Ki-Sun Kwon

Irreversible Oxidation of the Active-site Cysteine of Peroxiredoxin to Cysteine Sulfonic Acid for Enhanced Molecular Chaperone Activity

The thiol (–SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (–SO2H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (–SO3H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H2O2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO3H) by mass spectrometry. Tsa1p-SO3H was not an autoxidation product of Tsa1p-SO2H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO3H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO3H multimer lost its peroxidase activity, it gained ∼4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO3H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO3H is a marker of cumulative oxidative stress in cells.

Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1-/- fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27(kip1), a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27(kip1) and to decrease the stability of p27(kip1). VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27(kip1) stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27(kip1) from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27(kip1) stability via regulating JAB1.

Thioredoxin modulates activator protein 1 (AP-1) activity and p27Kip1 degradation through direct interaction with Jab1

Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.

The native strains in the hydrophobic core and flexible reactive loop of a serine protease inhibitor: crystal structure of an uncleaved α1-antitrypsin at 2.7 å

BACKGROUND The protein alpha1-antitrypsin is a prototype member of the serpin (serine protease inhibitor) family and is known to inhibit the activity of neutrophil elastase in the lower respiratory tract. Members of this family undergo a large structural rearrangement upon binding to a target protease, involving cleavage of the reactive-site loop. This loop is then inserted into the main body of the enzyme following the opening of a central beta sheet, leading to stabilization of the structure. Random mutageneses of alpha1-antitrypsin identified various mutations that stabilize the native structure and retard the insertion of the reactive-site loop. Structural studies of these mutations may reveal the mechanism of the conformational change. RESULTS We have determined the three-dimensional structure of an uncleaved alpha1-antitrypsin with seven such stabilizing mutations (hepta alpha1-antitrypsin) at 2.7 A resolution. From the comparison of the structure with other serpin structures, we found that hepta alpha1-antitrypsin is stabilized due to the release of various strains that exist in native wild type alpha1-antitrypsin, including unfavorable hydrophobic interactions in the central hydrophobic core. The reactive-site loop of hepta alpha1-antitrypsin is an extended strand, different from that of the previously determined structure of another uncleaved alpha1-antitrypsin, and indicates the inherent flexibility of the loop. CONCLUSIONS The present structural study suggests that the uncleaved alpha1-antitrypsin has many folding defects which can be improved by mutations. These folding defects seem to be utilized in a coordinated fashion in the regulation of the conformational switch of alpha1-antitrypsin. Some of the defects, represented by the Phe51 region and possibly the Met374 and the Thr59 regions, are part of the sheet-opening mechanism.

Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation of p21Cip1.

ABSTRACT p21Cip1 is an inhibitor of cell cycle progression that promotes G1-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21Cip1 downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21Cip1, promoting p21Cip1 nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21Cip1 revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21Cip1 translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21Cip1.

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.

Effect of glycosylation on the stability of α1-antitrypsin toward urea denaturation and thermal deactivation

The effects of glycosylation on the stability of human alpha1-antitrypsin were investigated. The transition midpoints in urea-induced equilibrium unfolding of a non-glycosylated recombinant, a yeast version of glycosylated, and human plasma alpha1-antitrypsin were 1.8 M, 2.2 M, and 2.5 M at 25 degrees C, respectively. Kinetic analyses of unfolding and refolding revealed that glycosylation retarded the unfolding without affecting the refolding rate significantly, suggesting that the stability increase is due to the stabilization of the native state as opposed to the destabilization of the unfolded state. In thermal deactivation, which is a heat-induced aggregation process, the unglycosylated recombinant alpha1-antitrypsin was deactivated most easily, which was followed in order by the yeast, and the plasma form. The results indicate that glycosylation confers the increase in stability of alpha1-antitrypsin, and that the oligomannose sugars present on the yeast form produce a less stable molecule than the complex type sugars on the plasma form. It appears that the effect of glycosylation on the enhancement of thermal resistance is exerted through the increase in conformational stability. However, a stable recombinant variant (Phe 51 --> Cys) that showed the same conformational stability as the plasma form was less resistant to thermal denaturation than the plasma alpha1-antitrypsin. The results suggest that the existence of carbohydrate moiety per se as well as the conformational stability contribute to the kinetic stability of alpha1-antitrypsin toward aggregation.

Crystal structure of an uncleaved α1-antitrypsin reveals the conformation of its inhibitory reactive loop

The crystal structure of a recombinant human α 1‐antitrypsin, in the uncleaved and uncomplexed state, has been determined by X‐ray crystallographic methods and refined to an R‐factor of 18.4% for 8.0–3.46 Å data with good stereochemistry. This structure provides the first view at the inhibitory loop and the central β‐sheet A of the uncleaved α 1‐antitrypsin. The reactive loop takes a distorted helical conformation and no pre‐insertion of two residues in the reactive loop into the β‐sheet A is observed. The present structure is largely in agreement with the model predicted by Engh, Wright, and Huber [Prot. Eng. 3 (1990) 469–477].Abstract The crystal structure of a recombinant human α1-antitrypsin, in the uncleaved and uncomplexed state, has been determined by X-ray crystallographic methods and refined to an R-factor of 18.4% for 8.0–3.46 A data with good stereochemistry. This structure provides the first view at the inhibitory loop and the central β-sheet A of the uncleaved α1-antitrypsin. The reactive loop takes a distorted helical conformation and no pre-insertion of two residues in the reactive loop into the β-sheet A is observed. The present structure is largely in agreement with the model predicted by Engh, Wright, and Huber [Prot. Eng. 3 (1990) 469–477].

TRIM32 Protein Sensitizes Cells to Tumor Necrosis Factor (TNFα)-induced Apoptosis via Its RING Domain-dependent E3 Ligase Activity against X-linked Inhibitor of Apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

Evidences for correlation between the reduced VCAM-1 expression and hyaluronan synthesis during cellular senescence of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) undergo cellular senescence during in vitro expansion culture, which accompanies the loss of migration and homing abilities. In this study, we analyzed expression levels of several surface markers of human MSCs at different passages of expansion culture. It has been shown that expression of vascular cell adhesion molecule-1 (VCAM-1) was most markedly decreased among the tested markers in the senescent MSCs. Interestingly the reduced VCAM-1 expression could be restored by applying hyaluronan, a major glycosaminoglycan ligand of CD44, to the culture. It was found that the hyaluronan level in extracellular and pericellular matrices was greatly reduced in the senescent MSCs, mainly due to the decreased expression of hyaluronan synthases, suggesting a correlation between the reduced VCAM-1 expression and hyaluronan synthesis. In fact, when hyaluronan synthases were knock-downed by siRNA transfection, the VCAM-1 expression was also reduced. Our results indicate that VCAM-1 expression in the senescent MSCs was down-regulated because of the reduced synthesis of hyaluronan. Thus, we suggest that hyaluronan supplementation in expansion culture of MSCs would compensate adverse effects induced by its decreased synthesis and subsequently enhance cell adhesion and migration abilities.