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1.
J Biol Chem ; 292(14): 5695-5704, 2017 04 07.
Article in English | MEDLINE | ID: mdl-28223361

ABSTRACT

The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.


Subject(s)
Adenosine Triphosphate/chemistry , Endopeptidase Clp/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Protein Unfolding , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Catalytic Domain , Endopeptidase Clp/genetics , Endopeptidase Clp/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism
2.
Anal Biochem ; 509: 73-78, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27387056

ABSTRACT

A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer(318)) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer(318)-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer(318)-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer(318)-Atg13 upon ULK1 inhibition.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Protein-1 Homolog/metabolism , Autophagy-Related Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Array Analysis/methods , Animals , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay/methods , Heterografts , Humans , Mice , Neoplasm Transplantation , Phosphorylation
3.
Toxicol Res (Camb) ; 5(6): 1619-1628, 2016 Nov 01.
Article in English | MEDLINE | ID: mdl-30090462

ABSTRACT

The proteasome inhibitor bortezomib is associated with the development of peripheral neuropathy in patients, but the mechanism by which bortezomib can induce peripheral neuropathy is not fully understood. One study suggested that off-target inhibition of proteases other than the proteasome, particularly HtraA2/Omi, may be the underlying mechanism of the neuropathy. The same study also concluded that carfilzomib, a second proteasome inhibitor that is associated with less peripheral neuropathy in patients than bortezomib, showed no inhibition of HtrA2/Omi. The goal of the work described here was to determine whether either proteasome inhibitors truly affected HtrA2/Omi activity. A variety of methods were used to test the effects of both bortezomib and carfilzomib on HtrA2/Omi activity that included in vitro recombinant enzyme assays, and studies with the human neuroblastoma SH-SY5Y cell line and HtrA2/Omi-knockout mouse embryonic fibroblasts. The compound ucf-101 was used to assess the effects of specific HtrA2/Omi inhibition. In contrast to previously published data, our results clearly demonstrated that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity in recombinant enzyme assays at concentrations up to 100 µM, while the specific inhibitor ucf-101 did inhibit the enzyme. The proteasome inhibitors did not inhibit HtrA2/Omi activity in either SH-SY5Y cells or mouse embryonic fibroblasts, as determined by expression of the HtrA2/Omi substrates eIF4G1 and UCH-L1. Based on our biochemical and cell-based assays, we conclude that neither bortezomib nor carfilzomib inhibited HtrA2/Omi activity. Therefore, it is unlikely that bortezomib associated peripheral neuropathy is a direct result of off-target inhibition of HtrA2/Omi.

4.
J Biol Chem ; 289(33): 22648-22658, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-24966333

ABSTRACT

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.


Subject(s)
Multiprotein Complexes , Proteins , Ubiquitin-Activating Enzymes , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Catalytic Domain , Cell Line , Enzyme Activation , Humans , Models, Chemical , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Protein Binding , Protein Structure, Quaternary , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism
5.
J Am Chem Soc ; 131(46): 16751-7, 2009 Nov 25.
Article in English | MEDLINE | ID: mdl-19919143

ABSTRACT

The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.


Subject(s)
Tryptophan/chemistry , gamma-Crystallins/chemistry , gamma-Crystallins/radiation effects , Humans , Phenylalanine/chemistry , Phenylalanine/genetics , Spectrometry, Fluorescence , Tryptophan/genetics , Ultraviolet Rays , gamma-Crystallins/genetics
6.
Biochemistry ; 48(17): 3708-16, 2009 May 05.
Article in English | MEDLINE | ID: mdl-19358562

ABSTRACT

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated beta-sheet Greek key domains of beta- and gamma-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human gammaD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552-11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human gammaS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n - 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.


Subject(s)
Crystallins/chemistry , Evolution, Molecular , Lens, Crystalline/chemistry , Protein Folding , Tryptophan/chemistry , Ultraviolet Rays/adverse effects , gamma-Crystallins/chemistry , Crystallins/genetics , Crystallins/radiation effects , Crystallography, X-Ray , Electron Transport/genetics , Electron Transport/radiation effects , Humans , Lens, Crystalline/injuries , Lens, Crystalline/radiation effects , Mutagenesis , Protein Folding/radiation effects , Spectrometry, Fluorescence , Static Electricity , Sunlight/adverse effects , Tryptophan/genetics , Tryptophan/radiation effects , gamma-Crystallins/genetics , gamma-Crystallins/radiation effects
7.
Biochemistry ; 47(40): 10705-21, 2008 Oct 07.
Article in English | MEDLINE | ID: mdl-18795792

ABSTRACT

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.


Subject(s)
Spectrometry, Fluorescence/methods , Tryptophan/chemistry , gamma-Crystallins/chemistry , Humans , Protein Structure, Secondary
8.
Biochemistry ; 46(41): 11451-8, 2007 Oct 16.
Article in English | MEDLINE | ID: mdl-17892301

ABSTRACT

S-Adenosylhomocysteine hydrolase (SAHH) regulates biomethylation and homocysteine metabolism and thus is an attractive target in drug design studies. SAHH has been shown to be a copper binding protein in vivo; however, the structure and catalytic mechanism of SAHH exclude a role for Cu2+. In the present work, we studied the mechanism of inhibition of SAHH activity by Cu2+. The experimental results showed that Cu2+ inhibited SAHH activity in a noncompetitive manner. Binding of Cu2+ to SAHH resulted in the release of NAD+ cofactors, explaining the loss of the enzymatic activity of SAHH. Further investigation by an ESR probe and computational simulation suggested that Cu2+ could bind at the central channel and interrupt the subunit interactions of SAHH, resulting in a large decrease in affinity to the NAD+ cofactor. This effect of Cu2+ resembled that of enzyme mutations at the C-terminal domain or Asp244 [Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme, J. Biol. Chem. 275, 32147-32156]. The mechanism of action of Cu2+ on SAHH suggested a possible regulative role for Cu2+ on the intracellular activity of SAHH. This could be helpful in understanding the biological effects of copper compounds and suggest a potential coupling mechanism between biomethylation and the redox states of cells.


Subject(s)
Adenosylhomocysteinase/antagonists & inhibitors , Adenosylhomocysteinase/chemistry , Copper/pharmacology , NAD/metabolism , Apoenzymes/chemistry , Apoenzymes/metabolism , Drug Design , Female , Humans , Kinetics , Microwaves , Placenta/enzymology , Pregnancy , Protein Binding , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry
9.
Biochemistry ; 45(38): 11552-63, 2006 Sep 26.
Article in English | MEDLINE | ID: mdl-16981715

ABSTRACT

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.


Subject(s)
Crystallins/metabolism , Fluorescence , Tryptophan/metabolism , Amino Acid Sequence , Amino Acid Substitution , Crystallins/chemistry , Fluorescence Resonance Energy Transfer , Humans , Models, Molecular , Molecular Sequence Data , Mutant Proteins/metabolism , Mutation/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Quantum Theory , gamma-Crystallins
10.
J Inorg Biochem ; 98(6): 977-83, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15149804

ABSTRACT

S-Adenosylhomocysteine (AdoHcy) hydrolase regulates biomethylation and homocysteine metabolism. It has been proposed to be a copper binding protein playing an important role in copper transport and distribution. In the present work, the kinetics of binding and releasing of copper ions was studied using fluorescence method. The dissociation constant for copper ions with AdoHcy hydrolase was determined by fluorescence quenching titration and activity titration methods using ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and glycine as competitive chelators. The experimental results showed that copper ions bind to AdoHcy hydrolase with a K(d) of approximately 10(-11) M. The association rate constant was determined to be 7 x 10(6) M(-1)s(-1). The releasing of copper ions from the enzyme was found to be biphasic with a k(1) of 2.8 x 10(-3) s(-1) and k(2) of 1.7x10(-5) s(-1). It is suggested that copper ions do not bind to the substrate binding sites because the addition of adenine substrate did not compete with the binding of copper to AdoHcy hydrolase. Interestingly, it was observed that EDTA could bind to AdoHcy hydrolase with a dissociation constant of K(1) = 8.0 x 10(-5) M and result in an increased affinity (K(d) = approximately 10(-17) M) of binding of copper ions to the enzyme.


Subject(s)
Adenosylhomocysteinase/chemistry , Copper/chemistry , Adenosylhomocysteinase/metabolism , Copper/metabolism , Humans , Protein Binding
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