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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. [ABSTRACT FROM AUTHOR]

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging. Nuclear DNA damage downstream of mitochondrial ROS is often cited to contribute to cancer initiation and aging. However, here the authors show that although H2O2 induces DNA mutations when produced near DNA, it does not when released by mitochondria. [ABSTRACT FROM AUTHOR]

Forkhead box O (FOXO) transcription factors (TFs) are a subclass of the larger family of forkhead TFs. Mammalians express four members FOXO1, FOXO3, FOXO4, and FOXO6. The interest in FOXO function stems mostly from their observed role in determining lifespan, where in model organisms, increased FOXO activity results in extended lifespan. FOXOs act as downstream of several signaling pathway and are extensively regulated through post‐translational modifications. The transcriptional program activated by FOXOs in various cell types, organisms, and under various conditions has been described and has shed some light on what the critical transcriptional targets are in mediating FOXO function. At the cellular level, these studies have revealed a role for FOXOs in cell metabolism, cellular redox, cell proliferation, DNA repair, autophagy, and many more. The general picture that emerges hereof is that FOXOs act to preserve equilibrium, and they are important for cellular homeostasis. Here, we will first briefly summarize the general knowledge of FOXO regulation and possible functions. We will use genomic stability to illustrate how FOXOs ensure homeostasis. Genomic stability is critical for maintaining genetic integrity, and therefore preventing disease. However, genomic mutations need to occur during lifetime to enable evolution, yet their accumulation is believed to be causative to aging. Therefore, the role of FOXO in genomic stability may underlie its role in lifespan and aging. Finally, we will come up with questions on some of the unknowns in FOXO function, the answer(s) to which we believe will further our understanding of FOXO function and ultimately may help to understand lifespan and its consequences. [ABSTRACT FROM AUTHOR]

Despite the fact that 5-fluorouracil (5-FU) is the backbone for chemotherapy in colorectal cancer (CRC), the response rates in patients is limited to 50%. The mechanisms underlying 5-FU toxicity are debated, limiting the development of strategies to improve its efficacy. How fundamental aspects of cancer, such as driver mutations and phenotypic heterogeneity, relate to the 5-FU response remains obscure. This largely relies on the limited number of studies performed in pre-clinical models able to recapitulate the key features of CRC. Here, we analyzed the 5-FU response in patient-derived organoids that reproduce the different stages of CRC. We find that 5-FU induces pyrimidine imbalance, which leads to DNA damage and cell death in the actively proliferating cancer cells deficient in p53. Importantly, p53-deficiency leads to cell death due to impaired cell cycle arrest. Moreover, we find that targeting the Warburg effect in KRASG12D glycolytic tumor organoids enhances 5-FU toxicity by further altering the nucleotide pool and, importantly, without affecting non-transformed WT cells. Thus, p53 emerges as an important factor in determining the 5-FU response, and targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment. In p53-deficient colorectal cancer organoids, 5-fluorouracil induces pyrimidine imbalance, which causes DNA damage and cell death. Rewiring glucose metabolism through PDK inhibition by DCA enhances 5-FU toxicity in glycolytic p53-deficient organoids. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance has become one of the major threats to human health. Therefore, there is a strong need for novel antimicrobials with new mechanisms of action. The kingdom of fungi is an excellent source of antimicrobials for this purpose because it encompasses countless fungal species that harbor unusual metabolic pathways. Previously, we have established a library of secondary metabolites from 10,207 strains of fungi. Here, we screened for antimicrobial activity of the library against seven pathogenic bacterial strains and investigated the identity of the active compounds using ethyl acetate extraction, activity-directed purification using HPLC fractionation and chemical analyses. We initially found 280 antimicrobial strains and subsequently identified 17 structurally distinct compounds from 26 strains upon further analysis. All but one of these compounds, berkchaetoazaphilone B (BAB), were known to have antimicrobial activity. Here, we studied the antimicrobial properties of BAB, and found that BAB affected energy metabolism in both prokaryotic and eukaryotic cells. We conclude that fungi are a rich source of chemically diverse secondary metabolites with antimicrobial activity. [ABSTRACT FROM AUTHOR]

The transcription factor forkhead box protein P2 (FOXP2) is a highly conserved key regulator of embryonal development. The molecular mechanisms of how FOXP2 regulates embryonal development, however, remain elusive. Using RNA sequencing, we identified the Wnt signaling pathway as key target of FOXP2‐dependent transcriptional regulation. Using cell‐based assays, we show that FOXP2 transcriptional activity is regulated by the Wnt coregulator β‐catenin and that β‐catenin contacts multiple regions within FOXP2. Using nuclear magnetic resonance spectroscopy, we uncovered the molecular details of these interactions. β‐catenin contacts a disordered FOXP2 region with α‐helical propensity via its folded armadillo domain, whereas the intrinsically disordered β‐catenin N terminus and C terminus bind to the conserved FOXP2 DNA‐binding domain. Using RNA sequencing, we confirmed that β‐catenin indeed regulates transcriptional activity of FOXP2 and that the FOXP2 α‐helical motif acts as a key regulatory element of FOXP2 transcriptional activity. Taken together, our findings provide first insight into novel regulatory interactions and help to understand the intricate mechanisms of FOXP2 function and (mis)‐regulation in embryonal development and human diseases. Database: Expression data are available in the GEO database under the accession number GSE138938. [ABSTRACT FROM AUTHOR]

Objective A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. Methods pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). Results In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. Conclusion The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival. [ABSTRACT FROM AUTHOR]

Objectives: Considering the critical role of microRNAs (miRNAs) in regulation of cell activation, we investigated their role in circulating type-2 conventional dendritic cells (cDC2s) of patients with primary Sjögren's syndrome (pSS) compared to healthy controls (HC). Methods: CD1c-expressing cDC2s were isolated from peripheral blood. A discovery cohort (15 pSS, 6 HC) was used to screen the expression of 758 miRNAs and a replication cohort (15 pSS, 11 HC) was used to confirm differential expression of 18 identified targets. Novel targets for two replicated miRNAs were identified by SILAC in HEK-293T cells and validated in primary cDC2s. Differences in cytokine production between pSS and HC cDC2s were evaluated by intracellular flow-cytometry. cDC2s were cultured in the presence of MSK1-inhibitors to investigate their effect on cytokine production. Results: Expression of miR-130a and miR-708 was significantly decreased in cDC2s from pSS patients compared to HC in both cohorts, and both miRNAs were downregulated upon stimulation via endosomal TLRs. Upstream mediator of cytokine production MSK1 was identified as a novel target of miR-130a and overexpression of miR-130a reduced MSK1 expression in cDC2s. pSS cDC2s showed higher MSK1 expression and an increased fraction of IL-12 and TNF-α-producing cells. MSK1-inhibition reduced cDC2 activation and production of IL-12, TNF-α, and IL-6. Conclusions: The decreased expression of miR-130a and miR-708 in pSS cDC2s seems to reflect cell activation. miR-130a targets MSK1, which regulates pro-inflammatory cytokine production, and we provide proof-of-concept for MSK1-inhibition as a therapeutic avenue to impede cDC2 activity in pSS. [ABSTRACT FROM AUTHOR]

Genetic lipodystrophies are a group of rare syndromes associated with major metabolic complications – including severe insulin resistance, type 2 diabetes mellitus, and hypertriglyceridemia – which are classified according to the distribution of adipose tissue. Lipodystrophies can be present at birth or develop during life and can range from local to partial and general. With at least 18 different genes implicated so far, definite diagnosis can be challenging due to clinical and genetic heterogeneity. In an adult female patient with clinical and metabolic features of partial lipodystrophy we identified via whole genome sequencing (WGS) a single complex AGPAT2 allele [V67M;V167A], functionally equivalent to heterozygosity. AGPAT2 encodes for an acyltransferase implicated in the biosynthesis of triacylglycerol and glycerophospholipids. So far homozygous and compound heterozygous mutations in AGPAT2 have only been associated with generalized lipodystrophy. A SNP risk score analysis indicated that the index patient is not predisposed to lipodystrophy based on her genetic background. The partial phenotype in our patient is therefore more likely associated to the genetic variants in AGPAT2. To test whether the resulting double-mutant AGPAT2 protein is functional we analyzed its in vitro enzymatic activity via mass spectrometry. The resulting AGPAT2 double mutant is enzymatically inactive. Our data support the view that the current classification of lipodystrophies as strictly local, partial or generalized may have to be re-evaluated and viewed more as a continuum, both in terms of clinical presentation and underlying genetic causes. Better molecular understanding of lipodystrophies may lead to new therapies to treat adipose tissue dysfunction in common and rare diseases. [ABSTRACT FROM AUTHOR]

Abstract: Intestinal organoids accurately recapitulate epithelial homeostasis in vivo, thereby representing a powerful in vitro system to investigate lineage specification and cellular differentiation. Here, we applied a multi‐omics framework on stem cell‐enriched and stem cell‐depleted mouse intestinal organoids to obtain a holistic view of the molecular mechanisms that drive differential gene expression during adult intestinal stem cell differentiation. Our data revealed a global rewiring of the transcriptome and proteome between intestinal stem cells and enterocytes, with the majority of dynamic protein expression being transcription‐driven. Integrating absolute mRNA and protein copy numbers revealed post‐transcriptional regulation of gene expression. Probing the epigenetic landscape identified a large number of cell‐type‐specific regulatory elements, which revealed Hnf4g as a major driver of enterocyte differentiation. In summary, by applying an integrative systems biology approach, we uncovered multiple layers of gene expression regulation, which contribute to lineage specification and plasticity of the mouse small intestinal epithelium. [ABSTRACT FROM AUTHOR]

Organoid technology provides the possibility of culturing patient-derived colon tissue and colorectal cancers (CRCs) while maintaining all functional and phenotypic characteristics. Labeling stem cells, especially in normal and benign tumor organoids of human colon, is challenging and therefore limits maximal exploitation of organoid libraries for human stem cell research. Here, we developed STAR (stem cell Ascl2 reporter), a minimal enhancer/promoter element that reports transcriptional activity of ASCL2, a master regulator of LGR5+ intestinal stem cells. Using lentiviral infection, STAR drives specific expression in stem cells of normal organoids and in multiple engineered and patient-derived CRC organoids of different genetic makeup. STAR reveals that differentiation hierarchies and the potential for cell fate plasticity are present at all stages of human CRC development. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research into human adult stem cell biology. [ABSTRACT FROM AUTHOR]

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 19 (FGF19) is a gut-derived peptide hormone that is produced following activation of Farnesoid X Receptor (FXR). FGF19 is secreted and signals to the liver, where it contributes to the homeostasis of bile acid (BA), lipid and carbohydrate metabolism. FGF19 is a promising therapeutic target for the metabolic syndrome and cholestatic diseases, but enthusiasm for its use has been tempered by FGF19-mediated induction of proliferation and hepatocellular carcinoma. To inform future rational design of FGF19-variants, we have conducted temporal quantitative proteomic and gene expression analyses to identify FGF19-targets related to metabolism and proliferation. Mice were fasted for 16 hours, and injected with human FGF19 (1 mg/kg body weight) or vehicle. Liver protein extracts (containing “light” lysine) were mixed 1:1 with a spike-in protein extract from 13C6-lysine metabolically labelled mouse liver (containing “heavy” lysine) and analysed by LC-MS/MS. Our analyses provide a resource of FGF19 target proteins in the liver. 189 proteins were upregulated (≥ 1.5 folds) and 73 proteins were downregulated (≤ -1.5 folds) by FGF19. FGF19 treatment decreased the expression of proteins involved in fatty acid (FA) synthesis, i.e., Fabp5, Scd1, and Acsl3 and increased the expression of Acox1, involved in FA oxidation. As expected, FGF19 increased the expression of proteins known to drive proliferation (i.e., Tgfbi, Vcam1, Anxa2 and Hdlbp). Importantly, many of the FGF19 targets (i.e., Pdk4, Apoa4, Fas and Stat3) have a dual function in both metabolism and cell proliferation. Therefore, our findings challenge the development of FGF19-variants that fully uncouple metabolic benefit from mitogenic potential. [ABSTRACT FROM AUTHOR]

Growth factors inactivate the FOXO (forkhead box O) transcription factors through PI3K (phosphoinositide 3-kinase) and PKB (protein kinase B). By comparing microarray data from multiple model systems, we identified HBP1 (high-mobility group-box protein 1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), PI3K and PKB, whereas it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukaemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and ChIP, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by shRNA increased the proliferation of human fibroblasts in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analysing a transcriptomics dataset from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumours that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K/FOXO pathway and controls cell proliferation in response to growth factors. [ABSTRACT FROM AUTHOR]

Forkhead box O (FOXO) transcription factors are involved in the regulation of the cell cycle, apoptosis and metabolism. In model organisms, FOXO activity also affects stem cell maintenance and lifespan as well as age-related diseases, such as cancer and diabetes. Multiple upstream pathways regulate FOXO activity through post-translational modifications and nuclear-cytoplasmic shuttling of both FOXO and its regulators. The diversity of this upstream regulation and the downstream effects of FOXOs suggest that they function as homeostasis regulators to maintain tissue homeostasis over time and coordinate a response to environmental changes, including growth factor deprivation, metabolic stress (starvation) and oxidative stress. [ABSTRACT FROM AUTHOR]

Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO‐regulated target gene expression, we studied genome‐wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP‐seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre‐existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3‐mediated enhancer activation correlates with regulation of adjacent genes and pre‐existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. [ABSTRACT FROM AUTHOR]

In this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component. Using biochemical interaction studies, we found Ku70 to be necessary and sufficient for the interaction. FOXO4 mediates its tumor-suppressive function in part through transcrip-tional regulation of the cell cycle arrest p27kip1 gene. Immunoblotting, luciferase reporter assays, and flow cytometry showed that Ku70 inhibited FOXO4-mediated p27kip1 transcription and cell cycle arrest induction by >40%. In contrast, Ku70 RNAi but not control RNAi significantly increased p27kip1 transcription. In addition, in contrast to wild-type mouse embryonic stem (ES) cells, Ku70-/- ES cells showed significantly increased FOXO activity, which was rescued by Ku70 reexpression. Immunofluorescence studies demonstrated that Ku70 sequestered FOXO4 in the nucleus. Interestingly, the Ku70-FOXO4 interaction stoichiometry followed a nonlinear dose-response curve by hydrogen peroxide-generated oxidative stress. Low levels of oxidative stress increased interaction stoichiometry up to 75%, peaking at 50 u.M, after which dissociation occurred. Because the Ku70 ortholog in the roundworm Caenorhabditis elegans was shown to regulate life span involving C. elegans FOXO, our findings suggest a conserved critical Ku70 role for FOXO function toward coordination of a survival program, regulated by the magnitude of oxidative damage. [ABSTRACT FROM AUTHOR]

Cellular damage invoked by reactive oxygen species plays a key role in the pathobiology of cancer and aging. Forkhead box class O (FoxO) transcription factors are involved in various cellular processes including cell cycle regulation, apoptosis and resistance to reactive oxygen species, and studies in animal models have shown that these transcription factors are of vital importance in tumor suppression, stem cell maintenance and lifespan extension. Here we report that the activity of FoxO in human cells is directly regulated by the cellular redox state through a unique mechanism in signal transduction. We show that reactive oxygen species induce the formation of cysteine-thiol disulfide–dependent complexes of FoxO and the p300/CBP acetyltransferase, and that modulation of FoxO biological activity by p300/CBP-mediated acetylation is fully dependent on the formation of this redox-dependent complex. These findings directly link cellular redox status to the activity of the longevity protein FoxO. [ABSTRACT FROM AUTHOR]

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses. [ABSTRACT FROM AUTHOR]

cAMP is a potent inhibitor of cell proliferation in a variety of cell lines. Downregulation of cyclin D1 and upregulation of the cell cycle inhibitor p27Kip1 are two mechanisms by which cAMP may induce a G1-arrest. Here we show that cAMP inhibits proliferation of cells that constitutively express cyclin D1 or are deficient for Rb, demonstrating that changes in these cell cycle regulators do not account for the cAMP-induced growth effects in mouse embryo fibroblasts (MEFs). Interestingly, the antiproliferative effect of cAMP mimics the effect previously observed for FoxO transcription factors. These transcription factors are under negative control of protein kinase B (PKB). We show that in MEFs cAMP strongly induces transcriptional activation of FoxO4 through the inhibition of PKB. Accordingly, not only p27Kip1 but also the FoxO target MnSOD is upregulated by cAMP. Importantly, introduction of dominant-negative FoxO partially rescues cAMP-induced inhibition of proliferation. From these results we conclude that inhibition of PKB and subsequent activation of FoxO transcription factors mediates an antiproliferative effect of cAMP.Oncogene (2005) 24, 2087-2095. doi:10.1038/sj.onc.1208450 Published online 31 January 2005 [ABSTRACT FROM AUTHOR]

It is more than a decade since the discovery of the first forkhead-box (FOX) transcription factor in the fruit fly Drosophila melanogaster. In the intervening time, there has been an explosion in the identification and characterization of members of this family of proteins. Importantly, in the past few years, it has become clear that members of the FOX family have crucial roles in various aspects of immune regulation, from lymphocyte survival to thymic development. This review focuses on FOXP3, FOXN1, FOXJ1 and members of the FOXO subfamily and their function in the immune system. [ABSTRACT FROM AUTHOR]

Reversible cysteine oxidation plays an essential role in redox signaling by reversibly altering protein structure and function. Cysteine oxidation may lead to intra- and intermolecular disulfide formation, and the latter can drastically stabilize protein–protein interactions in a more oxidizing milieu. The activity of the tumor suppressor p53 is regulated at multiple levels, including various post-translational modification (PTM) and protein–protein interactions. In the past few decades, p53 has been shown to be a redox-sensitive protein, and undergoes reversible cysteine oxidation both in vitro and in vivo. It is not clear, however, whether p53 also forms intermolecular disulfides with interacting proteins and whether these redox-dependent interactions contribute to the regulation of p53. In the present study, by combining (co-)immunoprecipitation, quantitative mass spectrometry and Western blot we found that p53 forms disulfide-dependent interactions with several proteins under oxidizing conditions. Cysteine 277 is required for most of the disulfide-dependent interactions of p53, including those with 14-3-3θ and 53BP1. These interaction partners may play a role in fine-tuning p53 activity under oxidizing conditions. [ABSTRACT FROM AUTHOR]

Redox signaling is controlled by the reversible oxidation of cysteine thiols, a post-translational modification triggered by H2O2 acting as a second messenger. However, H2O2 actually reacts poorly with most cysteine thiols and it is not clear how H2O2 discriminates between cysteines to trigger appropriate signaling cascades in the presence of dedicated H2O2 scavengers like peroxiredoxins (PRDXs). It was recently suggested that peroxiredoxins act as peroxidases and facilitate H2O2-dependent oxidation of redox-regulated proteins via disulfide exchange reactions. It is unknown how the peroxiredoxin-based relay model achieves the selective substrate targeting required for adequate cellular signaling. Using a systematic mass-spectrometry-based approach to identify cysteine-dependent interactors of the five human 2-Cys peroxiredoxins, we show that all five human 2-Cys peroxiredoxins can form disulfide-dependent heterodimers with a large set of proteins. Each isoform displays a preference for a subset of disulfide-dependent binding partners, and we explore isoform-specific properties that might underlie this preference. We provide evidence that peroxiredoxin-based redox relays can proceed via two distinct molecular mechanisms. Altogether, our results support the theory that peroxiredoxins could play a role in providing not only reactivity but also selectivity in the transduction of peroxide signals to generate complex cellular signaling responses. [ABSTRACT FROM AUTHOR]

Compelling evidence shows the involvement of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc) pathogenesis. This study investigated whether microRNAs (miRNAs) are involved in the dysregulation of pDCs in SSc patients already at early stages. RNA from circulating pDCs was isolated from two independent cohorts of SSc patients with different disease phenotypes, and individuals with Raynaud's phenomenon, for microRNA profiling and RNA-sequencing analysis. Proteomic analysis was exploited to identify novel direct miRNA targets at the protein level. Twelve and fifteen miRNAs were differentially expressed in at least one group of patients compared to healthy controls in discovery cohort I and II, respectively. Of note, miR-126 and miR-139-5p were upregulated in both preclinical and definite SSc patients and correlated with the expression of type I interferon (IFN)-responsive genes. Toll-like receptor 9 (TLR9) stimulation of healthy pDCs upregulated the expression of both miRNAs, similarly to what was observed in patients. The proteomic analysis identified USP24 as a novel target of miR-139-5p. The expression level of USP24 was inversely correlated with miR-139-5p expression in SSc patients and induced by TLR9 stimulation in healthy pDCs. These findings demonstrated that the miRNA profile is altered in pDCs of SSc patients already at early stages of the disease and indicate their potential contribution to pDC activation observed in patients. [ABSTRACT FROM AUTHOR]

Although the molecular machinery controlling aging has been well studied in lower organisms, it is unclear whether these mechanisms are conserved in mammals. A recent study supports a role for an evolutionarily conserved transcriptional pathway in maintaining the unlimited lifespan of haematopoietic stem cells. [ABSTRACT FROM AUTHOR]

Oncogene-driven adaptation of metabolism during tumorigenesis includes steps that stimulate the uptake of nutrients, especially glucose and glutamine, to sustain cell growth and proliferation. Macropinocytosis, a clathrin- and caveolin-independent endocytotic process that had previously been linked to the action of oncogenic Ras and Src, is now shown to contribute to amino acid uptake via enhanced delivery of extracellular proteins to lysosomes. [ABSTRACT FROM AUTHOR]