Kyoung-Soo Choi

Human peroxiredoxin 1 and 2 are not duplicate proteins: the unique presence of CYS83 in Prx1 underscores the structural and functional differences between Prx1 and Prx2.

Human peroxiredoxins 1 and 2, also known as Prx1 and Prx2, are more than 90% homologous in their amino acid sequences. Prx1 and Prx2 are elevated in various cancers and are shown to influence diverse cellular processes. Although their growth regulatory role has traditionally been attributed to the peroxidase activity, the physiological significance of this function is unclear because the proteins are highly susceptible to inactivation by H2O2. A chaperone activity appears to emerge when their peroxidase activity is lost. Structural studies suggest that they may form a homodimer or doughnut-shaped homodecamer. However, little information is available whether human Prx1 and Prx2 are duplicative in structure and function. We noted that Prx1 contains a cysteine (Cys83) at the putative dimer-dimer interface, which is absent in Prx2. We studied the role of Cys83 in regulating the peroxidase and chaperone activities of Prx1, because the redox status of Cys83 might influence the oligomeric structure and consequently the functions of Prx1. We show that Prx1 is more efficient as a molecular chaperone, whereas Prx2 is better suited as a peroxidase enzyme. Substituting Cys83 with Ser83 (Prx1C83S) results in dramatic changes in the structural and functional characteristics of Prx1 in a direction similar to those of Prx2. Here we also report the first crystal structure of human Prx1 and the presence of the Cys83–Cys83 bond at the dimer-dimer interface of decameric Prx1. These findings are consistent with the hypothesis that human Prx1 and Prx2 possess unique functions and regulatory mechanisms and that Cys83 bestows a distinctive identity to Prx1.

Allyl Isothiocyanate Arrests Cancer Cells in Mitosis, and Mitotic Arrest in Turn Leads to Apoptosis via Bcl-2 Protein Phosphorylation

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and β-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G1 phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.

Role for Hepatic and Circulatory ST6Gal-1 Sialyltransferase in Regulating Myelopoiesis

Recent findings have established a role for the ST6Gal-1 sialyltransferase in modulating inflammatory cell production during Th1 and Th2 responses. ST6Gal-1 synthesizes the Sia(α2,6) to Gal(β1,4)GlcNAc linkage on glycoproteins on cell surfaces and in systemic circulation. Engagement of P1, one of six promoter/regulatory regions driving murine ST6Gal-1 gene expression, generates the ST6Gal-1 for myelopoietic regulation. P1 utilization, however, is restricted to the liver and silent in hematopoietic cells. We considered the possibility that myelopoiesis is responsive to the sialylation of liver-derived circulatory glycoproteins, such that reduced α2,6-sialylation results in elevated myelopoiesis. However, 2-dimensional differential in gel electrophoresis (2D-DIGE) analysis disclosed only minimal alterations in the sialylation of sera glycoproteins of ST6Gal-1-deficient mice when compared with wild-type controls, either at baseline or during an acute phase response when the demand for sialylation is greatest. Furthermore, sera from ST6Gal-1-deficient animals did not enhance myelopoietic activity in ex vivo colony formation assays. Whereas there was only minimal consequence to the α2,6-sialylation of circulatory glycoproteins, ablation of the P1 promoter did result in strikingly depressed levels of ST6Gal-1 released into systemic circulation. Therefore, we considered the alternative possibility that myelopoiesis may be regulated not by the hepatic sialyl glycoproteins, but by the ST6Gal-1 that was released directly into circulation. Supporting this, ex vivo colony formation was notably attenuated upon introduction of physiologic levels of ST6Gal-1 into the culture medium. Our data support the idea that circulatory ST6Gal-1, mostly of hepatic origin, limits myelopoiesis by a mechanism independent of hepatic sialylation of serum glycoproteins.

Changes in HIV-related behaviours over time and associations with rates of HIV-related services coverage among female sex workers in Sichuan, China

Objectives: To investigate changes in HIV-related behaviours among female sex workers (FSW) and associations with services coverage rates. Methods: Behavioural surveillance data from Sichuan, China, were analysed. A mapping exercise was conducted; FSW were recruited from randomly selected sex-work establishments in 19 sites in Sichuan, China, from 2003 (n = 7068), 2004 (n = 6875) and 2005 (n = 6833). Results: Site variations were substantial. The random effect pooled AOR comparing the prevalence of condom use with regular sex partners, possession of condoms, HIV-related knowledge, HIV antibody testing and services coverage rates in 2005 versus 2003 ranged from 1.42 to 20.35. The 95% CI of these pooled AOR all excluded 1.0; hence rejecting the null hypothesis that such OR were not different from 1.0. Most of the AOR of these evaluative parameters (indicator of improvement) for the 19 individual sites in 2004 and 2005 (vs 2003) were significantly associated with coverage rates (Spearmen correlation coefficients  =  0.35 to 0.67, p<0.05). Conclusions: Improvements were observed in relevant behaviours and coverage rates and the two were associated with each other.

Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus

ABSTRACT Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1gp-340, mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.

Analysis of Protein Redox Modification by Hypoxia

Abstract We examined hypoxia‐induced changes in global thiol proteome profile in human prostate cancer cells using a BIAM‐based display method. We analyzed the kinetics of protein thiol modification by using a pattern recognition algorithm, self‐organizing maps (SOM) clustering, and identified the BIAM‐labeled proteins by MALDI‐TOF and ESI‐tandem mass spectrometry. We found 99 out of 215 of total BIAM‐labeled proteins were affected by hypoxia treatment and, yet, with diverse patterns and kinetics of redox modification. Our study proved that proteomics analysis employing the BIAM‐labeling method can provide valuable information pertaining to global changes in the redox status of proteins in response to hypoxia.

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.

Prolidase Directly Binds and Activates Epidermal Growth Factor Receptor and Stimulates Downstream Signaling

Background: All known ligands of EGF receptor (EGFR) are characterized by the EGF motif and generated from transmembrane precursors. Results: Prolidase, a cytosolic dipeptidase devoid of EGF motif, binds and activates EGFR independent of its dipeptidase activity when present outside of cell. Conclusion: Prolidase is a novel EGFR ligand. Significance: This shows a new function of prolidase and new mechanism of EGFR activation. Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.

Neural network-based analysis of thiol proteomics data in identifying potential selenium targets.

Abstract Generation of a monomethylated selenium metabolite is critical for the anticancer activity of selenium. Because of its strong nucleophilicity, the metabolite can react directly with protein thiols to cause redox modification. Here, we report a neural network‐based analysis to identify potential selenium targets. A reactive thiol specific reagent, BIAM, was used to monitor thiol proteome changes on 2D gel. We constructed a dynamic model and evaluated the relative importance of proteins mediating the cellular responses to selenium. Information from this study will provide new clues to unravel mechanisms of anticancer action of selenium. High impact selenium targets could also serve as biomarkers to gauge the efficacy of selenium chemoprevention.

Capillary-LC-μESI-MS/MS and nano-LC-nano ESI-MS/MS analysis using a single binary pump capillary LC system: Applications in proteomics

Abstract We describe the use of a single pump capillary‐LC system (flow rate: 1–100 µL/min) to perform both capillary‐LC‐µESI‐MS/MS and nano‐LC‐nano ESI‐MS/MS (flow rate: 50–1000 nL/min) analysis and its applications in proteomics. A highly constant nanoflow (180 nL/min) delivery has been achieved by using a capillary binary‐pump with electronic flow control and a flow splitter with compensation for the viscosity changes during gradient elution. A column switching technique was successfully used in nano‐LC‐nanoESI‐MS/MS analysis to provide two flow paths, one for sample loading with a flow rate of 15 µL/min directly from the capillary pump, the other for nanoflow gradient elution with a flow rate of 180 nL/min after flow splitting. In comparison with the capillary‐LC‐µESI‐MS/MS at a flow rate of 3 µL/min, this nano‐LC‐nano ESI‐MS/MS analysis improved sensitivity by more than 50 times. It has been successfully used to identify protein spots from 2DGE, as well as protein bands from 1DGE, after in‐gel tryptic digestion. Similar to any commercial nano‐LC system, this novel nano‐LC configuration can be used to perform MudPIT analysis, as well as offline capillary‐SCX/nano‐RPLC‐nanoESI‐MS/MS analysis. Our data has shown the unnecessary need for either a second pump or an expensive nano‐pump to do nano‐LC analysis as most commercial nano‐LC system requires, and the increased sensitivity, functionality, and flexibility of a simple capillary‐LC system, which is very useful to typical analytical laboratories for multiple‐purpose usages of limited resources.