Glycogen synthesis and beyond, a comprehensive review of GSK3 as a key regulator of metabolic pathways and a therapeutic target for treating metabolic diseases.

Glycogen synthase kinase-3 (GSK3) is a highly evolutionarily conserved serine/threonine protein kinase first identified as an enzyme that regulates glycogen synthase (GS) in response to insulin stimulation, which involves GSK3 regulation of glucose metabolism and energy homeostasis. Both isoforms of GSK3, GSK3α, and GSK3ß, have been implicated in many biological and pathophysiological processes. The various functions of GSK3 are indicated by its widespread distribution in multiple cell types and tissues. The studies of GSK3 activity using animal models and the observed effects of GSK3-specific inhibitors provide more insights into the roles of GSK3 in regulating energy metabolism and homeostasis. The cross-talk between GSK3 and some important energy regulators and sensors and the regulation of GSK3 in mitochondrial activity and component function further highlight the molecular mechanisms in which GSK3 is involved to regulate the metabolic activity, beyond its classical regulatory effect on GS. In this review, we summarize the specific roles of GSK3 in energy metabolism regulation in tissues that are tightly associated with energy metabolism and the functions of GSK3 in the development of metabolic disorders. We also address the impacts of GSK3 on the regulation of mitochondrial function, activity and associated metabolic regulation. The application of GSK3 inhibitors in clinical tests will be highlighted too. Interactions between GSK3 and important energy regulators and GSK3-mediated responses to different stresses that are related to metabolism are described to provide a brief overview of previously less-appreciated biological functions of GSK3 in energy metabolism and associated diseases through its regulation of GS and other functions.

Applications of Protein Fragment Complementation Assays for Analyzing Biomolecular Interactions and Biochemical Networks in Living Cells.

Protein-protein interactions (PPIs) are indispensable for the dynamic assembly of multiprotein complexes that are central players of nearly all of the intracellular biological processes, such as signaling pathways, metabolic pathways, formation of intracellular organelles, establishment of cytoplasmic skeletons, etc. Numerous approaches have been invented to study PPIs both in vivo and in vitro, including the protein-fragment complementation assay (PCA), which is a widely applied technology to study PPIs and biomolecular interactions. PCA is a technology based on the expression of the bait and prey proteins in fusion with two complementary reporter protein fragments, respectively, that will reassemble when in close proximity. The reporter protein can be the enzymes or fluorescent proteins. Recovery of the enzymatic activity or fluorescent signal can be the indicator of PPI between the bait and prey proteins. Significant effort has been invested in developing many derivatives of PCA, along with various applications, in order to address specific questions. Therefore, a prompt review of these applications is important. In this review, we will categorize these applications according to the scenarios that the PCAs were applied and expect to provide a reference guideline for the future selection of PCA methods in solving a specific problem.

Proximity Labeling of Interacting Proteins: Application of BioID as a Discovery Tool.

Proteins perform biochemical functions by forming complexes, or protein-protein interactions (PPIs). Many different approaches such as phage display, yeast hybridization, etc. were developed to illustrate the PPIs, and disclose the composition and organization of protein complexes. However, none of these approaches are based on the real-time and in vivo PPI analysis. Proximity-dependent labeling (PDL) of interacting proteins has recently been proposed by taking advantage of several enzymes, which are capable of attaching the known reactive groups to the nearby proteins covalently. Among the PDL methods, BioID is the earliest and the most widely used one and has been upgraded from its prototype, making it an extremely convenient research tool. In this review, we describe the BioID technology development, its potential applications according to the nature of the target protein, and some recent efforts to circumvent the technical limitations. Moreover, some comparable PDL methods are introduced, including selective proteomic proximity labeling assay using tyramide, enzyme-mediated activation of radical sources, Proximity Labeling with Ascorbate Peroxidase, in vivo proximal labeling, etc., and we propose that systematic comparison of the working radius of these methods may be helpful to develop a tool box, from which the right method can be selected for a given target protein for PPI research.

Wnt/ß-Catenin Mediates AICAR Effect to Increase GATA3 Expression and Inhibit Adipogenesis.

A better understanding of the mechanism and manipulation of the tightly regulated cellular differentiation process of adipogenesis may contribute to a reduction in obesity and diabetes. Multiple transcription factors and signaling pathways are involved in the regulation of adipogenesis. Here, we report that the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) can activate AMPK in preadipocytes and thus increase the expression of GATA3, an anti-adipogenic factor. However, AICAR-increased GATA3 is mediated by the stimulation of Wnt/ß-catenin signaling in preadipocytes. Mechanistically, AICAR-activated AMPK inhibits GSK3ß through a phosphorylation process that stabilizes ß-catenin. This stabilized ß-catenin then translocates into nucleus where it interacts with T-cell factors (TCF), leading to the increased ß-catenin/TCF transcriptional activity that induces GATA3 expression. In addition, AICAR also relieves the repressing effect of the C-terminal-binding protein (CtBP) co-repressor by diverting CtBP away from the ß-catenin·TCF complex at the GATA3 promoter. The anti-adipogenic effect of GATA3 and AICAR is consistently attenuated by the disruption of Wnt/ß-catenin signaling. Furthermore, GATA3 suppresses key adipogenic regulators by binding to the promoters of these regulators, such as the peroxisome proliferator-activated receptor-γ (PPARγ) gene, and the disruption of Wnt/ß-catenin signaling reduces the GATA3 binding at the PPARγ promoter. In differentiated adipocytes, GATA3 expression inhibition is facilitated by the down-regulation of ß-catenin levels, the reduction in ß-catenin binding, and the increase in CtBP binding at the GATA3 promoter. Our findings shed light on the molecular mechanism of adipogenesis by suggesting that different regulation pathways and adipogenic regulators collectively modulate adipocyte differentiation through cross-talk.

The dual lives of bidirectional promoters.

The sequencing of the human genome led to many insights into gene organization and structure. One interesting observation was the high frequency of bidirectional promoters characterized by two protein encoding genes whose promoters are arranged in a divergent or "head-to-head" configuration with less than 2000 base pairs of intervening sequence. Computational estimates published by various groups indicate that nearly 10% of the coding gene promoters are arranged in such a manner and the extent of this bias is a unique feature of mammalian genomes. Moreover, as a class, head-to-head promoters appear to be enriched in specific categories of gene function. Here we review the structure, composition, genomic properties and functional classifications of genes controlled by bidirectional promoters and explore the biological implication of these features. This article is part of a Special Issue entitled: Chromatin in time and space.

Metabolic modulation of CtBP dimeric status impacts the repression of DNA damage repair genes and the platinum sensitivity of ovarian cancer.

Platinum drug-based chemotherapy plays a dominant role in OC (ovarian cancer) treatment. The expression of DNA damage repair (DDR) genes is critical in distinguishing drug-sensitive and drug-refractory patients, as well as in the development of drug resistance in long-term treated patients. CtBP is a highly expressed oncogene in OC and was found to repress DDR genes expression in our previous study. In the present study, the formation of CtBP dimers in live cells was studied, and the functional differences between monomeric and oligomeric CtBP were explored by CHIP-seq and RNA-seq. Besides, the dynamics of CtBP dimer formation in response to the metabolic modulation were investigated by the protein fragment complementation (PCA) assays. We show that dimerized CtBP, but not the dimerization-defective mutant, binds to and represses DDR gene expression in OC cells. Treatment of the mice tumors grown from engrafted OC cells by cisplatin disclosed that high-level CtBP expression promotes the CtBP dimerization and increases the therapeutic effect of cisplatin. Moreover, the CtBP dimerization is responsive to the intracellular metabolic status as represented by the free NADH abundance. Metformin was found to increase the dimerization of CtBP and potentiate the therapeutic effect of cisplatin in a CtBP dimerization-dependent manner. Our data suggest that the CtBP dimerization status is a potential biomarker to predict platinum drug sensitivity in patients with ovarian cancer and a target of metformin to improve the therapeutic effect of platinum drugs in OC treatment.

[Detection and clinical value of epithelial cellular adhesion molecule (EpCAM) mRNA positive circulating tumor cells in metastatic breast cancer].

OBJECTIVE: To test for circulating tumor cells (CTCs) relying on epithelial cellular adhesion molecule (EpCAM) expression in metastatic breast cancer by quantitative real-time reverse transcription-PCR. METHODS: In the study,47 metastatic breast cancer patients were evaluated by quantitative real-time PCR for detecting EpCAM mRNA. In addition, analyses were carried out for their correlation with patients' clinicopathologic features, response, and the time to progression (TTP). RESULTS: The sensitivity of EpCAM mRNA in the metastatic breast cancer patients was about 40%. However, the specificity of EpCAM mRNA for 20 healthy controls was 100%. TTP was calculated, and compared with that between EpCAM mRNA-positive and EpCAM mRNA-negative groups. For the retrospective study, the median TTP was 7.1 months and 11.1 months (P=0.013) for patients with EpCAM mRNA-positive and EpCAM mRNA-negative, respectively, after the first cycle chemotherapy. Moreover, a statistically significant correlation was demonstrated between EpCAM mRNA and TTP in patients who underwent the first or the second-line chemotherapy (P=0.018), but there was no significance in the patients pretreated with two or more previous chemotherapy lines (P=0.471). CONCLUSION: This study provides evidence of the presence of EpCAM mRNA in approximately 40% of patients with metastatic breast cancer. There is a strong correlation between EpCAM mRNA results after the first cycle therapy and TTP in metastatic breast cancer patients, and EpCAM mRNA positive after chemotherapy may predict shorter TTP.

HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro.

OBJECTIVE: Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. METHODS: The peripheral blood mononuclear cells (PBMCs) from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase (LDH) releasing assay. RESULTS: Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio, E:T), but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%). CONCLUSION: The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.

[Concurrent versus sequential systemic chemotherapy and whole brain radiation therapy for brain matastases in non-small-cell lung cancer patients].

OBJECTIVE: To evaluate the efficacy, survival and toxicity in patients with brain metastases from non-small cell lung cancer (NSCLC), treated with concurrent systemic chemotherapy and whole brain radiation therapy (WBRT) or sequential systemic chemotherapy/WBRT. METHODS: A total of 60 NSCLC patients with brain metastases were divided into two groups in this prospective clinical study: concurrent systemic chemotherapy and WBRT group (concurrent group) and sequential systemic chemotherapy/WBRT group (sequential group). RESULTS: Of 59 assessable patients, the overall response rate was 22.0%, and the brain response rate was 35.6%; the median progression-free survival time was 3.0 months, and the overall 1- and 2-year survival rates were 55% and 24.4%, respectively, with a median survival time of 16.0 months. The overall response rate was 20.0% in the concurrent group and 24.1% in sequential group (P > 0.05). The brain response rates of 43.3% in concurrent group and 27.6% in sequential group were also not significantly different (P > 0.05). The median progression-free survival time for the patients in the concurrent group was 3.0 months versus 4.0 months in the sequential group, and the median survival time was 16.0 months versus 13.0 months (all P > 0.05). The 1- and 2-year survival rates were 58.5% and 37.2% versus 52.9% and 18.9%, respectively, with a significant difference in the 2-year survival rate between the two groups (P = 0.011). In the sequential group, leukopenia was more frequent during chemotherapy than that in the concurrent group (P = 0.029). CONCLUSION: Concurrent systemic chemotherapy and WBRT is effective with tolerable adverse events in treating brain metastasis from NSCLC with an encouraging survival, and deserves further large sample and randomized multicenter clinical trials.

[Dendritic cells infected by recombinant adeno-associated virus with CEA gene to induce antigen-specific CTL response to CD44(+)CD24(-/low) cells from MCF-7 breast cancer cell line].

OBJECTIVE: To infect dendritic cells (DC) by recombinant human adeno-associated virus (rh-AAV) vector with CEA gene to generate antigen-specific CTL cells in vitro and to assay the CEA specific cytotoxic T lymphocyte(CTL), response to CD44(+)CD24(-/low) breast cancer stem cells. METHODS: Peripheral blood mononuclear cells were induced to generate DCs by cytokines interleukin-4(IL-4), granulocyte-macrophage colony-stimulating factor(GM-CSF)and tumor necrosis factor-alpha(TNF-α) while T lymphocytes were cultured with cytokines interleukin-2(IL-2). DCs were infected by CEA gene containing rh-AAV. After being matured, DCs were co-cultured with T cells to generate CTL cells. CD44(+)CD24(-/low) breast cancer stem cells were sorted out from MCF-7 and MDA-MB-231 cells. CEA specific CTL response to CD44(+)CD24(-/low) breast cancer stem cells was assayed by MTT method. RESULTS: The percentages of CD44(+)CD24(-/low) breast cancer stem cells subsets in MCF-7 and MDA-MB-231 were 5.1% and 76.3% respectively. DCs transfected with CEA gene could induce CEA antigen CTLs response. The killing ratio of MCF-7 cells in CEA gene transfection group was 46.5% plusmn; 15.0% with significant difference (P=0.009) as compared with that of CEA gene untransfection group. As for CD44(+)CD24(-/low) breast cancer stem cells subset from MCF-7 cells, CEA gene transfection group resulted in a higher killing ratio of 44.7% ± 28.2% as compared with that of CEA untransfection group. While the inhibitory rate of non-breast cancer stem cells subset from MCF-7 cells in CEA gene transfection group was 50.6% ± 22.2% (P=0.05). CTL cells generated by DCs transfected with CEA gene did not show inhibitory activity to MDA-MB-231 breast cancer cells (without CEA expression) as compared with CEA gene untransfection group. It was the same in CD44(+)CD24(-/low) breast cancer stem cells subset and non-breast cancer stem cells subset from MDA-MB-231 breast cancer cells (P>0.05). CONCLUSION: DCs infected by rh-AAV with CEA gene could induce antigen-specific CTL response to kill CEA expressing breast cancer cells involved in CD44(+)CD24(-/low) breast cancer stem cells subset. It suggests that immune therapy might be a potential treatment of breast cancer stem cells.

[Randomized clinical case-control trial for the comparison of docetaxel plus thiotepa versus docetaxel plus capecitabine in patients with metastatic breast cancer].

OBJECTIVE: To evaluate the efficacy and safety of docetaxel plus thiotepa(TXT/TSPA) and docetaxel plus capecitabine(TXT/CAPE) in patients with metastatic breast cancer. METHODS: The patients were randomized to give intravenous TXT 35 mg/m2 on days 1 and 8 plus intravenous TSPA 60-65 mg/m(2) on day 1 every 3 weeks, or intravenous TXT 35 mg/m(2) on days 1 and 8 plus oral CAPE 1 000 mg/m(2) twice daily on days 1 to 14 every 3 weeks, at least 2 cycles applied. RESULTS: TXT/TSPA group (22 patients) and TXT/CAPE group (24 patients) had consistent baseline. Docetaxel thiotepa group (21 cases) and docetaxel combined with capecitabine group (22 cases) were evaluated for their clinical responses, which showed that 2 of the 21 (9.52%) from TXT/TSPA group and 6 of the 22 (27.27%) from TXT/CAPE group had achieved partial remission; 11 of the 21 (52.38%) from TXT/TSPA group versus 7 of the 22 (31.82%) from TXT/CAPE group for stable diseases; 8 of the 21 (38.10%) from TXT/TSPA group versus and 9 of the 22 (40.91%) from TXT/CAPE group for progressive diseases, respectively. The disease control rate was 61.90% (13/21) and 59.09% (13/22) for TXT/TSPA and TXT/CAPE groups, the median progression-free survival(PFS) was 7.9 months [95% confidence interval(CI) 0.77 to 15.03] from TXT/TSPA group versus 8.3 months (95% CI 4.01 to 11.79) from TXT/CAPE group. One year survival rate was 88.20% for TXT/TSPA versus 81.00% for TXT/CAPE group, respectively. P values all exceeded 0.05, and the two groups showed no difference. No chemotherapy-related deaths occurred. Myelosuppression was the major side effect. The adverse events of grades 3 to 4 respectively occurred in TXT/TSPA and TXT/CAPE groups:leucocytopenia was 45.45% vs. 26.09%; neutropenia 45.45% vs. 21.74%; thrombocytopenia 9.09% vs. 0%; hand-foot syndrome 0% vs. 13.04%. P values all exceeded 0.05, and the two groups showed no difference. CONCLUSION: Combination of docetaxel and thiotepa in the treatment of metastatic breast cancer has some curative effect and adverse reactions can be tolerated. It can be used as an economical and effective rescue plan.

Mobilization of Peripheral Blood Stem Cells Using Regimen Combining Docetaxel with Granulocyte Colony-stimulating Factor in Breast Cancer Patients.

OBJECTIVE: To evaluate the effectiveness and safety of the mobilization of peripheral blood hematopoietic stem cells by combining docetaxel with granulocyte colony-stimulating factor (G-CSF) in breast cancer patients. METHODS: A total of 57 breast cancer patients were treated with docetaxel 120 mg/m(2). When the white blood cell (WBC) count decreased to 1.0×10(9)/L, patients were given G-CSF 5 µg/kg daily by subcutaneous injection until the end of apheresis. Peripheral blood mononuclear cells (MNC) were isolated by Cobe Spectra Apheresis System. The percentage of CD34(+) cell was assayed by flow cytometry. RESULTS: At a median 6 of days (range 3-8) after the administration of docetaxel, the median WBC count decreased to 1.08×10(9)/L (range 0.20-2.31). The median duration of G-CSF mobilization was 3 days (range 2-7). The MNC collection was conducted 8-12 days (median 10 days) after docetaxel treatment. The median MNC was 5.35×10(8)/kg (range 0.59-14.07), the median CD34(+) cell count was 2.43×10(6)/kg (range 0.16-16.69). The CD34(+) cell count was higher than 1.00×10(6)/kg in 47 of 57 cases (82.46%) and higher than 2.00×10(6)/kg in 36 cases (63.16%). The CD34(+) cell count was higher than 2.00×10(6)/kg in 27 collections (23.68%). The MNC count and the CD34(+) cell count were correlated with the bottom of WBC after docetaxel chemotherapy (r=0.364, 0.502, P=0.005, 0.000). The CD34(+) cell count was correlated with the MNC count (r=0.597, P=0.000). The mobilization and apheresis were well tolerated in all patients. Mild perioral numbness and numbness of hand or feet were observed in 3 cases. No serious adverse events were reported. CONCLUSION: Mobilization of peripheral blood hematopoietic stem cell by combining docetaxel with G-CSF was effective and safety in breast cancer patients.

From impossible to possible: the lessons from the control of recent COVID-19 outbreaks in China.

The COVID-19 pandemic has catastrophically impacted the world. Before the success in vaccination, this virus shows no sign of stop spreading. Nearly all the countries have implemented stringent approaches to slow down the transmission of the virus, but the virus still caused over 2 million deaths and the number is increasing. Therefore, preventing the virus spreading is still necessary to protect most people, especially the ones with pre-conditions. Mainland China has successfully eradicated the COVID-19 virus infection in Wuhan in 2020. After that, several small-scale outbreaks occurred in many cities in China, but none of these COVID-19 virus infections caused the widespread. In this review, we would like to give a detailed presentation of the approaches that were implemented by the China government to suppress the virus spreading by considering the unique characteristics of this virus and the paths of the virus transmission. Both the pros and cons of these strategies will also be analyzed. The experiences and lessons learned during the virus-fighting in China, expectedly, will be a useful source of reference for other regions in overcoming the threat caused by the COVID-19 virus.

Applications of the CRISPR-Cas system for infectious disease diagnostics.

INTRODUCTION: Rapid and accurate diagnostic approaches are essential for impeding the spread of infectious diseases. This review aims to summarize current progress of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems in the applications for diagnostics of infectious diseases including the ongoing COVID-19 epidemic. AREAS COVERED: In this review, we discuss class 2 CRISPR-Cas biosensing systems-based diagnostics in various emerging and reemerging infectious diseases, CRISPR-Cas systems have created a new era for early diagnostics of infectious diseases, especially with the discovery of the collateral cleavage activity of Cas12 and Cas13. We mainly focus on different CRISPR-Cas effectors for the detection of pathogenic microorganisms as well as provide a detailed explanation of the pros and cons of CRISPR-Cas biosensing systems. In addition, we also introduce future research perspectives. EXPERT COMMENTARY: However, further improvement of newly discovered systems and engineering existing ones should be developed to increase the specificity, sensitivity or stability of the diagnostic tools. It may be a long journey to finish the clinical transition from research use. CRISPR-Cas approaches will emerge as more promising and robust tools for infectious disease diagnosis in the future.

Peripheral cytotoxic T lymphocyte predicts first-line progression free survival in HER2-positive advanced breast cancer.

BACKGROUND: The role of peripheral blood lymphocyte (pBL) in breast cancer has long been studied. However, the predictive role of pBL in advanced breast cancer (ABC) is poorly understood. METHODS: A total of 303 patients with ABC were consecutively recruited at our center between January 2015 and September 2019. At baseline, pBL subtypes were detected in all patients with 229 blood samples available for circulating tumor DNA (ctDNA) detection. pBL was analyzed through flow cytometry. ctDNA-based gene mutations were detected using next generation sequencing. The cutoff value of pCTL was estimated by X-tile software. Progression free survival (PFS) was estimated by Kaplan-Meier curve and Cox hazard proportion regression model, with difference detection by log-rank test. RESULTS: Median follow-up time of the study was 21.0 months. The median age of diagnosis was 52.0 years. Among the pBL subtypes, only pCTL level was found predictive for PFS in the HER2+ patients whom received anti-HER2 therapy (13.1 vs. 5.6 months, P = 0.001). However, the predictive role of pCTL was not found in HR-positive (P = 0.716) and TNBC (P = 0.202). pCTL high associated with suppressive immune indictors including lower CD4/CD8 ratio (P = 0.004) and high level of Treg cell (P = 0.004). High occurrence of FGFR1 amplification which has been reported as immune suppressor was also found in HER2+ patients with pCTL high (22.2% vs. 4.3%, P = 0.048). CONCLUSIONS: Higher pCTLs level associated with shorter PFS and FGFR1 mutation in HER2+ ABC patients.

Nuclear HKII-P-p53 (Ser15) Interaction is a Prognostic Biomarker for Chemoresponsiveness and Glycolytic Regulation in Epithelial Ovarian Cancer.

In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

Active chromatin hub of the mouse alpha-globin locus forms in a transcription factory of clustered housekeeping genes.

RNA polymerases can be shared by a particular group of genes in a transcription "factory" in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse alpha-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active alpha1 and alpha2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse alpha-globin cluster. In fetal liver cells, the active alpha1 and alpha2 genes, but not the inactive zeta gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse alpha-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active alpha1 and alpha2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the zeta gene promoter, which is specifically repressed during development, is much lower than that at the alpha1 and alpha2 promoters. Thus, the mouse alpha-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate alpha-globin gene expression.

BioID: A Proximity-Dependent Labeling Approach in Proteomics Study.

Biological activities are mainly executed by proteins and in most of the occasions these activities are accomplished by protein complexes or through protein-protein interactions (PPI). So it is critical to reveal how the protein complexes are organized and demonstrate the PPIs involved in the biological processes. In addition to the traditional biochemical approaches, proximity-dependent labeling (PDL) has recently been proposed to identify the interacting partners of a given protein. PDL requires the fusion expression of the target protein with an enzyme which catalyzes the attachment of a reactive molecule to the interacting partners in a distance-dependent manner. Further analysis of all the proteins that are modified by the reactive molecule discloses the identity of these proteins which are presumed to be interacting partners of the target protein. BioID is one of those representative PDL methods with the most widely applications. The enzyme used in BioID is a biotin ligase BirA which catalyzes the biotinylation of target protein with the presence of biotin. Through streptavidin-mediated pull-down and mass spectrometry analysis, the interacting protein candidates of a given protein can be obtained.

Nuclear localization of Desmoplakin and its involvement in telomere maintenance.

The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.