SB1 Proteomic Analysis of Manganese Regulation of Neisseria gonorrhoeae

Hsing-Ju Wu1, Kuan-Tin Pan1, He-Hsuan Hsiao1, Chen-Wen Yao3, Alastair G. McEwan2, Michael P. Jennings2 and Andrew H-J. Wang1

1Core Facilities for Proteomics Research, Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2School of Molecular and Microbial Sciences & Centre for Metals in Biology, The University of Queensland, Brisbane, Australia; 3Chinese Herbal Medicine Research Center, Tri-Service General Hospital, Taipei, Taiwan

 

Neisseria gonorrhoeae is an important human pathogen which causes gonorrhoea and pelvic inflammatory disease. It is a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain. These factors would suggest that this bacterium would require defense systems to respond to toxic oxygen species. In the previous studies, we have shown that the accumulation of manganese (Mn) and Mn(II) uptake system, MntABC, in N. gonorrhoeae protected against killing by superoxide anion, and was independent of superoxide dismutase activity. Also, investigation of a regulatory role for Mn(II) in N. gonorrhoeae has revealed that a key virulence factor, pili, is repressed by Mn via a PerR-independent post-transcriptional mechanism. To provide a more comprehensive view of the regulatory network and its molecular mechanism, the shotgun proteomic approach, i.e. one dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with 1D liquid chromatography (LC) - tandem mass spectrometry (MS/MS) and the quantitative method, i.e., isotope coded affinity tag (ICAT) were performed. N. gonorrhoeae cells were grown in the presence and absence of Mn and cell lysates were fractionated into cytoplasmic, inner membrane and outer membrane components. These results revealed that 109 proteins were differentially regulated at the post transcriptional level under conditions of increased or decreased Mn. The Mn-regulated proteins have a broad range of functions including oxidative stress defence (i.e. superoxide dismutase, azurin, bacterioferritin), cellular metabolism, protein synthesis, RNA processing, cell division, pilin and the proteins involved in the pilus assembly, such as PilC1 and PilQ. This confirms our previous study and may explain how the expression of pili was downregulated when cells were grown in the Mn supplement. Taken together, these data give us a proteomic view of Mn regulation and provided us with leads to correlate protection against oxidative stress with pilus formation and surface protein expression.

 

 

SB2 Localizomics study in TPA-induced HL-60 cells differentiation into macrophages.

Yu-Wei Chang1, Shing-Chuan Chang2, Hsueh-Fen Juan1,2*

1Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan
2Department of Life Science, National Taiwan University, Taipei, Taiwan

 

The human myeloid leukemia cell line HL-60 is a useful model to study the molecular mechanisms of differentiation therapy in M3 subtype acute promyelocytic leukemia (APL) and hematopoietic development of macrophage in decades. 12-O-tetradecanoy-Phorbol-13-Acetate (TPA), acting via its cellular receptor protein kinase C (PKC), induce HL-60 cells to differentiate into macrophages and lose their proliferative activity. Many approaches such as DNA microarray and proteomics study have been used to find out what kinds of mechanisms are involved in the TPA-induced HL-60 differentiation process. Here we show the results of localizomics study, a very important part of proteomics study, by extracting cytoplasimc and membrane proteins from TPA-treated HL-60 cells and then following by 2DGE and mass spectrometry techniques. We found that three down-regulated and four up-regulated proteins localized in cytoplasm and eight up-regulated proteins localized in membrane. Using transmembrane domain prediction programs and subcellular localization prediction program, pWolf, 60% proteins are found in mitochondria as non-transmembrane proteins, and 40% proteins are found in lysosome and ER as non-transmembrane and transmembrane proteins, respectively. As the results showed above, we suggest that proteins in mitochondria may play an important role in the process of differentiation.