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author:("Yang, youyang")
1.  Manganese and Zinc Regulate Virulence Determinants in Borrelia burgdorferi 
Infection and Immunity  2013;81(8):2743-2752.
Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn2+) and zinc (Zn2+), that influenced regulation of RpoS. The intracellular Mn2+ level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn2+ repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn2+. Conversely, a decrease of intracellular Mn2+ by deletion of the Mn2+ transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn2+ affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn2+ levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn2+ and Zn2+ appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn2+ and Zn2+ play a critical role in regulation of virulence in bacteria.
doi:10.1128/IAI.00507-13
PMCID: PMC3719580  PMID: 23690398
2.  Role of eIF3a in regulating cisplatin sensitivity and nucleotide excision repair of nasopharyngeal carcinomas 
Oncogene  2011;30(48):4814-4823.
Translational control at the initiation step has been recognized as a major and important regulatory mechanism of gene expression. eIF3a, a putative subunit of eIF3 complex, has recently been shown to play an important role in regulating translation of a subset of mRNAs and found to correlate with prognosis of cancers. In this study, using nasopharyngeal carcinoma (NPC) cells as a model system we tested the hypothesis that eIF3a negatively regulates synthesis of nucleotide excision repair (NER) proteins and, thus, NER activities and cellular response to treatments with DNA damaging agents such as cisplatin. We found that a cisplatin-sensitive subclone S16 isolated from a NPC cell line CNE2 via limited dilution has increased eIF3a expression. Knocking down its expression in S16 cells increased cellular resistance to cisplatin, NER activity, and synthesis of NER proteins XPA, XPC, RAD23B, and RPA32. Altering eIF3a expression also changed cellular response to cisplatin and UV treatment in other NPC cell lines. Taken together, we conclude that eIF3a plays an important role in cisplatin response and NER activity of nasopharyngeal carcinomas by suppressing synthesis of NER proteins.
doi:10.1038/onc.2011.189
PMCID: PMC3165083  PMID: 21625209
cisplatin sensitivity; eIF3a; nasopharyngeal carcinoma; nucleotide excision repair; translational control
3.  Role of Transmembrane Segment 5 and Extracellular Loop 3 in the Homodimerization of Human ABCC1† 
Biochemistry  2010;49(51):10854-10861.
Resistance to multiple anticancer agents is a major obstacle in the successful treatment of cancers. Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCC1 has been shown to be a major contributor of multidrug resistance (MDR) in both laboratory cell line models and the clinical setting. ABCC1 has been thought to function as a homodimer with a putative dimerization domain located in the first 281 amino acid residues, including MSD0 and L0 domains. In this study, we further mapped in detail the dimerization site and placed it in TM5 and ECL3 in MSD0 using co-expression and co-immunoprecipitation of a series of deletion constructs. TM5 and ECL3 in one subunit appear to interact with TM5 and ECL3 in the opposing subunit in a sequence-independent manner, but their physical location together with the hydrophobicity of TM5 and the length of ECL3 appears to be important contributors to the dimerization ability of ABCC1.
doi:10.1021/bi101350x
PMCID: PMC3095655  PMID: 21090806
4.  BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation 
Biochimica et biophysica acta  2010;1797(11):1795-1806.
BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state.
doi:10.1016/j.bbabio.2010.07.006
PMCID: PMC2933961  PMID: 20655869
mitochondria; calcium; BAX; BID; Bcl-xL; permeability transition
5.  Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers 
Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.
PMCID: PMC3039422  PMID: 21331354
Human fatty acid synthase (FASN); palmitate; gemcitabine; radiation treatments; treatment resistance; pancreatic cancers
6.  Role of fatty acid synthase in gemcitabine and radiation resistance of pancreatic cancers 
Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.
PMCID: PMC3039422  PMID: 21331354
Human fatty acid synthase (FASN); palmitate; gemcitabine; radiation treatments; treatment resistance; pancreatic cancers
7.  Characterization and analyses of multidrug resistance-associated protein 1 (MRP1/ABCC1) polymorphisms in Chinese population 
Pharmacogenetics and genomics  2009;19(3):206-216.
Multidrug resistance (MDR) is one of the major obstacles for successful cancer chemotherapy. Over-expression of ATP-binding cassette (ABC) transporters such as MRP1/ABCC1 has been suggested to cause MDR. In this study, we explored the distribution frequencies of four common single nucleotide polymorphisms (SNPs) of MRP1/ABCC1 in a mainland Chinese population and investigated whether these SNPs affect the expression and function of the MRP1/ABCC1. We found that the allelic frequencies of Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A) and Arg1058Gln (3173G>A) in mainland Chinese were 0.5%, 1.4%, 5.8% and 0.5%, respectively. These four SNPs were recreated by site-directed mutagenesis and tested for their effect on MRP1/ABCC1 expression and MDR function in HEK293 and CHO-K1 cells lines. We found that none of these mutations had any effect on MRP1/ABCC1 expression and trafficking, but that Arg723Gln mutation significantly reduced MRP1/ABCC1-mediated resistance to daunorubicin, doxorubicin, etoposide, vinblastine and vincristine. The Cys43Ser mutation did not affect all tested drugs resistance. On the other hand, the Thr73Ile mutation reduced resistance to methotrexate and etoposide while the Arg1058Gln mutation increased the response of two anthracycline drugs and etoposide in HEK293 and CHO-K1 cells as well as vinblastine and methotrexate in CHO-K1 cells. We conclude that the allelic frequency of the Arg723Gln mutation is relatively higher than other SNPs in mainland Chinese population and therefore this mutation significantly reduces MRP1/ABCC1 activity in MDR.
doi:10.1097/FPC.0b013e328323f680
PMCID: PMC2667206  PMID: 19214144
ABC transporter; MDR; Multidrug resistance-associated protein 1 (MRP1/ABCC1); drug resistance; genetic polymorphism
8.  Effect of cysteine mutagenesis on the function and disulfide bond formation of human ABCG2 
ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily. Its over-expression causes multidrug resistance in cancer chemotherapy. Based on its apparent half size in sequence when compared to other traditional ABC transporters, ABCG2 has been thought to exist and function as a homodimer linked by inter-molecular disulfide bonds. However, recent evidence suggests that ABCG2 may exist as a higher form of oligomers due to non-covalent interactions. In this study, we attempted to create a cysless mutant ABCG2 as a tool for further characterization of this molecule. We found, however, that the cysless mutant ABCG2 is well expressed but not functional. Mapping of the cysteine residues showed that three cysteine residues (C284, C374, and C438) are required concurrently for the function of ABCG2 and potentially for intra-molecular disulfide bond formation. We also found that the cysteine residues (C592, C603, and C608) in the third extracellular loop are involved in forming inter-molecular disulfide bonds and that mutation of these residues does not affect the expression or drug transport activity of human ABCG2. Thus, we conclude that C284, C374, and C438, which may be involved in intra-molecular disulfide bond formation, are concurrently required for ABCG2 function whereas C592, C603, and C608, potentially involved in inter-molecular disulfide bond formation, are not required.
doi:10.1124/jpet.108.138115
PMCID: PMC2632310  PMID: 18430864
9.  A Novel Two Mode-Acting Inhibitor of ABCG2-Mediated Multidrug Transport and Resistance in Cancer Chemotherapy 
PLoS ONE  2009;4(5):e5676.
Background
Multidrug resistance (MDR) is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.
Methods/Preliminary Findings
Using rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl)-2-[(6-{[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-yl)sulfanyl]acetamide), that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.
Conclusion/Significance
Unlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.
doi:10.1371/journal.pone.0005676
PMCID: PMC2682573  PMID: 19479068
10.  Regulation of expression by promoters versus internal ribosome entry site in the 5′-untranslated sequence of the human cyclin-dependent kinase inhibitor p27kip1 
Nucleic Acids Research  2005;33(12):3763-3771.
p27kip1 regulates cell proliferation by binding to and inhibiting the activity of cyclin-dependent kinases and its expression oscillates with cell cycle. Recently, it has been suggested from studies using the traditional dicistronic DNA assay that the expression of p27kip1 is regulated by internal ribosome entry site (IRES)-mediated translation initiation, and several RNA-binding protein factors were thought to play some role in this regulation. Considering the inevitable drawbacks of the dicistronic DNA assay, which could mislead a promoter activity or alternative splicing to IRES as previously demonstrated, we decided to reanalyze the 5′-untranslated region (5′-UTR) sequence of p27kip1 and test whether it contains an IRES element or a promoter using more stringent methods, such as dicistronic RNA and promoterless dicistronic and monocistronic DNA assays. We found that the 5′-UTR sequence of human p27kip1 does not have any significant IRES activity. The previously observed IRES activities are likely generated from the promoter activities present in the 5′-UTR sequences of p27kip1. The findings in this study indicate that transcriptional regulation likely plays an important role in p27kip1 expression, and the mechanism of regulation of p27 expression by RNA-binding factors needs to be re-examined. The findings in this study also further enforce the importance that more stringent studies, such as promoterless dicistronic and monocistronic DNA and dicistronic RNA tests, are required to safeguard any future claims of cellular IRES.
doi:10.1093/nar/gki680
PMCID: PMC1174905  PMID: 16006622

Results 1-10 (10)