Jeehyeon Bae

MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain.

MCL-1 (myeloid cellleukemia-1) is an antiapoptotic BCL-2 family protein discovered as an early induction gene during myeloblastic leukemia cell differentiation. This survival protein has the BCL-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region. We identified a short splicing variant of the MCL-1mRNA in the human placenta encoding a protein, termed MCL-1 short (MCL-1S), with an altered C terminus as compared with the full-length MCL-1 long (MCL-1L), leading to the loss of BH1, BH2, and the transmembrane domains. Analysis of the human MCL-1 gene indicated that MCL-1S results from the splicing out of exon 2 during mRNA processing. MCL-1S, unlike MCL-1L, does not interact with diverse proapoptotic BCL-2-related proteins in the yeast two-hybrid system. In contrast, MCL-1S dimerizes with MCL-1L in the yeast assay and coprecipitates with MCL-1L in transfected mammalian cells. Overexpression of MCL-1S induces apoptosis in transfected Chinese hamster ovary cells, and the MCL-1S action was antagonized by the antiapoptotic MCL-1L. Thus, the naturally occurring MCL-1S variant represents a new proapoptotic BH3 domain-only protein capable of dimerizing with the antiapoptotic MCL-1L. The fate of MCL-1-expressing cells could be regulated through alternative splicing mechanisms and interactions of the resulting anti- and proapoptotic gene products.

Thyrostimulin, a heterodimer of two new human glycoprotein hormone subunits, activates the thyroid-stimulating hormone receptor

Human thyrotropin (TSH), luteotropin (LH), follitropin (FSH), and chorionic gonadotropin are members of the heterodimeric glycoprotein hormone family. The common alpha subunit forms noncovalent heterodimers with different beta subunits. Two novel human glycoprotein hormonelike genes, alpha2 (A2) and beta5 (B5), recently have been identified. Using a yeast two-hybrid assay, the two subunits were found as potential heterodimerization partners. Immunological analyses confirmed the heterodimerization of A2 and B5 in transfected cells and their colocalization in the anterior pituitary. Recombinant A2/B5 heterodimeric glycoproteins, purified using cation exchange and size fractionation chromatography, activated human TSH receptors, but not LH and FSH receptors, and showed high affinity to TSH receptors in a radioligand receptor assay. The heterodimer also stimulated cAMP production and thymidine incorporation by cultured thyroid cells and increased serum thyroxine levels in TSH-suppressed rats in vivo. This new heterodimeric glycoprotein hormone was named as thyrostimulin based on its thyroid-stimulating activity. The expression of thyrostimulin in the anterior pituitary known to express TSH receptors suggested a paracrine mechanism. The present discovery of a new ligand based on genomic approaches could facilitate the understanding of the physiological roles of extra-thyroid TSH receptor systems and the structural-functional basis of receptor signaling by related glycoprotein hormones.

FOXL2 Interacts with Steroidogenic Factor-1 (SF-1) and Represses SF-1-Induced CYP17 Transcription in Granulosa Cells

Mutations in FOXL2 are responsible for blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) type I, in which affected women exhibit premature ovarian failure. FOXL2-null mice showed defects in granulosa cell development during folliculogenesis. We screened a rat ovarian yeast two-hybrid cDNA library to identify FOXL2-interacting proteins and found steroidogenic factor-1 (SF-1). Here, we show that human FOXL2 and SF-1 proteins interact in human granulosa cells and that FOXL2 negatively regulates the transcriptional activation of a steroidogenic enzyme, CYP17, by SF-1. Furthermore, FOXL2 mutants found in blepharophimosis-ptosis-epicanthus inversus syndrome type I patients lost the ability to repress CYP17 induction mediated by SF-1. Chromatin immunoprecipitation and EMSA results further revealed that FOXL2 inhibited the binding of SF-1 to the CYP17 promoter, whereas the FOXL2 mutants failed to block this interaction. Therefore, this study identifies a novel regulatory role for FOXL2 on a key steroidogenic enzyme and provides a possible mechanism by which mutations in FOXL2 disrupt normal ovarian follicle development.

Tankyrase 1 Interacts with Mcl-1 Proteins and Inhibits Their Regulation of Apoptosis

Mcl-1L (myeloid cell leukemia-1 long) is an antiapoptotic Bcl-2 family protein discovered as an early induction gene during leukemia cell differentiation. Previously, we identified Mcl-1S (short) as a short splicing variant of theMcl-1 gene with proapoptotic activity. To identify Mcl-1-interacting proteins, we performed yeast two-hybrid screening and found cDNAs encoding tankyrase 1. This protein possesses poly(ADP-ribose) polymerase activity and presumably facilitates the turnover of substrates following ADP-ribosylation. In yeast and mammalian cells, tankyrase 1 interacts with both Mcl-1L and Mcl-1S, but does not bind to other Bcl-2 family proteins tested. Analysis of truncated tankyrase 1 mutants indicated that the first 10 ankyrin repeats are involved in interaction with Mcl-1. In the N terminus of Mcl-1, a stretch of 25 amino acids is sufficient for binding to tankyrase 1. Overexpression of tankyrase 1 antagonizes both Mcl-1L-mediated cell survival and Mcl-1S-induced cell death. Furthermore, coexpression of tankyrase 1 with Mcl-1L or Mcl-1S decreased the levels of Mcl-1 proteins. Although tankyrase 1 down-regulates Mcl-1 protein expression, no ADP-ribosylation of Mcl-1 was detected. In contrast, overexpression of Mcl-1 proteins suppressed the ADP-ribosylation of the telomeric repeat binding factor 1, another tankyrase 1-interacting protein. Thus, interaction of Mcl-1L and Mcl-1S with tankyrase 1 could serve as a unique mechanism to decrease the expression of these Bcl-2 family proteins, thereby leading to the modulation of the apoptosis pathway.

Laparoscopic and laparotomic staging in stage I epithelial ovarian cancer: a comparison of feasibility and safety.

The aim of this study was to compare laparoscopic and laparotomic surgical staging in patients with stage I epithelial ovarian cancer in terms of feasibility and safety. A retrospective chart review was undertaken of all patients with apparent stage I epithelial ovarian cancer who underwent laparoscopic (laparoscopy group) or laparotomic (laparotomy group) surgical staging at the Center for Uterine Cancer, National Cancer Center, Korea, between January 2001 and August 2006. During the study period, 19 patients underwent laparotomic surgical staging and 17 patients underwent laparoscopic surgical staging. No cases were converted from laparoscopy to laparotomy. The two groups were similar in terms of age, body mass index, procedures performed, number of lymph nodes retrieved, and operating time. The laparoscopy group had less estimated blood loss (P= 0.001), faster return of bowel movement (P< 0.001), and a shorter postoperative hospital stay (P= 0.002) compared to the laparotomy group. Transfusions were required only in two laparotomy patients, and postoperative complications occurred only in four laparotomy patients. However, two patients with stage IA grade 1 and 2 disease in laparoscopy group had recurrence with one patient dying of disease. The accuracy and adequacy of laparoscopic surgical staging were comparable to laparotomic approach, and the surgical outcomes were more favorable than laparotomic approach. However, the oncologic safety of laparoscopic staging was not certain. This is the first report on the possible hazards of laparoscopic staging in early-stage ovarian cancer. In the absence of a large prospective trial, this technique should be performed cautiously.

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis.

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.

Escherichia coli ribonuclease III activity is downregulated by osmotic stress: consequences for the degradation of bdm mRNA in biofilm formation

During the course of experiments aimed at identifying genes with ribonuclease III (RNase III)‐dependent expression in Escherichia coli, we found that steady state levels of bdm mRNA were dependent on cellular concentrations of RNase III. The half‐lives of adventitiously overexpressed bdm mRNA and the activities of a transcriptional bdm‘–’cat fusion were observed to be dependent on cellular concentrations of RNase III, indicating the existence of cis‐acting elements in bdm mRNA responsive to RNase III. In vitro and in vivo cleavage analyses of bdm mRNA identified two RNase III cleavage motifs, one in the 5′‐untranslated region and the other in the coding region of bdm mRNA, and indicated that RNase III cleavages in the coding region constitute a rate‐determining step for bdm mRNA degradation. We also discovered that downregulation of the ribonucleolytic activity of RNase III is required for the sustained elevation of RcsB‐induced bdm mRNA levels during osmotic stress and that cells overexpressing bdm form biofilms more efficiently. These findings indicate that the Rcs signalling system has an additional regulatory pathway that functions to modulate bdm expression and consequently, adapt E. coli cells to osmotic stress.

Effective delivery of anti-miRNA DNA oligonucleotides by functionalized gold nanoparticles

MicroRNAs (miRNAs) are gaining recognition as essential regulators involved in many biological processes, and they are emerging as therapeutic targets for treating disease. Here, we introduce a method for effective delivery of anti-miRNA oligonucleotides (AMOs) using functionalized gold nanoparticles (AuNPs). To demonstrate the ability of AMOs to silence miRNA, we selected miR-29b, which is known to downregulate myeloid cell leukemia-1 (MCL-1), a factor responsible for promoting cell survival. We first generated AuNPs coated with cargo DNA, which was then coupled to complementary DNA linked to an antisense miR-29b sequence. When the AuNPs were delivered into HeLa cells, MCL-1 protein and mRNA levels were increased significantly. Furthermore, apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inhibited, proving that AMOs targeting miR-29b were effectively delivered by our innovative AuNP. In addition, we provided evidence that AuNP could deliver other AMOs against miR-21 into two independent cell lines, KGN and 293T, suggesting that the AuNP conjugates can be versatile for any AMO and cell type.

p19ras Interacts with and Activates p73 by Involving the MDM2 Protein

p73β is a structural and functional homologue of p53, a tumor suppressor gene. In this study, we identified a novel p73β-binding protein, p19ras, by the yeast two-hybrid screening method. Alternative splicing of the proto-oncogene H-ras pre-mRNA has led to two distinct transcripts, p19ras and p21ras. In both endogenous and overexpressed systems, we confirmed that p19ras binds to full-length p73β in vivo and in vitro. Coexpression of p19ras with p73β stimulated the transcriptional activity of p73β. Ras proteins are known to be small membrane-localized guanine nucleotide-binding proteins. However, unlike other Ras proteins, p19ras is localized in the nucleus and the cytosol and its interaction with p73β occurred exclusively in the nucleus. Oncogenic MDM2 (mouse double minutes 2) is a known repressor of p73 transcriptional activity. In this study, when p19ras was bound to MDM2, it further inhibited the association of MDM2 to the p73β protein. In addition, p19ras abolished MDM2-mediated transcriptional repression of p73β. Therefore, this study presents a novel pathway of Ras signaling that occurs in the nucleus, involving p19ras and p73β. Furthermore, a p19ras-mediated novel regulatory mechanism of p73 involving the MDM2 protein is described.

Sumoylation of Forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory Gene

Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to the transcriptional activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles.