe familyFlaviviridaeconsists of four genera,Flavivirus,Pestivirus,Pegivirus, andHepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of theFlaviviridaeviruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamicsin vivo. Taken together, our findings indicate that the recombinantFlaviviridaeviruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals againstFlaviviridaeviruses.IMPORTANCEThe construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinantFlaviviridaeviruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable toin vitroandin vivoexperiments, suggesting that these recombinantFlaviviridaeviruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals againstFlaviviridaeviruses.

scholarly journals Monitoring the stabilities of a mixture of peptides by mass-spectrometry-based techniques

2019 ◽  
Vol 25 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Daniel R Fuller ◽  
Christopher R Conant ◽  
Tarick J El-Baba ◽  
Zhichao Zhang ◽  
Kameron R Molloy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Transition State ◽  
Elevated Temperatures ◽  
Proteolytic Enzymes ◽  
Side Chain ◽  
Free Energies ◽  
Dissociation Kinetics ◽  
Penultimate Proline ◽  
The Stability

Biomolecular degradation plays a key role in proteostasis. Typically, proteolytic enzymes degrade proteins into smaller peptides by breaking amino acid bonds between specific residues. Cleavage around proline residues is often missed and requires highly specific enzymes for peptide processing due to the cyclic proline side-chain. However, degradation can occur spontaneously (i.e. in the absence of enzymes). In this study, the influence of the first residue on the stability of a series of penultimate proline containing peptides, with the sequence Xaa–Pro–Gly–Gly (where Xaa is any amino acid), is investigated with mass spectrometry techniques. Peptides were incubated as mixtures at various solution temperatures (70℃ to 90℃) and were periodically sampled over the duration of the experiment. At elevated temperatures, we observe dissociation after the Xaa–Pro motif for all sequences, but at different rates. Transition state thermochemistry was obtained by studying the temperature-dependent kinetics and although all peptides show relatively small differences in the transition state free energies (∼95 kJ/mol), there is significant variability in the transition state entropy and enthalpy. This demonstrates that the side-chain of the first amino acid has a significant influence on the stability of the Xaa–Pro sequence. From these data, we demonstrate the ability to simultaneously measure the dissociation kinetics and relative transition state thermochemistries for a mixture of peptides, which vary only in the identity of the N-terminal amino acid.

Sign in / Sign up

Export Citation Format

Share Document