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The genomic RNAs of influenza A viruses are associated with the viral polymerase subunits (PB1, PB2, PA) and nucleoprotein (NP), forming ribonucleoprotein complexes (RNPs). Transcription/replication of the viral genome occurs in the nucleus of infected cells. A role for Hsp90 in nuclear import and assembly of newly synthetized RNA-polymerase subunits has been proposed. Here we report that the p23 cochaperone of Hsp90, which plays a major role in glucocorticoid receptor folding and function, associates with influenza virus polymerase. We show that p23 is not essential for viral multiplication in cultured cells but relocalizes to the nucleus in influenza virus-infected cells, which may alter some functions of p23 and Hsp90. Moreover, we show that influenza virus infection inhibits glucocorticoid receptor-mediated gene transactivation, and that this negative effect can occur through a p23-independent pathway. Viral-induced inhibition of the glucocorticoid receptor response might be of significant importance regarding the physiopathology of influenza infections in vivo. [ABSTRACT FROM AUTHOR]

Background: To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for coregulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. Results: We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. Conclusions: The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its cochaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses. [ABSTRACT FROM AUTHOR]

Nuclear receptors (NRs) form one of the largest superfamilies of transcription factors in metazoans. MicroRNAs (miRNAs) are small non-coding RNAs that bind the 3′ untranslated region (3′UTR) of target mRNAs to reduce their stability and/or translation. miRNAs can directly regulate the protein output of target NR mRNAs, and, conversely, the expression of miRNAs can be modulated by NRs at the transcriptional level. At least one NR also regulates the posttranscriptional maturation of miRNAs by interacting with miRNA processing factors via NR co-regulators. Moreover, miRNAs regulate NR signaling by targeting the mRNAs of NR co-regulators and target genes. This complex set of interactions also leads to an extensive network of feedback and feedforward regulatory loops. [ABSTRACT FROM AUTHOR]

Abstract: Steroid hormone receptors (SHRs) are notorious intracellular travellers, transiting among different cellular compartments as they mature, are subjected to regulation and exert their biological functions. Understanding the processes governing the intracellular traffic of SHRs is important, since their unbalanced or erroneous localization could lead to the development of diseases. In this review, we not only explore the functions of the heat-shock protein 90 (Hsp90) molecular chaperone machine for the intracellular transport of SHRs, but also for the regulation of their nuclear mobility, for their recycling and for the regulation of their transcriptional output. [Copyright &y& Elsevier]

The steroid hormone oestrogen can signal through several receptors and pathways. Although the transcriptional responses mediated by the nuclear oestrogen receptors (ER) have been extensively characterized, the changes in gene expression elicited by signalling through the membrane-associated ER GPR30 have not been studied. We show here for ER-negative human breast cancer cells that the activation of GPR30 signalling by oestrogen or by hydroxytamoxifen (OHT), an ER antagonist but GPR30 agonist, induces a transcription factor network, which resembles that induced by serum in fibroblasts. The most strongly induced gene, CTGF, appears to be a target of these transcription factors. We found that the secreted factor connective tissue growth factor (CTGF) not only contributes to promote proliferation but also mediates the GPR30-induced stimulation of cell migration. These results provide a framework for understanding the physiological and pathological functions of GPR30. As the activation of GPR30 by OHT also induces CTGF in fibroblasts from breast tumour biopsies, these pathways may be involved in promoting aggressive behaviour of breast tumours in response to endogenous oestrogens or to OHT being used for endocrine therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Tamoxifen has been used for more than 40 years to treat breast tumors that are dependent on the hormone estrogen for their growth. However, resistance and recurrence of the tumors during the course of the treatment are common. Understanding the mechanisms that drive tamoxifen resistance and discovering new biomarkers for early detection are keys for designing appropriate personalized therapies. Here, we show that low levels of SPRED2 may be useful as a novel biomarker of tamoxifen resistance. We found that SPRED2 deficiency causes a hyperactivation of the mitogen-activated protein kinases (MAPKs) ERK1/ERK2, which in turn enhances estrogen signaling and diminishes the toxic effects of tamoxifen on breast cancer cells. Treatment with the ERK1/2 inhibitor, ulixertinib, could restore their sensitivity to tamoxifen. Therefore, we propose that patients with estrogen-dependent breast cancer characterized by low expression levels of SPRED2 may be candidates for a combination therapy with tamoxifen and ulixertinib. Breast cancer is the number one cause of cancer-related mortality in women worldwide. Most breast tumors depend on the expression of the estrogen receptor α (ERα) for their growth. For this reason, targeting ERα with antagonists such as tamoxifen is the therapy of choice for most patients. Although initially responsive to tamoxifen, about 40% of the patients will develop resistance and ultimately a recurrence of the disease. Thus, finding new biomarkers and therapeutic approaches to treatment-resistant tumors is of high significance. SPRED2, an inhibitor of the MAPK signal transduction pathway, has been found to be downregulated in various cancers. In the present study, we found that SPRED2 is downregulated in a large proportion of breast-cancer patients. Moreover, the knockdown of SPRED2 significantly increases cell proliferation and leads to tamoxifen resistance of breast-cancer cells that are initially tamoxifen-sensitive. We found that resistance occurs through increased activation of the MAPKs ERK1/ERK2, which enhances the transcriptional activity of ERα. Treatment of SPRED2-deficient breast cancer cells with a combination of the ERK 1/2 inhibitor ulixertinib and 4-hydroxytamoxifen (4-OHT) can inhibit cell growth and proliferation and overcome the induced tamoxifen resistance. Taken together, these results indicate that SPRED2 may also be a tumor suppressor for breast cancer and that it is a key regulator of cellular sensitivity to 4-OHT. [ABSTRACT FROM AUTHOR]

Background: The estrogen receptor α (ERα) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERa in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. Methodology/Principal Findings: We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERα-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERα primarily activates and represses genes, respectively. The combined treatments with the anti-estrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. Conclusions/Significance: The activating signal dictates both target gene selection and regulation by ERα, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERα-dependent carcinomas. [ABSTRACT FROM AUTHOR]

Abstract: The glucocorticoid receptor is a known regulator of a variety of physiological processes. Its mode of action is well defined: upon hormone binding, it undergoes a conformational change, translocates to the nucleus and modulates the transcription of target genes. Molecular chaperones have a widely recognized role in the folding of newly made proteins, but their participation in further maturation of folded proteins to their active states and beyond tends to be underestimated. This review presents the current knowledge on how the Hsp70 and Hsp90 chaperone machines help to shape the responses to glucocorticoids. We discuss the contributions of these molecular chaperones to folding, activation, intracellular transport, transcriptional regulation, and decay of the glucocorticoid receptor. [Copyright &y& Elsevier]

Abstract: The abundant molecular chaperone Hsp90 functions in association with co-chaperones including p23 to promote the folding and maturation of a subset of cytosolic proteins. “Fluorescence recovery after photobleaching” (FRAP) experiments showed that the dynamics of p23 in live cells is dictated by Hsp90. Since Hsp90 is present in large excess over p23, the mobility of Hsp90 could conceivably be quite different. To facilitate the analysis and to allow a direct comparison with p23, we developed a 2-color FRAP technique. Two test proteins are expressed as fusion proteins with the two spectrally separable fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP). The 2-color FRAP technique is powerful for the concomitant recording of two proteins located in the same area of a cell, two components of the same protein complex, or mutant and wild-type versions of the same protein under identical experimental conditions. 2-color FRAP of Hsp90 and p23 is virtually indistinguishable, consistent with the notion that they are both engaged in a multitude of large protein complexes. However, when Hsp90–p23 complexes are disrupted by the Hsp90 inhibitor geldanamycin, p23 moves by free diffusion while Hsp90 maintains its low mobility because it remains bound in remodeled multicomponent complexes. [Copyright &y& Elsevier]

Those that efface themselves in the action tend to be forgotten. But molecular chaperones are always there, often serving as equal partners. Because of their intrinsic functional frailty, a large number of signaling molecules have come to depend on molecular chaperones, notably the Hsp90 chaperone machine. This applies to the subset of nuclear receptors that converts steroid hormone signals to transcriptional outputs. Steroid receptors appear to rely on the Hsp90 machine for folding, regulation of the allosteric switch and recycling. This review discusses the complexities of the chaperone machinery and the diversity of regulatory options afforded by this assistance for hormone action. [Copyright &y& Elsevier]

Nuclear receptors (NRs) are a large family of transcription factors. One hallmark of this family is the ligand-binding domain (LBD), for its primary sequence, structure, and regulatory function. To date, NRs have been found exclusively in animals and sponges, which has led to the generally accepted notion that they arose with them. We have overcome the limitations of primary sequence searches by combining sequence profile searches with structural predictions at a genomic scale, and have discovered that the heterodimeric transcription factors Oaf1/Pip2 of the budding yeast Saccharomyces cerevisiae contain putative LBDs resembling those of animal NRs. Although the Oaf1/Pip2 LBDs are embedded in an entirely different architecture, the regulation and function of these transcription factors are strikingly similar to those of the mammalian NR heterodimer peroxisome proliferator-activated receptor α/retinoid X receptor (PPARα/RXR). We demonstrate that the induction of Oaf1/Pip2 activity by the fatty acid oleate depends on oleate's direct binding to the Oaf1 LBD. The alteration of two amino acids in the predicted ligand-binding pocket of Oaf1 abolishes both ligand binding and the transcriptional response. Hence, LBDs may have arisen as allosteric switches, for example, to respond to nutritional and metabolic ligands, before the animal and fungal lineages diverged. [ABSTRACT FROM AUTHOR]

Abstract: p23 is a component of the Hsp90 molecular chaperone machine. It binds and stabilizes the ATP-bound dimeric form of Hsp90. Since Hsp90 binds protein substrates in the ATP conformation, p23 has been proposed to stabilize Hsp90–substrate complexes. In addition, p23 can also function as a molecular chaperone by itself and even possesses an unrelated enzymatic activity. Whether it fulfills the latter functions in cells while bound to Hsp90 remains unknown and is difficult to extrapolate from cell-free biochemical experiments. Using the “fluorescence recovery after photobleaching” (FRAP) technology, I have examined the dynamics of human p23, expressed as a fusion protein with the green fluorescent protein (GFP), in living human HeLa cells. GFP-p23 is distributed throughout the cell, and its mobility is identical in the cytoplasm and in the nucleus. When the Hsp90 interaction is disrupted either with the Hsp90 inhibitor geldanamycin or by introduction of point mutations into p23, the mobility of p23 is greatly accelerated. Under these conditions, its intracellular movement may be diffusion-controlled. In contrast, when wild-type p23 is able to bind Hsp90, a more complex FRAP behavior is observed, suggesting that it is quantitatively bound in Hsp90 complexes undergoing a multitude of other interactions. [Copyright &y& Elsevier]

Cell death and survival play a key role in the immune system as well as during development. The control mechanisms that balance cell survival against cell death are not well understood. Here we report a novel strategy used by a single protein to regulate chronologically cell survival and death. The interferon-induced protein kinase PKR acts as a molecular clock by using catalysis-dependent and -independent activities to temporally induce cell survival prior to cell death. We show that the proapoptotic protein PKR surprisingly activates a survival pathway, which is mediated by NF-κB to delay apoptosis. Cell death is then induced by PKR through the phosphorylation of eIF-2α. This unique temporal control might serve as a paradigm for other kinases whose catalytic activity is not required for all of their functions. [ABSTRACT FROM AUTHOR]

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin-related co-chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone-dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone-dependent reporter gene activation by as much as 20-fold at limiting hormone concentrations, and this potentiation is readily blocked by co-expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone-binding affinity that requires both the Hsp90-binding ability and the prolyl isomerase activity of FKBP52. [ABSTRACT FROM AUTHOR]

PKR, a member of the eukaryotic initiation‐factor 2α (eIF‐2α) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co‐chaperone p23 bind to PKR through its N‐terminal double‐stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF‐2α. A short‐term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf–MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates. [ABSTRACT FROM AUTHOR]

Examines the in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast. Complementation of N-terminal beta-galactosidase coexpressed on alpha-peptide in Escherichia coli. Exploitation of plasmid cloning on blue-white screening method; Expression of individual subunits of protein complex.

Simple Summary: Here, we review the literature on Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) function and regulation and its role in cancer. Elevated expression of AP4 was detected in tumors of various organs and is associated with poor patient survival. AP4 is encoded by a Myc target gene and mediates cell fate decisions by regulating multiple processes, such as cell proliferation, epithelial-mesenchymal transition, stemness, apoptosis, and cellular senescence. Thereby, AP4 may be critical for tumor initiation and progression. In this review article, we summarize published evidence showing how AP4 functions as a transcriptional activator and repressor of a plethora of direct target genes in various physiological and pathological conditions. We also highlight the complex interactions of AP4 with c-Myc, N-Myc, p53, lncRNAs, and miRNAs in feed-back loops, which control AP4 levels and mediate AP4 functions. In the future, a better understanding of AP4 may contribute to improved prognosis and therapy of cancer. Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) is a basic-helix-loop-helix-leucine-zipper transcription factor that was first identified as a protein bound to SV40 promoters more than 30 years ago. Almost 15 years later, AP4 was characterized as a target of the c-Myc transcription factor, which is the product of a prototypic oncogene that is activated in the majority of tumors. Interestingly, AP4 seems to represent a central hub downstream of c-Myc and N-Myc that mediates some of their functions, such as proliferation and epithelial-mesenchymal transition (EMT). Elevated AP4 expression is associated with progression of cancer and poor patient prognosis in multiple tumor types. Deletion of AP4 in mice points to roles of AP4 in the control of stemness, tumor initiation and adaptive immunity. Interestingly, ex vivo AP4 inactivation results in increased DNA damage, senescence, and apoptosis, which may be caused by defective cell cycle progression. Here, we will summarize the roles of AP4 as a transcriptional repressor and activator of target genes and the contribution of protein and non-coding RNAs encoded by these genes, in regulating the above mentioned processes. In addition, proteins interacting with or regulating AP4 and the cellular signaling pathways altered after AP4 dysregulation in tumor cells will be discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: A high pathological complete response in the neoadjuvant setting is directly associated with a better overall response. A favorable prognosis is achieved when preoperative chemo or endocrine therapy succeeds in achieving a high pathological complete response (total eradication of tumors in the breast and the lymph nodes). Approximately 70% of breast cancers are ER-positive. The growth and progression of ER-positive breast cancers are critically dependent on estrogen receptor signaling. Although endocrine therapies (tamoxifen, an aromatase inhibitor, and fulvestrant) in ER-positive breast cancers are the backbone of adjuvant setting, the efficacy of such therapies in terms of achieving a pathological complete response is not encouraging in the neoadjuvant setting. Similar results are observed following targeted therapies in a neoadjuvant setting. Reviewing the literature in the context of different therapies of ER-positive breast cancers in the neoadjuvant setting, here we propose two hypothetical strategies to induce apoptosis based on the background of genomic alterations in the tumor tissues. A strong association of pCR (pathological complete response) with disease-free survival or overall survival is clinically desirable. The association of pCR with disease-free survival or overall survival in ER+/HER2−breast cancers following neoadjuvant systemic therapy (NAT) or neoadjuvant endocrine therapy (NET) is relatively low as compared to the other two subtypes of breast cancers, namely triple-negative and HER2+ amplified. On the bright side, a neoadjuvant model offers a potential opportunity to explore the efficacy of novel therapies and the associated genomic alterations, thus providing a rare personalized insight into the tumor's biology and the tumor cells' response to the drug. Several decades of research have taught us that the disease's biology is a critical factor determining the tumor cells' response to any therapy and hence the final outcome of the disease. Here we propose two scenarios wherein apoptosis can be induced in ER+/HER2− breast cancers expressing wild type TP53 and RB genes following combinations of BCL2 inhibitor, MDM2 inhibitor, and cell-cycle inhibitor. The suggested combinations are contextual and based on the current understanding of the cell signaling in the ER+/HER2− breast cancers. The two combinations of drugs are (1) BCL2 inhibitor plus a cell-cycle inhibitor, which can prime the tumor cells for apoptosis, and (2) BCL2 inhibitor plus an MDM2 inhibitor. [ABSTRACT FROM AUTHOR]

miR-22 is one of the most abundant miRNAs in the liver and alterations of its hepatic expression have been associated with the development of hepatic steatosis and insulin resistance, as well as cancer. However, the pathophysiological roles of miR-22-3p in the deregulated hepatic metabolism with obesity and cancer remains poorly characterized. Herein, we observed that alterations of hepatic miR-22-3p expression with non-alcoholic fatty liver disease (NAFLD) in the context of obesity are not consistent in various human cohorts and animal models in contrast to the well-characterized miR-22-3p downregulation observed in hepatic cancers. To unravel the role of miR-22 in obesity-associated NAFLD, we generated constitutive Mir22 knockout (miR-22KO) mice, which were subsequently rendered obese by feeding with fat-enriched diet. Functional NAFLD- and obesity-associated metabolic parameters were then analyzed. Insights about the role of miR-22 in NAFLD associated with obesity were further obtained through an unbiased proteomic analysis of miR-22KO livers from obese mice. Metabolic processes governed by miR-22 were finally investigated in hepatic transformed cancer cells. Deletion of Mir22 was asymptomatic when mice were bred under standard conditions, except for an onset of glucose intolerance. However, when challenged with a high fat-containing diet, Mir22 deficiency dramatically exacerbated fat mass gain, hepatomegaly, and liver steatosis in mice. Analyses of explanted white adipose tissue revealed increased lipid synthesis, whereas mass spectrometry analysis of the liver proteome indicated that Mir22 deletion promotes hepatic upregulation of key enzymes in glycolysis and lipid uptake. Surprisingly, expression of miR-22-3p in Huh7 hepatic cancer cells triggers, in contrast to our in vivo observations, a clear induction of a Warburg effect with an increased glycolysis and an inhibited mitochondrial respiration. Together, our study indicates that miR-22-3p is a master regulator of the lipid and glucose metabolism with differential effects in specific organs and in transformed hepatic cancer cells, as compared to non-tumoral tissue. [ABSTRACT FROM AUTHOR]

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation. Hop, also known as Stip1 or Sti1, facilitates substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Characterization of proteostasis-related pathways in STIP1 knock-out cell lines reveals that in eukaryotes Stip1 modulates the balance between protein folding and degradation. [ABSTRACT FROM AUTHOR]

We have developed a software, Cell Database, for archiving records about cells stored in liquid nitrogen tanks. Once installed on a web server, the database is accessed through a standard web browser. This user-friendly and self-explanatory application is independent of computer platform and periodic upgrades of a commercial software. Our web application allows import of data from other database programs and adaptation to different tank formats, types of samples, and archiving needs. [ABSTRACT FROM AUTHOR]

The HSP90 paralog TRAP1 was discovered more than 20 years ago; yet, a detailed understanding of the function of this mitochondrial molecular chaperone remains elusive. The dispensable nature of TRAP1 in vitro and in vivo further complicates an understanding of its role in mitochondrial biology. TRAP1 is more homologous to the bacterial HSP90, HtpG, than to eukaryotic HSP90. Lacking co-chaperones, the unique structural features of TRAP1 likely regulate its temperature-sensitive ATPase activity and shed light on the alternative mechanisms driving the chaperone's nucleotide-dependent cycle in a defined environment whose physiological temperature approaches 50 °C. TRAP1 appears to be an important bioregulator of mitochondrial respiration, mediating the balance between oxidative phosphorylation and glycolysis, while at the same time promoting mitochondrial homeostasis and displaying cytoprotective activity. Inactivation/loss of TRAP1 has been observed in several neurodegenerative diseases while TRAP1 expression is reported to be elevated in multiple cancers and, as with HSP90, evidence of addiction to TRAP1 has been observed. In this review, we summarize what is currently known about this unique HSP90 paralog and why a better understanding of TRAP1 structure, function, and regulation is likely to enhance our understanding of the mechanistic basis of mitochondrial homeostasis. [ABSTRACT FROM AUTHOR]