Rapamycin is an immunosuppressant drug used to prevent organ rejection in transplant patients. In this study, we investigated the metabolic effects of rapamycin in an obese animal model, KK/HlJ mice. Mice were treated with a daily intraperitoneal injection of rapamycin at 2 mg/kg or vehicle for 42 days on a high-fat diet. Treated mice lost body weight and adiposity, reduced weight gain and retroperitoneal and epididymal fat pads/body weight, decreased serum leptin and plasma triglyceride levels and had lower liver fat concentration. However, treated mice had higher serum insulin levels and food intake. Dissection of rapamycin-treated mice revealed a marked reduction in fatty liver scores and fat cell size in retroperitoneal and epididymal adipocytes. Moreover, Western blot analysis revealed that rapamycin treatment resulted in decreasing adipophilin expression, as a marker of lipid accumulation, and reducing phosphorylation of mTOR downstream targets S6K1 compared to control group. Unfortunately, rapamycin-treated animals showed a marked decline in glucose tolerance as judged by the 180-min. area under the curve for plasma glucose levels, paralleled by increased generation of plasma reactive oxygen species. These results suggest that continual rapamycin administration may help to prevent diet-induced obesity, while prolonged use of rapamycin may exacerbate glucose intolerance.The mammalian target of the rapamycin (mTOR) signalling pathway performs an important function in the regulation of cell growth, proliferation and nutrient signals [1,2]. Recent studies have demonstrated that mTOR is involved in specific pathological responses including obesity, diabetes, and cancer [2,3]. Consistent with the development of these diseases, the activation of the mTOR pathway is evident in insulin-resistant obese rats maintained on a high-fat diet [4].Rapamycin (also known as sirolimus), an anti-fungal macrolide, is a uniquely specific mTOR kinase inhibitor [5]. Rapamycin blocks mTOR Complex1 (mTORC1) function by forming a gain-of-function inhibitory complex with the immunophilin FK506 binding protein 1A (FKBP12) that inhibits progression through involvement in the G1 phase of the cell cycle [6]. Subsequently, rapamycin was shown to have potent immunosuppressive and antiproliferative effects [7]. As an immunosuppressant drug, it is used to prevent organ rejection following kidney, liver, and heart transplants [8,9]. In addition, because of its antiproliferative effects, rapamycin and its analogues have been shown to be effective and novel anticancer agents [10,11]. Moreover, rapamycin has recently been used to coat cardiac stents, preventing stenosis [12,13].However, despite evidence suggesting a role by mTOR in regulating metabolic syndromes [14,15], the extent of its role has not been fully explored. In this study, we aimed to clarify how rapamycin administration inhibition of the mTOR pathway affects the relationship between metabolic syndrome and diabetes. We planned to achieve this by examining the effect of ra...

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.As the most common sexually transmitted disease of nonviral origin in humans, trichomoniasis caused by infection with the protozoan parasite Trichomonas vaginalis is an important risk factor for the transmission of human immunodeficiency virus (33). Along with increasing numbers of drug-resistant clinical T. vaginalis isolates (7, 9), trichomoniasis is emerging as a major threat to public health.Iron availability, which periodically varies in the human vagina, where the parasite colonizes, regulates the cytoadherence of T. vaginalis, possibly through controlling the expression of several adhesion proteins in transcription initiation and protein trafficking steps (2,11,18,35). Although only a few T. vaginalis genes have been characterized in detail, the most common example seems to suggest that T. vaginalis uses a conserved initiator (Inr) sequence as the sole core promoter element to regulate the basal transcription of protein-coding genes via interaction of the Inr with a unique Inr-binding protein, IBP39, to recruit ␣-ammanitin-resistant RNA polymerase II (17,20,22,30). This basal transcription machinery significantly differs from that of higher eukaryotic systems, in which the ␣-ammanitin-sensitive RNA polymerase II machinery exhibits considerable diversity both in the core promoter context and in the components of the promoter-recognition TFIID complex (15,24,31). Thus, the transcription efficiency of a particular type II...

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 ϳ32-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.

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.Trichomonas vaginalis is a protozoan parasite that causes the most common sexually transmitted disease of nonviral origin in humans. The disease poses an imminent threat to public health as revealed by recent findings that transmission of the human immunodeficiency virus increases in patients with trichomoniasis (1). The parasite persistently inhabits the human urogenital tract without an alternating life stage outside of the host. Cytoadherence, which is crucial for T. vaginalis to establish an infection, has been shown to involve multiple surface adhesion proteins and lipophosphoglycans (2-4). The iron supply, which undergoes periodic fluctuations in the human vagina, is one of the principle determinants modulating cytoadherence of the parasite toward human vaginal epithelial cells (5, 6), possibly through transcriptional regulation of some of the adhesion protein (ap) 2 genes, especially those in the ap65 family (7, 8), which encode proteins identical to malic enzymes (9, 10). Iron has also been implicated in modulating phenotypic variation of the parasite as well as its resistance to complement lysis (11, 12). These observations underscore the importance of iron in modulating expression of parasite virulence.Gene transcription in T. vaginalis is monocistronic with only a few intron-containing genes capable of undergoing RNA splicing (13). Transcription initiation by RNA polymerase II is thus a key step in controlling expression of the protein coding genes in the parasite. Using transcription of the ap65-1 gene as a model system, we...

Background: Myb1 mediates transcription suppression of an ap65-1 gene. Results: TvCyP1 may accelerate conformational changes in Myb1 to enable its release from certain vesicles. Conclusion: TvCyP1 is essential for moving Myb1 toward the nucleus. Significance: This study elucidates a key step in nuclear translocation of Myb1 and provides tools to study the physiological role of TvCyP1.

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.

in Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg 75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.Trichomonas vaginalis is a p arasitic protozoan that survives only as trophozoites in the human urogenital tract 1 . Trichomoniasis has long held the position as the most common sexually transmitted disease of nonviral origin 2 . The infection often manifests mild symptoms or is asymptomatic, but it can sometimes cause adverse outcomes during pregnancy, ranging from preterm deliveries or low birth weights to abortions and stillbirths 3,4 . Trichomoniasis is also recognized as a risk factor for the elevated transmission of the human immunodeficiency virus (HIV) and papillomaviruses, and the development of progressive cervical and prostate cancers 5-8 . Trichomoniasis can be easily cured by metronidazole, a drug commonly used for gram-negative bacterial infections and luminal giardiasis and entamebiasis, but reports of drug-resistant clinical isolates have been increasing over the years 3 . The infection is often overlooked, and transmission of T. vaginalis is difficult to control 9 , rendering this neglected parasite an emerging threat to public health.Among environmental factors, iron was shown to modulate the virulence of this parasite via its effects on the transcription of myriad genes 10-12 . Accordingly, iron was shown to regulate expression levels, protein trafficking, and promoter entry of three transcription factors, Myb1, Myb2, and Myb3, which i...

Background: Iron induces the immediate nuclear influx of Myb3 in T. vaginalis. Results: Iron triggered a cAMP-mediated signaling that resulted in the phosphorylation and ubiquitination of Myb3 to accelerate its nuclear influx. Conclusion: Iron triggers signal transduction to activate a rapid nuclear influx of Myb3. Significance: This work revealed a novel role of iron in poorly studied signal transduction in the parasite.