Cheolju Lee

ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.

Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

Channel-Mediated Tonic GABA Release from Glia

Tonic Inhibition Neuronal inhibition has recently drawn much attention; however, the mechanisms involved in tonic release of and the cellular source of the neurotransmitter involved, γ-aminobutyric acid (GABA), have been difficult to pin down. Lee et al. (p. 790, published online 23 September) showed that tonic release of GABA in the cerebellum occurs through the Bestrophin 1 anion channel of cerebellar astrocytes and Bergmann glial cells. These results confirm that glia can serve as a source of GABA for tonic inhibition of neurons and provide more evidence for interactions between neurons and glia cells that have implications for our understanding of brain-signaling mechanisms. The neurotransmitter GABA is tonically released from cells through an anion channel with an unusually large pore. Synaptic inhibition is based on both tonic and phasic release of the inhibitory transmitter γ-aminobutyric acid (GABA). Although phasic GABA release arises from Ca2+-dependent exocytosis from neurons, the mechanism of tonic GABA release is unclear. Here we report that tonic inhibition in the cerebellum is due to GABA being released from glial cells by permeation through the Bestrophin 1 (Best1) anion channel. We demonstrate that GABA directly permeates through Best1 to yield GABA release and that tonic inhibition is eliminated by silencing of Best1. Glial cells express both GABA and Best1, and selective expression of Best1 in glial cells, after preventing general expression of Best1, fully rescues tonic inhibition. Our results identify a molecular mechanism for tonic inhibition and establish a role for interactions between glia and neurons in mediating tonic inhibition.

Phosphorylation of EZH2 Activates STAT3 Signaling via STAT3 Methylation and Promotes Tumorigenicity of Glioblastoma Stem-like Cells

Glioblastoma multiforme (GBM) displays cellular hierarchies harboring a subpopulation of stem-like cells (GSCs). Enhancer of Zeste Homolog 2 (EZH2), the lysine methyltransferase of Polycomb repressive complex 2, mediates transcriptional repression of prodifferentiation genes in both normal and neoplastic stem cells. An oncogenic role of EZH2 as a transcriptional silencer is well established; however, additional functions of EZH2 are incompletely understood. Here, we show that EZH2 binds to and methylates STAT3, leading to enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. The EZH2-STAT3 interaction preferentially occurs in GSCs relative to non-stem bulk tumor cells, and it requires a specific phosphorylation of EZH2. Inhibition of EZH2 reverses the silencing of Polycomb target genes and diminishes STAT3 activity, suggesting therapeutic strategies.

Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s−1. The disulfide bond–mediated conformation switch results in a metastable form that is locally strained by ∼3 kcal mol−1. On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

Protein glycation: creation of catalytic sites for free radical generation.

Abstract: In a glycation reaction, α‐dicarbonyl compounds such as deoxyglucosone, methylglyoxal, and glyoxal are more reactive than the parent sugars with respect to their ability to react with amino groups of proteins to form inter‐ and intramolecular cross‐links of proteins, stable end products called advanced Maillard products or advanced end products (AGEs). The AGEs, which are irreversibly formed, accumulate with aging, atherosclerosis, and diabetes mellitus, and are especially associated with long‐lived proteins such as collagens, lens crystallins, and nerve proteins. It was suggested that the formation of AGEs not only modifies protein properites but also induces biological damage in vivo. In this report, we summerize results obtained from our studies for (1) identifying the structure of the cross‐linked radical species formed in the model system—the reaction between α‐dicarbonyl methylglyoxal with amino acids, and (2) the reactivity of the radical center of the protein created by the similar reaction. These results indicate that glycation of protein generates active centers for catalyzing one‐electron oxidation‐reduction reactions. This active center, which exhibits enzyme‐like character, is suggested to be the cross‐linked Schiff‐based radical cation of the protein. It mimics the characteristics of the metal‐catalyzed oxidation system. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free redicals.

Oxidation-Reduction Properties of Methylglyoxal-modified Protein in Relation to Free Radical Generation

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with l-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995)J. Biol. Chem. 270, 28228–28233) or withN α-acetyl-l-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochromec to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochromec in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulatedin vivo provide stable active sites for catalyzing the formation of free radicals.

Structure of Human FIH-1 Reveals a Unique Active Site Pocket and Interaction Sites for HIF-1 and von Hippel-Lindau

The master switch of cellular hypoxia responses, hypoxia-inducible factor 1 (HIF-1), is hydroxylated by factor inhibiting HIF-1 (FIH-1) at a conserved asparagine residue under normoxia, which suppresses transcriptional activity of HIF-1 by abrogating its interaction with transcription coactivators. Here we report the crystal structure of human FIH-1 at 2.8-Å resolution. The structural core of FIH-1 consists of a jellyroll-like β-barrel containing the conserved ferrous-binding triad residues, confirming that FIH-1 is a member of the 2-oxoglutarate-dependent dioxygenase family. Except for the core structure and triad residues, FIH-1 has many structural deviations from other family members including N- and C-terminal insertions and various deletions in the middle of the structure. The ferrous-binding triad region is highly exposed to the solvent, which is connected to a prominent groove that may bind to a helix near the hydroxylation site of HIF-1. The structure, which is in a dimeric state, also reveals the putative von Hippel-Lindau-binding site that is distinctive to the putative HIF-1-binding site, supporting the formation of the ternary complex by FIH-1, HIF-1, and von Hippel-Lindau. The unique environment of the active site and cofactor-binding region revealed in the structure should allow design of selective drugs that can be used in ischemic diseases to promote hypoxia responses.

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer–related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R2 > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

Identification of S100A8 and S100A9 as serological markers for colorectal cancer.

In search of novel serological protein biomarkers for human colorectal cancer (CRC), we analyzed CRC tissues using two-dimensional difference in-gel electrophoresis (2D-DIGE) on a narrow range IPG strip (pH 5.5-6.7). By comparing tumor tissues with matched normal tissues in a pairwise manner (n = 6), we identified 34 up-regulated and 17 down-regulated spots with intensity changes greater than 2-fold (Students t-test, p < 0.05). Expression of both mRNA and protein levels of four proteins, adenosylhomocysteinase, Nm23-H1, S100A8 and S100A9, in CRC tissues was further evaluated by semiquantitative RT-PCR and Western blot analysis. The results revealed that all four proteins were elevated in the tumor tissues. We also confirmed, by immunohistochemistry, that adenosylhomocysteinase and Nm23-H1 were overexpressed in tumor cell cytoplasm and that S100A8 and S100A9 proteins were strongly expressed in tumor infiltrating immune cells. Western blot analysis with fractionated plasma samples showed that S100A8 and S100A9 were significantly increased in the plasma of CRC patients (n = 77) and colorectal adenoma patients (n = 11), compared to healthy controls (n = 21). The area under a receiver operating characteristic (ROC) curve was 0.91 for S100A8 and 0.89 for S100A9, which was superior to the established tumor marker carcinoembryonic antigen with 0.78 for the area under the ROC curve. Some patients with inflammatory diseases such as pancreatitis also showed elevated levels of the proteins. Importantly, in comparison to the control group, both proteins showed a remarkable change at the early stage of cancer. Therefore, we suggest S100A8 and S100A9 as candidates for serological biomarkers in combination with other serum markers that aid CRC diagnosis.

Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IkappaB kinase beta (IKKbeta) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-kappaB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-kappaB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFalpha-stimulated NF-kappaB activation. The E(T/S)GE motif, which is found only in the IKKbeta subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-kappaB activity, and up-regulated the expression of NF-kappaB target genes. Ectopic expression of KEAP1 decreased the expression of IKKbeta, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFalpha was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-kappaB signaling through the inhibition of IKKbeta phosphorylation and the mediation of autophagy-dependent IKKbeta degradation.