scholarly journals Characterization of the Structural Determinants of the Ubiquitin-Dependent Proteasomal Degradation of Human Hepatic Tryptophan 2,3-Dioxygenase

2021 ◽  
Author(s):  
Yi Liu ◽  
Sung Mi Kim ◽  
YongQiang Wang ◽  
Shay Karkashon ◽  
Ariel Lewis-Ballester ◽  
...  
Keyword(s):  
Proteasomal Degradation ◽  
Site Directed Mutagenesis ◽  
Liver Protein ◽  
Lysosomal Degradation ◽  
Structural Determinants ◽  
The Poor ◽  
Structural Relevance ◽  
Positively Charged

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ~2.3 h, relative to an average t1/2 of ~2–3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation (UPD) pathway. Additionally, we identified 15 ubiquitination K sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here we further established autophagic lysosomal degradation (ALD) as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.

scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

2019 ◽  
Author(s):  
Timothy S. Strutzenberg ◽  
Ruben Garcia-Ordonez ◽  
Scott Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Chemical Probes ◽  
Structural Determinants ◽  
Synthetic Ligand ◽  
Hdx Ms

ABSTRACTMembers of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism1 to inflammation2 and cancer3. As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology4. However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and site-directed mutagenesis to collectively reveal the structural determinants of RORγ ligand-dependent activation, critical for designing full agonists for application in cancer immunotherapy. Combined these observations support a model of receptor activation to more accurately describe RORγ pharmacology. Likewise, this ‘bump-and-hole’ inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Timothy S Strutzenberg ◽  
Ruben D Garcia-Ordonez ◽  
Scott J Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Small Molecules ◽  
Structural Dynamics ◽  
Site Directed Mutagenesis ◽  
Chemical Probes ◽  
Structural Determinants ◽  
X Ray ◽  
Synthetic Ligand ◽  
Hdx Ms

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it’s unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals The Role of Surface Chemistry and Polyethylenimine Grafting in the Removal of Cr (VI) by Activated Carbons from Cashew Nut Shells

2021 ◽  
Vol 7 (1) ◽  
pp. 27
Author(s):  
Victoria A. Smith ◽  
Juster F. A. Rivera ◽  
Ruby Bello ◽  
Elena Rodríguez-Aguado ◽  
Mohammed R. Elshaer ◽  
...  
Keyword(s):  
Surface Characterization ◽  
Activated Carbons ◽  
Carbon Surface ◽  
Maximum Efficiency ◽  
Cashew Nut ◽  
Average Maximum ◽  
Carboxylic Groups ◽  
Positively Charged

Activated carbons prepared from cashew nut shells and modified by grafting polyethylenimine onto the surface were tested for removal of Cr (VI). The removal efficiency of carbons without and with polyethylenimine decreased with an increase in pH, with maximum efficiency found at pH 2. The average maximum adsorption capacities of carbons were calculated to be 340 ± 20 mg/g and 320 ± 20 mg/g for unmodified and modified carbons, respectively. Surface characterization of carbons revealed that C–O functionalities are actively involved in both polyethylenimine grafting and Cr (VI) removal. Moreover, lactone groups and amides, formed by polyethylenimine grafting, seemingly undergo acid hydrolysis with formation of phenol and carboxylic groups. Considering that Cr (III) is the only form of chromium found on the surface of both carbons, the reduction mechanism is deduced as the predominant one. Here Cr (VI), majorly present as HCrO4¯, is attracted to the positively charged carbon surface, reduced to Cr (III) by phenol groups, and adsorbed inside the pores. The mechanism of Cr (VI) removal appears to be similar for unmodified and modified carbons, where the smaller adsorption capacity of the latter one can be related to steric hindrance and pore inaccessibility.

Characterization of Recombinant von Willebrand Factors Mutated on Cysteine 509 or 695

1996 ◽  
Vol 76 (03) ◽  
pp. 453-459 ◽  
Author(s):  
Virginie Siguret ◽  
Anne-Sophie Ribba ◽  
Olivier Christophe ◽  
Ghislaine Chérel ◽  
Bernadette Obert ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
High Molecular Weight Multimers

SummaryThe interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be “locked” into active or inactive conformations in regard to the binding to platelet GPIb receptor.

The Functional Role of Positively Charged Amino Acid Side Chains in α-Bungarotoxin Revealed by Site-Directed Mutagenesis of a His-Tagged Recombinant α-Bungarotoxin†

Biochemistry ◽  
1999 ◽  
Vol 38 (24) ◽  
pp. 7847-7855 ◽  
Author(s):  
Julie A. Rosenthal ◽  
Mark M. Levandoski ◽  
Belle Chang ◽  
Jerald F. Potts ◽  
Qing-Luo Shi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Functional Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Side Chains ◽  
Amino Acid Side Chains ◽  
Positively Charged

scholarly journals Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

2011 ◽  
Vol 437 (2) ◽  
pp. 243-253 ◽  
Author(s):  
Anatoli Tchigvintsev ◽  
Alexander U. Singer ◽  
Robert Flick ◽  
Pierre Petit ◽  
Greg Brown ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Saturation Curve ◽  
Significant Activity ◽  
Active Site Residues ◽  
Living Organisms ◽  
Hydrolysis Of

Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å=0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.

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