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1.  Human ABCG2: structure, function, and its role in multidrug resistance 
Human ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily and is known to contribute to multidrug resistance (MDR) in cancer chemotherapy. Among ABC transporters that are known to cause MDR, ABCG2 is particularly interesting for its potential role in protecting cancer stem cells and its complex oligomeric structure. Recent studies have also revealed that the biogenesis of ABCG2 could be modulated by small molecule compounds. These modulators, upon binding to ABCG2, accelerate the endocytosis and trafficking to lysosome for degradation and effectively reduce the half-life of ABCG2. Hence, targeting ABCG2 stability could be a new venue for therapeutic discovery to sensitize drug resistant human cancers. In this report, we review recent progress on understanding the structure, function, biogenesis, as well as physiological and pathophysiological functions of ABCG2.
PMCID: PMC3325772  PMID: 22509477
Human ABCG2; structure; function; multidrug resistance; ATP-binding cassette; cancer; chemotherapy
2.  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
3.  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
4.  Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker 
Human fatty acid synthase (FASN) is a 270-kDa cytosolic dimeric enzyme that is responsible for palmitate synthesis. FASN is slowly emerging and rediscovered as a marker for diagnosis and prognosis of human cancers. Recent studies showed that FASN is an oncogene and inhibition of FASN effectively and selectively kill cancer cells. With recent publications of the FASN crystal structure and the new development of FASN inhibitors, targeting FASN opens a new window of opportunity for metabolically combating cancers. In this article, we will review critically the recent progresses in understanding the structure, function, and the role of FASN in cancers and pharmacologically targeting FASN for human cancer treatment.
PMCID: PMC2919769  PMID: 20706604
Fatty acid synthase; inhibitors; drug resistance; cancer; prognosis; diagnosis
5.  Biochemistry, molecular biology, and pharmacology of fatty acid synthase, an emerging therapeutic target and diagnosis/prognosis marker 
Human fatty acid synthase (FASN) is a 270-kDa cytosolic dimeric enzyme that is responsible for palmitate synthesis. FASN is slowly emerging and rediscovered as a marker for diagnosis and prognosis of human cancers. Recent studies showed that FASN is an oncogene and inhibition of FASN effectively and selectively kill cancer cells. With recent publications of the FASN crystal structure and the new development of FASN inhibitors, targeting FASN opens a new window of opportunity for metabolically combating cancers. In this article, we will review critically the recent progresses in understanding the structure, function, and the role of FASN in cancers and pharmacologically targeting FASN for human cancer treatment.
PMCID: PMC2919769  PMID: 20706604
Fatty acid synthase; inhibitors; drug resistance; cancer; prognosis; diagnosis
6.  14-3-3σ, the double-edged sword of human cancers 
14-3-3σ is a member of a highly conserved family of 14-3-3 proteins that are present in all eukaryotic organisms. 14-3-3σ has been considered as a tumor suppressor with reduced expression in some human cancers while its increased expression causes resistance to anticancer agents and radiation that cause DNA damages. The increased expression of 14-3-3σ may also predict poor prognosis in some human cancers. Thus, 14-3-3σ may play an important role as a double-edged sword in human cancers, which may attribute to its property as a molecular chaperone by binding to various protein ligands important to many cellular processes such as cell cycle checkpoint regulation and apoptosis in response to DNA damages. In this article, we will review recent studies and progresses in understanding 14-3-3σ as a double-edged sword in human cancers.
PMCID: PMC2780041  PMID: 19956445
14-3-3σ; tumorigenesis; metastasis; prognosis; drug resistance; expression regulation
7.  EIF3i Promotes Colon Oncogenesis by Regulating COX-2 Protein Synthesis and β-Catenin Activation 
Oncogene  2013;33(32):4156-4163.
SUMMARY
Translational control of gene expression has recently been recognized as an important mechanism controlling cell proliferation and oncogenesis and it mainly occurs in the initiation step of protein synthesis that involves multiple eukaryotic initiation factors (eIFs). Many eIFs have been found to have aberrant expression in human tumors and the aberrant expression may contribute to oncogenesis. However, how these previously considered house-keeping proteins are potentially oncogenic remains elusive. In this study, we investigated the expression of eIF3i in human colon cancers, tested its contribution to colon oncogenesis, and determined the mechanism of eIF3i action in colon oncogenesis. We found that eIF3i expression was up-regulated in both human colon adenocarcinoma and adenoma polyps as well as in model inducible colon tumorigenic cell lines. Over-expression of ectopic eIF3i in intestinal epithelial cells causes oncogenesis by directly up-regulating synthesis of COX-2 protein and activates the β-catenin/TCF4 signaling pathway that mediates the oncogenic function of eIF3i. Together, we conclude that eIF3i is a proto-oncogene that drives colon oncogenesis by translationally up-regulating COX-2 and activating β-catenin signaling pathway. These findings imply that protooncogenic eIFs likely exert their tumorigenic function by regulating/altering the synthesis level of down-stream tumor suppressor or oncogenes.
doi:10.1038/onc.2013.397
PMCID: PMC3962800  PMID: 24056964
eIF3i; COX-2; β-catenin; translational control; colon cancer; RNA-binding
8.  Over-expression of asparagine synthetase and matrix metalloproteinase 19 confers cisplatin sensitivity in nasopharyngeal carcinoma cells 
Molecular cancer therapeutics  2013;12(10):2157-2166.
Platinum-based concurrent chemo-radiotherapy is considered a standard treatment approach for locoregionally advanced nasopharyngeal carcinoma (NPC). However, only a minority of patients benefit from this treatment regimen compared to radiotherapy alone. Identification of a set of molecular markers predicting sensitivity of platinum-based chemotherapy may contribute to personalized treatment of NPC patients for better clinical outcome with less toxicity. Previously, we generated a cisplain sensitive NPC cell line, S16, by clonal selection from CNE-2 cells and found that eIF3a is up-regulated and contributes to cisplatin sensitivity by down-regulating the synthesis of NER proteins. In this study, we conducted a gene expression profiling analysis and found three other genes, asparagine synthetase (ASNS), choriogonadotropin α subunit (CGA), and matrix metalloproteinase 19 (MMP19), that are up-regulated in the cisplatin-sensitive S16 cells compared with the CNE-2 cells. However, only ASNS and MMP19, but not CGA, contributes to cisplatin sensitivity by potentiating cisplatin-induced DNA damage and apoptosis. Thus, ASNS and MMP19, along with eIF3a, are sensitivity factors for cisplatin treatment and may serve as potential candidate molecular markers for predicting cisplatin sensitivity of advanced nasopharyngeal carcinoma.
doi:10.1158/1535-7163.MCT-12-1190
PMCID: PMC3795908  PMID: 23956056
9.  Translational regulation of RPA2 via internal ribosomal entry site and by eIF3a 
Carcinogenesis  2013;34(6):1224-1231.
RPA2 is a subunit of a trimeric replication protein A (RPA) complex important for DNA repair and replication. Although it is known that RPA activity is regulated by post-translational modification, whether RPA expression is regulated and the mechanism therein is currently unknown. eIF3a, the largest subunit of eIF3, is an important player in translational control and has been suggested to regulate translation of a subset of messenger RNAs important for tumorigenesis, metastasis, cell cycle progression, drug response and DNA repair. In the present study, we show that RPA2 expression is regulated at translational level via internal ribosome entry site (IRES)-mediated initiation in response to DNA damage. We also found that eIF3a suppresses RPA2 synthesis and inhibits its cellular IRES activity by directly binding to the IRES element of RPA2 located at −50 to −150 bases upstream of the translation start site. Taken together, we conclude that RPA2 expression is translationally regulated via IRES and by eIF3a and that this regulation is partly accountable for cellular response to DNA damage and survival.
doi:10.1093/carcin/bgt052
PMCID: PMC3670257  PMID: 23393223
10.  Critical Residue That Promotes Protein Dimerization: A Story of Partially Exposed Phe25 in 14-3-3σ 
Many proteins exist and function as oligomers. While hydrophobic interactions have been recognized as the major driving force for oligomerization, detailed molecular mechanisms for the assembly are unknown. Here, we used 14-3-3σ as a model protein and investigated the role of hydrophobic residues at the dimeric interface using MD simulations and coimmunoprecipitations. We found that a half-exposed and half-buried residue in the interface, Phe25, plays a more important role in promoting homodimerization than the hydrophobic core residues by organizing both favorable hydrophobic and hydrophilic interactions. Phe25 is critical in packing and stabilizing hydrophobic core residues. We conclude that the structural stability of hydrophobic cores is critical for a stable homodimer complex and this stable property can be bestowed by residues outside of hydrophobic core. The important organizing activity of Phe25 for homodimerization of 14-3-3σ originates from its unique physical location, rigidity, size, and hydrophobicity. Thus, hydrophobic residues that are not deeply buried at the oligomeric interface may play important but different roles from the buried core residues and they may promote oligomerization by organizing co-operativity of core and other residues for favorable hydrophobic and electrostatic interactions.
doi:10.1021/ci200212y
PMCID: PMC3322420  PMID: 21870863
11.  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
12.  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
13.  Identification of novel small molecule inhibitors of the XPA protein using in silico based screening 
ACS chemical biology  2010;5(10):953-965.
The nucleotide excision repair pathway catalyzes the removal of bulky adduct damage from DNA and requires the activity of more than 30 individual proteins and complexes. A diverse array of damage can be recognized and removed by the NER pathway including UV-induced adducts and intrastrand adducts induced by the chemotherapeutic compound cisplatin. The recognition of DNA damage is complex and involves a series of proteins including the xeroderma pigmentosum group A and C proteins and the UV-damage DNA binding protein. The xeroderma pigmentosum group A protein is unique in the sense that it is required for both transcription coupled and global genomic nucleotide excision repair. In addition, xeroderma pigmentosum group A protein is required for the removal of all types of DNA lesions repaired by nucleotide excision repair. Considering its importance in the damage recognition process, the minimal information available on the mechanism of DNA binding and the potential that inhibition of xeroderma pigmentosum group A protein could enhance the therapeutic efficacy of platinum based anticancer drugs, we sought to identify and characterize small molecule inhibitors of the DNA binding activity of the xeroderma pigmentosum group A protein. In silico screening of a virtual small molecule library resulted in the identification of a class of molecules confirmed to inhibit the xeroderma pigmentosum group A protein-DNA interaction. Biochemical analysis of inhibition with varying DNA substrates revealed a common mechanism of xeroderma pigmentosum group A protein DNA binding to single-stranded DNA and cisplatin-damaged DNA.
doi:10.1021/cb1000444
PMCID: PMC2955790  PMID: 20662484
14.  Dynamic vs Static ABCG2 Inhibitors to Sensitize Drug Resistant Cancer Cells 
PLoS ONE  2010;5(12):e15276.
Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in multidrug resistance and protecting cancer stem cells. ABCG2-knockout had no apparent adverse effect on the development, biochemistry, and life of mice. Thus, ABCG2 is an interesting and promising target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and for eliminating cancer stem cells. Previously, we reported a novel two mode-acting ABCG2 inhibitor, PZ-39, that induces ABCG2 degradation in addition to inhibiting its activity. In this manuscript, we report our recent progresses in identifying two different groups of ABCG2 inhibitors with one inhibiting only ABCG2 function (static) and the other induces ABCG2 degradation in lysosome in addition to inhibiting its function (dynamic). Thus, the inhibitor-induced ABCG2 degradation may be more common than we previously anticipated and further investigation of the dynamic inhibitors that induce ABCG2 degradation may provide a more effective way of sensitizing ABCG2-mediated MDR in cancer chemotherapy.
doi:10.1371/journal.pone.0015276
PMCID: PMC2998423  PMID: 21151870
15.  Role of 14-3-3σ in poor prognosis and in radiation and drug resistance of human pancreatic cancers 
BMC Cancer  2010;10:598.
Background
Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3σ mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis.
Methods
Western blot analysis was used to determine 14-3-3σ protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3σ was established and the effect of 14-3-3σ over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays.
Results
We found that 14-3-3σ protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3σ protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3σ in a pancreatic cancer cell line caused resistance to γ-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest.
Conclusion
The increased level of 14-3-3σ protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3σ may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients.
doi:10.1186/1471-2407-10-598
PMCID: PMC2991307  PMID: 21040574
17.  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
18.  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
19.  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
20.  Use of comparative proteomics to identify potential resistance mechanisms in cancer treatment 
Cancer treatment reviews  2007;33(8):741-756.
Drug resistance is a major problem in successful cancer chemotherapy. Many molecular mechanisms that are responsible for drug resistance are known whereas others have yet to be discovered. Determining the exact mechanism activated in a particular case (clinical or laboratory) is a difficult task. Recently, proteomics has been applied to investigate drug resistance mechanisms in model cancer cell lines. As a result, novel mechanisms of resistance have been discovered and known mechanisms of resistance confirmed. In this paper, we wish to review recent developments and progresses in the application of proteomic tools to identify known and novel drug resistance mechanisms in drug-selected model cancer cell lines. Our combined analyses of multiple proteomic studies of various drug resistant cancer cell lines revealed that many mechanisms of resistance likely exist in any given drug-selected cancer cell line and that common mechanisms of resistance may be selected in a spectrum of cancer cell lines. These observations suggest that combination therapies targeting multiple mechanisms to sensitize drug resistant cancers may be necessary to eradicate cancers in the future.
doi:10.1016/j.ctrv.2007.07.018
PMCID: PMC2203306  PMID: 17854999
proteomics; 2-dimensional gel electrophoresis; mass spectrometry; drug resistance; cancer chemotherapy
21.  Dimerization of Human XPA and Formation of XPA2-RPA Protein Complex† 
Biochemistry  2002;41(43):13012-13020.
XPA plays an important role in the DNA damage recognition during human nucleotide excision repair. Here we report that the XPA is a homodimer either in the free state or as a complex with human RPA in solution under normal conditions. The human XPA protein purified from baculovirus-infected sf21 insect cells has a molecular mass of 36 317 Da, as determined by mass spectroscopy. However, the apparent molecular mass of XPA determined by the native gel filtration chromatography was about 71 kDa, suggesting that XPA is a dimer. This observation was supported by a native PFO-PAGE and fluorescence spectroscopy analysis. XPA formed a dimer (XPA2) in a broad range of XPA and NaCl concentrations, and the dimerization was not due to the disulfide bond formation. Furthermore, a titration analysis of the binding of XPA to the human RPA indicated that it was the XPA2 that formed the complex with RPA. Finally, the difference between the mass spectrometric and the calculated masses of XPA implies that the protein contains posttranslational modifications. Taken together, our data suggest that the dimerization of XPA may play an important role in the DNA damage recognition of nucleotide excision repair.
PMCID: PMC1450105  PMID: 12390028
22.  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
23.  Regulation of ribonucleotide reductase M2 expression by the upstream AUGs 
Nucleic Acids Research  2005;33(8):2715-2725.
Ribonucleotide reductase catalyzes a rate-limiting reaction in DNA synthesis by converting ribonucleotides to deoxyribonucleotides. It consists of two subunits and the small one, M2 (or R2), plays an essential role in regulating the enzyme activity and its expression is finely controlled. Changes in the M2 level influence the dNTP pool and, thus, DNA synthesis and cell proliferation. M2 gene has two promoters which produce two major mRNAs with 5′-untranslated regions (5′-UTRs) of different lengths. In this study, we found that the M2 mRNAs with the short (63 nt) 5′-UTR can be translated with high efficiency whereas the mRNAs with the long (222 nt) one cannot. Examination of the long 5′-UTR revealed four upstream AUGs, which are in the same reading frame as the unique physiological translation initiation codon. Further analysis demonstrated that these upstream AUGs act as negative cis elements for initiation at the downstream translation initiation codon and their inhibitory effect on M2 translation is eIF4G dependent. Based on the findings of this study, we conclude that the expression of M2 is likely regulated by fine tuning the translation from the mRNA with a long 5′-UTR during viral infection and during the DNA replication phase of cell proliferation.
doi:10.1093/nar/gki569
PMCID: PMC1097769  PMID: 15888728
24.  EIF3 p170, a Mediator of Mimosine Effect on Protein Synthesis and Cell Cycle Progression 
Molecular Biology of the Cell  2003;14(9):3942-3951.
l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influence translation initiation. P170, a putative subunit of eIF3, has been suggested to be nonessential for eIF3 function to form preinitiation complexes and it may function as a regulator for translation of a subset of mRNAs. In this article, we tested this hypothesis and investigated whether eIF3 p170 mediates mimosine effect on mRNA translation. We found that p170 translation was dramatically reduced by mimosine due to its iron-chelating function. The decreased expression of p170 by mimosine caused diminished de novo synthesis of tyrosinated α-tubulin and elevated translation of p27 before cell cycle arrest. These observations suggest that p170 is likely an early response gene to mimosine treatment and a mediator for mimosine effect on mRNA translation. The effect of p170 on the synthesis of tyrosinated α-tubulin and p27 in a reciprocal manner also suggests that p170 functions as a regulator for mRNA translation.
doi:10.1091/mbc.E02-12-0784
PMCID: PMC196594  PMID: 12972576
25.  Regulation of Gene Expression by Internal Ribosome Entry Sites or Cryptic Promoters: the eIF4G Story 
Molecular and Cellular Biology  2002;22(21):7372-7384.
As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5′ untranslated region (5′-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5′-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5′-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPβ. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5′-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5′-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.
doi:10.1128/MCB.22.21.7372-7384.2002
PMCID: PMC135655  PMID: 12370285

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