The WWOX tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically-relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically-distinct E66/Y85 duo at structurally-equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, introduction of E66R/Y85W double-substitution within the WW2 domain not only results in gain-of-function but the resulting engineered domain, hereinafter referred to as WW2_RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease.
WW-ligand thermodynamics; WW domain chaperone; WW domain engineering; WW tandem domains
YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery (ITC) and circular dichroism (CD) in combination with molecular modeling (MM) and molecular dynamics (MD), we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, non-consensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II (PPII) helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a non-bulky and flexible glycine one-residue C-terminal to the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, arguing that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.
YAP2 transcriptional regulator; WBP1 and WBP2 proline-rich proteins; WW-ligand thermodynamics; Isothermal titration calorimetry; Circular dichroism; Molecular modeling; Molecular dynamics
This study elucidates the role of E6-associated protein, E6-AP (a dual function steroid hormone receptor coactivator and ubiquitin-protein ligase) in the regulation of PI3K-Akt signaling pathway, prostate gland growth and proliferation. Here, we report the generation of transgenic mice and prostate cancer cell line, LNCaP cells that overexpress E6-AP protein. Using these models we show that the levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells suggesting that E6-AP regulates the PI3K-Akt signaling pathway. The prostate glands in our transgenic mice are ~20% larger and produce preneoplastic lesions at the age of 18 months. Our data also suggest that E6-AP modulates PI3K-Akt signaling pathway by both androgen-independent and -dependent mechanisms. In the androgen-independent mechanism, E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. Further, we show that E6-AP, a known coactivator of AR, amplifies the androgen-dependent activation of PI3K-Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased cell size and proliferation. Overall our data suggests that E6-AP regulates both the positive and negative modulators of the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and decreased apoptosis.
Yes-associated protein (YAP) is a well characterized transcriptional coactivator that interacts with various transcription factors and modulates their transcriptional activities. Phosphorylation of YAP by specific kinases regulates its cellular distribution and transcriptional activation functions. Sequestration of phosphorylated YAP in cytoplasm results in the reduction of transcription from its target genes. Since, YAP has been characterized as a coactivator of estrogen (ER) and progesterone (PR) receptors, we examined the immunohistochemical expression profile of YAP and correlation of YAP expression with that of ER and PR in normal (40 samples) and tumor breast (226 samples) from microarray tissue samples using immunohistochemistry. Here we show that YAP expression is significantly reduced in invasive carcinoma samples compared to normal breast tissues, which express high levels of YAP (YAP was positive for 45.1% of invasive carcinoma samples vs. 82.5% of normal samples p<.0001). Furthermore, our data shows that reduced expression of YAP in invasive carcinoma samples is significantly associated with ER negativity (YAP was negative for 59.9% in ER negative vs. 38.9% in ER positive invasive carcinoma samples, p=0.007) and PR negativity (YAP was negative for 60.1% in PR negative vs. 28.9% in PR positive, p=0.0004). Among invasive carcinoma samples, 42.9% were YAP, ER and PR negative, whereas only 7.5% were found to be YAP, ER and PR positive. On the contrary, 20 out of 23 (87%) normal breast tissues that were positive for ER and PR were also positive for YAP. These data suggest that YAP may act as a tumor suppressor in invasive breast carcinomas and it can also be used as a molecular marker for ER and PR negative breast tumors.
Yes-associated protein; YAP; estrogen and progesterone receptors; breast cancers
Background and Aims
Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO.
Materials and methods
Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO.
Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P < 0.05) of EPO was observed in the medium from Huh-7 cell line.
Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.
EPO; erythropoietin; CHO; Chinese hamster ovary cell line; Huh-7; Human hepatoma cell line; PCR; polymerase chain reaction
Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression, fatigue, and "flu-like" symptoms. Herbal plants have been used for centuries against different diseases including viral diseases and have become a major source of new compounds to treat bacterial and viral diseases.
The present study was design to study the antiviral effect of Glycyrrhizin (GL) against HCV. For this purpose, HCV infected liver cells were treated with GL at non toxic doses and HCV titer was measured by Quantitative real time RT-PCR.
Results and Discussion
Our results demonstrated that GL inhibit HCV titer in a dose dependent manner and resulted in 50% reduction of HCV at a concentration of 14 ± 2 μg. Comparative studies were made with interferon alpha to investigate synergistic effects, if any, between antiviral compound and interferon alpha 2a. Our data showed that GL exhibited synergistic effect when combined with interferon. Moreover, these results were verified by transiently transfecting the liver cells with HCV 3a core plasmid. The results proved that GL dose dependently inhibit the expression of HCV 3a core gene both at mRNA and protein levels while the GAPDH remained constant.
Our results suggest that GL inhibit HCV full length viral particles and HCV core gene expression or function in a dose dependent manner and had synergistic effect with interferon. In future, GL along with interferon will be better option to treat HCV infection.
Hepatitis C Virus (HCV) has two envelop proteins E1 and E2 which is highly glycosylated and play an important role in cell entry. Inhibition of virus at entry step is an important target to find antiviral drugs against HCV. Glanthus Nivalis Agglutinin (GNA) is a mannose binding lectin which has tendency for specific recognition and reversible binding to the sugar moieties of a wide variety of glycoproteins of enveloped viruses.
In the present study, HCV pseudoparticles (HCVpp) for genotype 3a were produced to investigate the ability of GNA to block the HCV entry. The results demonstrated that GNA inhibit the infectivity of HCVpp and HCV infected serum in a dose-dependent manner and resulted in 50% reduction of virus at 1 ± 2 μg concentration. Molecular docking of GNA and HCV glycoproteins (E1 and E2) showed that GNA inhibit HCV entry by binding N-linked glycans.
These results demonstrated that targeting the HCV glycans is a new approach to develop antiviral drugs against HCV.
HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH4Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH4Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH4Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH4Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH4Cl showed more than 50% reduction of virus infectivity at 50 μM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV.
Hepatitis C virus (HCV) causes acute and chronic hepatitis which can eventually lead to permanent liver damage, hepatocellular carcinoma and death. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. In this review, we summarize molecular virology, replication and immune responses against HCV and discussed how HCV escape from adaptive and humoral immune responses. This advance knowledge will be helpful for development of vaccine against HCV and discovery of new medicines both from synthetic chemistry and natural sources.
HCV; replication; HCV entry; immune responses
Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design.
Hepatitis C is a major health problem affecting 270 million individuals in world including Pakistan. Current treatment regimen, interferon alpha and ribavirin only cure half of patients due to side effects and high cost.
In the present study Silybum marianum (Milk thistle) seeds were collected, extracted and analyzed against HCV 3a core gene by transiently transfecting the liver cells with HCV core plasmid. Our results demonstrated that Silymarin (SM) dose dependently inhibit the expression or function of HCV core gene at a non toxic concentration while the GAPDH remained constant. To identify the active ingredient, SM was fractioned by thin layer chromatography (TLC), column chromatography and HPLC. Purified fractions were tested for HCV core gene and western blotting results showed that two factions of SM (S1 and S2) inhibit HCV 3a core expression or function in liver cells
Our results suggest SM and its fractions (S1 and S2) inhibit HCV core gene of 3a genotype and combination of SM and its fractions with interferon will be a better option to treat HCV infection
Steroid hormone receptors (SHR) belong to a large family of ligand-activated transcription factors that perform their biological functions by enhancing the transcription of specific target genes. The transactivation functions of SHRs are regulated by a specialized group of proteins called coactivators. The SHR coactivators represent a growing class of proteins with various enzymatic activities that serve to modify the chromatin to facilitate the transcription of SHR target genes. The ubiquitin-proteasome pathway enzymes have also been added to the growing list of enzymatic activities that are recruited to the SHR target gene promoters during transcription. One such ubiquitin-proteasome pathway enzyme to be identified and characterized as a SHR coactivator was E6-associated protein (E6-AP). E6-AP is a hect (homologous to E6-associated protein carboxy-terminal domain) domain containing E3 ubiquitin ligase that possesses two independent separable functions; a coactivation function and an ubiquitin-protein ligase activity. Being a component of the ubiquitin-proteasome pathway, it is postulated that E6-AP may orchestrate the dynamics of steroid hormone receptor-mediated transcription by regulating the degradation of the transcriptional complexes. E6-AP has also been shown to be involved in the regulation of various aspects of reproduction such as prostate and mammary gland development. Furthermore, it has been demonstrated that E6-AP expression is down-regulated in breast and prostate tumors and that the expression of E6-AP is inversely associated with that of estrogen and androgen receptors. This review summarizes our current knowledge about the structures, molecular mechanisms, spatiotemporal expression patterns and biological functions of E6-AP.
Estrogen drives both transcriptional activation and proteolysis of estrogen receptor α (ERα; encoded by ESR1). Here we observed variable and overlapping ESR1 mRNA levels in 200 ERα-negative and 50 ERα-positive primary breast cancers examined, which suggests important posttranscriptional ERα regulation. Our results indicate that Src cooperates with estrogen to activate ERα proteolysis. Inducible Src stimulated ligand-activated ERα transcriptional activity and reduced ERα t1/2. Src and ERα levels were inversely correlated in primary breast cancers. ERα-negative primary breast cancers and cell lines showed increased Src levels and/or activity compared with ERα-positive cancers and cells. ERα t1/2 was reduced in ERα-negative cell lines. In both ERα-positive and -negative cell lines, both proteasome and Src inhibitors increased ERα levels. Src inhibition impaired ligand-activated ERα ubiquitylation and increased ERα levels. Src siRNA impaired ligand-activated ERα loss in BT-20 cells. Pretreatment with Src increased ERα ubiquitylation and degradation in vitro. These findings provide what we believe to be a novel link between Src activation and ERα proteolysis and support a model whereby crosstalk between liganded ERα and Src drives ERα transcriptional activity and targets ERα for ubiquitin-dependent proteolysis. Oncogenic Src activation may promote not only proliferation, but also estrogen-activated ERα loss in a subset of ERα-negative breast cancers, altering prognosis and response to therapy.
We investigated the role of the ubiquitin-conjugating enzyme UBCH7 in nuclear receptor transactivation. Using transient transfection assays, we demonstrated that UBCH7 modulates the transcriptional activity of progesterone receptor (PR) and glucocorticoid, androgen, and retinoic acid receptors in a hormone-dependent manner and that the ubiquitin conjugation activity of UBCH7 is required for its ability to potentiate transactivation by steroid hormone receptors (SHR). However, UBCH7 showed no significant effect on the transactivation functions of p53 and VP-16 activation domain. Depletion of endogenous UBCH7 protein by small interfering RNAs suggests that UBCH7 is required for the proper function of SHR. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of UBCH7 onto estrogen receptor- and PR-responsive promoters. Additionally, we show that UBCH7 and E6-associated protein (E6-AP) synergistically enhance PR transactivation. We also demonstrate that UBCH7 interacts with steroid receptor coactivator 1 (SRC-1) and that UBCH7 coactivation function is dependent on SRC-1. Taken together, our results reveal the possible role of UBCH7 in steroid receptor transactivation and provide insights into the mechanism of action of UBCH7 in receptor function.
Sex steroid hormones, estrogen, progesterone and androgen, play pivotal roles in sex differentiation and development, and in reproductive functions and sexual behavior. Studies have shown that sex steroid hormones are the key regulators in the development and progression of endocrine-related cancers, especially the cancers of the reproductive tissues. The actions of estrogen, progesterone and androgen are mediated through their cognate intracellular receptor proteins, the estrogen receptors (ER), the progesterone receptors (PR) and the androgen receptor (AR), respectively. These receptors are members of the nuclear receptor (NR) superfamily, which function as transcription factors that regulate their target gene expression. Proper functioning of these steroid receptors maintains the normal responsiveness of the target tissues to the stimulations of the steroid hormones. This permits the normal development and function of reproductive tissues. It can be inferred that factors influencing the expression or function of steroid receptors will interfere with the normal development and function of the target tissues, and may induce pathological conditions, including cancers. In addition to the direct contact with the basal transcription machinery, nuclear receptors enhance or suppress transcription by recruiting an array of coactivators and corepressors, collectively named coregulators. Therefore, the mutation or aberrant expression of sex steroid receptor coregulators will affect the normal function of the sex steroid receptors and hence may participate in the development and progression of the cancers.
nuclear receptors; steroid receptors; coregulators; coactivators; corepressors; cancer
Progesterone, an ovarian steroid hormone, plays a key role in the development and function of the mammary gland, as it also does in the uterus and the ovary. The action of progesterone is mediated through its intracellular cognate receptor, the progesterone receptor (PR), which functions as a transcription factor that regulates gene expression. As with other nuclear receptors, coregulators (coactivators and corepressors) recruited by the liganded or unliganded PR, either to enhance or to suppress transcription activity, modulate the function of the PR. Mutation or aberrant expression of the coregulators might thus affect the normal function of the PR and hence disrupt the normal development of the mammary gland, which may lead to breast cancer.
breast cancer; coactivator; corepressor; progesterone receptor
The E6-associated protein (E6-AP), although originally identified as a ubiquitin ligase, has recently been shown to function as a coactivator of steroid receptor-dependent gene expression in in vitro assays. In order to determine whether E6-AP acts as a coactivator in vivo, physiological parameters associated with male and female sex steroid action were assessed in the E6-AP null mouse. Gonadal size was reduced in E6-AP null male and female mice in comparison to wild-type controls in conjunction with reduced fertility in both genders. Consistent with this observation, defects in sperm production and function, as well as ovulation were observed. In comparison to wild-type controls, induction of prostate gland growth induced by testosterone and uterine growth by estradiol were significantly reduced. In contrast, estrogen and progesterone-stimulated growth of virgin mammary gland was not compromised by E6-AP ablation despite E6-AP expression in this tissue. This latter finding contrasts with the impaired estrogen and progesterone-induced mammary gland development observed previously for steroid receptor coactivator type 1 (SRC-1) and SRC-3 female knockout mice. Taken together, these results are consistent with a role for E6-AP in mediating a subset of steroid hormone actions in vivo. Nevertheless, differences observed between SRC and E6-AP knockout phenotypes indicate that these two families of steroid receptor coactivators are not functionally equivalent and supports the hypothesis that coactivators contribute to tissue-specific steroid hormone action.
In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitin-protein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway.