Jung-Hsiang Tai

Quantitative phosphoproteomics studies using stable isotope dimethyl labeling coupled with IMAC-HILIC-nanoLC-MS/MS for estrogen-induced transcriptional regulation.

17β-Estradiol (E2) regulates transcriptional activity partly by inducing protein-kinase cascades, leading to the phosphorylation of estrogen receptors (ERs) and other functional proteins. Many of these phosphorylation events are also modulated by growth factors. To gain an insight into E2-modulated protein phosphorylation, we applied quantitative phosphoproteomics to investigate global changes in protein phosphorylation induced by E2 in MCF-7 cells. Proteomic analyses using stable isotope dimethyl labeling coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography (IMAC-HILIC) fractionation and nanoLC-MS/MS identified and quantified 2857 unique phosphorylation sites in 1338 phosphoproteins from 1 mg of total cellular protein. In addition to S118 of ERα, a 30-min E2 treatment significantly altered the status of 403 phosphorylation sites, including 112 novel sites. Interestingly, the substrate motifs for ERK1/2 were largely enriched in both the up-regulated and down-regulated phosphorylation sites. An increase in the phosphorylation on either the T202 or Y204 sites of ERK1 was observed after E2 treatment, while dual phosphorylation on both sites were not detected, implying that a feedback loop to deactivate MAPK signaling was achieved during a 30-min E2 treatment. In contrast, the PKA and CKII substrate motifs were majorly enriched among the up-regulated phosphorylation sites. Western blot analysis confirmed that E2 increased the phosphorylation level of S226 within a CKII motif of HSP90β by a factor of 2- to 3-fold without changing the total protein expression level. E2 also up-regulated phosphorylations of S255 in HSP90β and S353 within a CKII motif of HSP90α. These results indicated that E2 may modulate gene transcription by affecting the stability, function, and activity of many regulators through a HSP90 phosphorylation-mediated chaperoning process. This study, using a quantitative, multidimensional separation phosphoproteomic approach that required a relatively low amount of cells, provides new insights into the diversity, variability, and dynamic nature of the protein phosphorylation/dephosphorylation elicited by E2.

Multifarious Transcriptional Regulation of Adhesion Protein Gene ap65-1 by a Novel Myb1 Protein in the Protozoan Parasite Trichomonas vaginalis

ABSTRACT The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.

Activation of Multifarious Transcription of an Adhesion Protein ap65-1 Gene by a Novel Myb2 Protein in the Protozoan Parasite Trichomonas vaginalis

Multifarious transcription of the adhesion protein ap65-1 gene in the human pathogen, Trichomonas vaginalis, is critically regulated by the coordination of two similar but opposite oriented DNA regulatory regions, MRE-1/MRE-2r and MRE-2f, both of which are binding sites for multiple Myb-like proteins. In the present study, MRE-1/MRE-2r was demonstrated to be composed of multiple overlapping promoter elements, among which the entire region is required for growth-related ap65-1 transcription, and the 5′-MRE-1 antagonizes the suppressive activity of the 3′-MRE-2r in iron-inducible transcription. The recombinant Myb2 protein derived from a previously identified myb2 gene was demonstrated to recognize distinct sequence contexts in MRE-2r and MRE-2f, whereas Myb2 in the nuclear lysate preferentially binds to MRE-2f to MRE-2r. Iron repletion resulted in persistent repression of the myb2 gene, and temporal activation/deactivation of Myb2 promoter entry, which was also activated by prolonged iron depletion. The hemagglutinintagged Myb2 when overexpressed during iron-depleted conditions facilitated basal and growth-related ap65-1 transcription to a level that was achieved in iron-replete cells, whereas ironinducible ap65-1 transcription was abolished with knockdown of Myb2. These findings demonstrated that Myb2 is involved in activation of growth-related and iron-inducible transcription of the ap65-1 gene, possibly through differential promoter selection in competition with other Myb proteins.

Transcriptional regulation of an iron-inducible gene by differential and alternate promoter entries of multiple Myb proteins in the protozoan parasite Trichomonas vaginalis.

ABSTRACT Iron-inducible transcription of a malic enzyme gene (also reputed to be ap65-1) in Trichomonas vaginalis was previously shown to involve a Myb1 repressor and a Myb2 activator, each of which may preferentially select two closely spaced promoter sites, MRE-1/MRE-2r, which comprises overlapping promoter elements, and MRE-2f. In the present study, an iron-inducible ∼32-kDa Myb3 nuclear protein was demonstrated to bind only the MRE-1 element. Changes in the iron supply, which produced antagonistic effects on the levels of Myb2 and Myb3 expression, also resulted in temporal and alternate entries of Myb2 and Myb3 into the ap65-1 promoter. Repression or activation of basal and iron-inducible ap65-1 transcription was detected in transfected cells when Myb3 was, respectively, substantially knocked down or overexpressed. In the latter case, increased Myb3 promoter entry was detected with concomitant decrease in Myb2 promoter entry under specific conditions, while Myb3 promoter entry was inhibited under all test conditions in cells overexpressing Myb2. In contrast, concomitant promoter entries by Myb2 and Myb3 diminished in cells overexpressing Myb1, except that Myb3 promoter entry was slightly affected under prolonged iron depletion. Together, these results suggest that Myb2 and Myb3 may coactivate basal and iron-inducible ap65-1 transcription against Myb1 through conditional and competitive promoter entries.

NMR structural analysis of DNA recognition by a novel Myb1 DNA-binding domain in the protozoan parasite Trichomonas vaginalis

The transcription regulator, tvMyb1, is the first Myb family protein identified in Trichomonas vaginalis. Using an electrophoretic mobility shift assay, we defined the amino-acid sequence from Lys35 to Ser141 (tvMyb135–141) as the minimal DNA-binding domain, encompassing two Myb-like DNA-binding motifs (designated as R2 and R3 motifs) and an extension of 10 residues at the C-terminus. NMR solution structures of tvMyb135–141 show that both the R2 and R3 motifs adopt helix-turn-helix conformations while helix 6 in the R3 motif is longer than its counterpart in vertebrate Myb proteins. The extension of helix 6 was then shown to play an important role in protein stability as well as in DNA-binding activity. The structural basis for the tvMyb135–141/DNA interaction was investigated using chemical shift perturbations, residual dipolar couplings, DNA specificity data and data-driven macromolecular docking by HADDOCK. Our data indicate that the orientation between R2 and R3 motifs dramatically changes upon binding to DNA so as to recognize the DNA major groove through a number of key contacts involving residues in helices 3 and 6. The tvMyb135–141/DNA complex model furthers our understanding of DNA recognition by Myb proteins and this approach could be applied in determining the complex structures involving proteins with multiple domains.

Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T. vaginalis

SummaryThe 4.6-kb double-stranded (ds) RNA of Trichomonas vaginalis virus (TVV)-T1 has been shown to encode two overlapping genes, cap and pol. In this study, a serum for specifically detecting viral cap gene product was raised against a recombinant protein, and sera for specifically detecting pol gene product were raised against synthetic oligopeptides. A 75-kDa major protein and a 160-kDa minor protein were detected by anti-CAP serum in a TVV-T1 sample, indicating that the 75-kDa protein is the viral capsid protein. The 160-kDa protein alone was also detected by two distinct anti-POL sera, indicating that the pol gene is expressed as a CAP-POL fusion protein. These results suggest that the TVV-T1 genome is arranged into a cap-pol organization in a manner similar to that of viruses in family Totiviridae.

Involvement of multiple DNA elements in iron‐inducible transcription of the ap65‐1 gene in the protozoan parasite Trichomonas vaginalis

A reputed iron‐responsive region, which contains multiple nuclear protein‐binding DNA sequences, was shown previously to regulate iron‐inducible transcription of the ap65‐1 gene in the protozoan pathogen, Trichomonas vaginalis. These DNA sequences include two overlapping MYB recognition elements (MRE‐1/MRE‐2r) and three abutted T‐tract elements. Additional nuclear protein‐binding DNA sequences flanking the 5′ (AGTGAAGTGA) and 3′ (MRE‐2f) of the iron‐responsive region were identified in the present study. A stable promoter assay and primer extension revealed that transcriptional activity of the ap65‐1 promoter is iron inducible as well as growth related, being lowest in the early logarithmic phase and highest in the mid‐logarithmic phase. Subsequent mutational analysis of individual DNA elements of the ap65‐1 promoter suggests that closely spaced T‐tract elements together with an intervening GAAGGAAG sequence within the iron‐responsive region are most critical for regulation of overall transcriptional activity, whereas an additional AGTGAAGTGA and MRE‐2f together with an upstream T‐rich region are required for optimal iron‐inducible activity, and the MRE‐1/MRE‐2r overlap is only involved in growth‐related activity. These observations suggest that expression of the ap65‐1 gene is dynamically regulated under various growth conditions via interactions among multiple DNA regulatory elements.

Identification and characterization of a type III Trichomonas vaginalis virus in the protozoan pathogen Trichomonas vaginalis

A type III Trichomonas vaginalis virus, which may be involved in transcriptional regulation of the major surface protein gene P270 of the protozoan pathogen Trichomonas vaginalis, was purified and characterized in the present study. The complete 4844-base-pair complementary DNA sequence of the viral genome reveals overlapping cap and pol genes with a putative ribosomal frame-shifting signal within the overlap region. The type III virus is related more closely to the type II virus than to the type I virus in the sequence of its ribosomal frameshift signal and in its capsid protein. Phylogenetic analysis revealed that these viruses could be grouped in the same clade as a genus distantly related to other genera in the family Totiviridae. Virus-induced P270 gene expression was only evident in Trichomonas vaginalis cells infected with either a type II or type III virus, but not with a type I virus. These findings suggest that transcription of the P270 gene is likely regulated by viral factors common to type II and type III viruses and thus provides important information for future investigation of virus-host interactions.

Molecular basis of the recognition of the ap65-1 gene transcription promoter elements by a Myb protein from the protozoan parasite Trichomonas vaginalis

Iron-inducible transcription of the ap65-1 gene in Trichomonas vaginalis involves at least three Myb-like transcriptional factors (tvMyb1, tvMyb2 and tvMyb3) that differentially bind to two closely spaced promoter sites, MRE-1/MRE-2r and MRE-2f. Here, we defined a fragment of tvMyb2 comprising residues 40–156 (tvMyb240–156) as the minimum structural unit that retains near full binding affinity with the promoter DNAs. Like c-Myb in vertebrates, the DNA-free tvMyb240–156 has a flexible and open conformation. Upon binding to the promoter DNA elements, tvMyb240–156 undergoes significant conformational re-arrangement and structure stabilization. Crystal structures of tvMyb240–156 in complex with promoter element-containing DNA oligomers showed that 5′-a/gACGAT-3′ is the specific base sequence recognized by tvMyb240–156, which does not fully conform to that of the Myb binding site sequence. Furthermore, Lys49, which is upstream of the R2 motif (amino acids 52–102) also participates in specific DNA sequence recognition. Intriguingly, tvMyb240–156 binds to the promoter elements in an orientation opposite to that proposed in the HADDOCK model of the tvMyb135–141/MRE-1-MRE-2r complex. These results shed new light on understanding the molecular mechanism of Myb–DNA recognition and provide a framework to study the molecular basis of transcriptional regulation of myriad Mybs in T. vaginalis.

Characterization of a bi-directional promoter for divergent transcription of a PHD-zinc finger protein gene and a ran gene in the protozoan pathogen Giardia lamblia

We showed previously that transcription of the ran gene in Giardia lamblia is regulated by an AT‐rich initiator. In the present study, the ran initiator was found to regulate transcription of a neighbouring PHD zinc‐finger protein gene. Deletion and scanning mutagenesis of the phd promoter in a firefly luciferase reporter system showed that the promoter activity is determined by multiple single‐stranded T‐tract DNA elements distributed into a distal domain spanning the ran initiator (–134/–103) and a proximal domain (–88/–48) spanning phd messenger RNA (mRNA) start sites (–74, –55 and –53 relative to the first ATG). The promoter activity is repressed by the single T‐tract element on a non‐template strand of the ran initiator, and is activated by closely spaced T‐tract elements on the opposite strand. The T‐tract elements in the phd and ran initiators compete for similar ssDNA binding proteins. Mutation of –47/–42 resulted in dramatic reduction of luciferase activity without changing luciferase mRNA levels, indicating the potential involvement of a regulatory mechanism in PHD protein translation. These findings suggest that G. lamblia uses multiple copies of a T‐tract element as both core and distal elements in regulating transcription initiation, and that expression of the phd gene is regulated at multiple levels.