The atypical CDK activator RingoA/Spy1 regulates exit from quiescence in neural stem cells.

In the adult mammalian brain, most neural stem cells (NSCs) are held in a reversible state of quiescence, which is essential to avoid NSC exhaustion and determine the appropriate neurogenesis rate. NSCs of the mouse adult subependymal niche provide neurons for olfactory circuits and can be found at different depths of quiescence, but very little is known on how their quiescence-to-activation transition is controlled. Here, we identify the atypical cyclin-dependent kinase (CDK) activator RingoA as a regulator of this process. We show that the expression of RingoA increases the levels of CDK activity and facilitates cell cycle entry of a subset of NSCs that divide slowly. Accordingly, RingoA-deficient mice exhibit reduced olfactory neurogenesis with an accumulation of quiescent NSCs. Our results indicate that RingoA plays an important role in setting the threshold of CDK activity required for adult NSCs to exit quiescence and may represent a dormancy regulator in adult mammalian tissues.

Synthesis and Biological Activity of a VHL-Based PROTAC Specific for p38α.

We report a series of small molecule proteolysis-targeting chimeras (PROTACs) that target the protein kinase p38α for degradation. These PROTACs are based on a ligand of the VHL E3 ubiquitin ligase, which is linked to an ATP competitive inhibitor of p38α. We provide evidence that these compounds can induce the specific degradation of p38α, but not p38ß and other related kinases, at nanomolar concentrations in several mammalian cell lines. We also show that the p38α-specific PROTACs are soluble in aqueous solutions and therefore suitable for their administration to mice. Systemic administration of the PROTACs induces p38α degradation only in the liver, probably due to the PROTAC becoming inactivated in that organ, but upon local administration the PROTACs induce p38α degradation in mammary tumors. Our compounds provide an alternative to traditional chemical inhibitors for targeting p38α signaling in cultured cells and in vivo.

MK2 degradation as a sensor of signal intensity that controls stress-induced cell fate.

Cell survival in response to stress is determined by the coordination of various signaling pathways. The kinase p38α is activated by many stresses, but the intensity and duration of the signal depends on the stimuli. How different p38α-activation dynamics may impact cell life/death decisions is unclear. Here, we show that the p38α-signaling output in response to stress is modulated by the expression levels of the downstream kinase MK2. We demonstrate that p38α forms a complex with MK2 in nonstimulated mammalian cells. Upon pathway activation, p38α phosphorylates MK2, the complex dissociates, and MK2 is degraded. Interestingly, transient p38α activation allows MK2 reexpression, reassembly of the p38α-MK2 complex, and cell survival. In contrast, sustained p38α activation induced by severe stress interferes with p38α-MK2 interaction, resulting in irreversible MK2 loss and cell death. MK2 degradation is mediated by the E3 ubiquitin ligase MDM2, and we identify four lysine residues in MK2 that are directly ubiquitinated by MDM2. Expression of an MK2 mutant that cannot be ubiquitinated by MDM2 enhances the survival of stressed cells. Our results indicate that MK2 reexpression and binding to p38α is critical for cell viability in response to stress and illustrate how particular p38α-activation patterns induced by different signals shape the stress-induced cell fate.

Optimal linker length for small molecule PROTACs that selectively target p38α and p38ß for degradation.

We report the design of hetero-bifunctional small molecules that selectively target p38α and p38ß for degradation. These proteolysis targeted chimeras (PROTACs) are based on an ATP competitive inhibitor of p38α and p38ß, which is linked to thalidomide analogues to recruit the Cereblon E3 ubiquitin ligase complex. Compound synthesis was facilitated by the use of a copper catalyzed "click" reaction. We show that optimization of the linker length and composition is crucial for the degradation-inducing activity of these PROTACs. We provide evidence that these chemical compounds can induce degradation of p38α and p38ß but no other related kinases at nanomolar concentrations in several mammalian cell lines. Accordingly, the PROTACs inhibit stress and cytokine-induced p38α signaling. Our compounds contribute to understanding the development of PROTACs, and provide a useful tool to investigate functions of the p38 MAPK pathway and its involvement in diseases.

Tris-Acetate Polyacrylamide Gradient Gels for the Simultaneous Electrophoretic Analysis of Proteins of Very High and Low Molecular Mass.

Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris-acetate buffer and 3-15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10-500 kDa. We show that this system is highly sensitive, it has good resolution and high reproducibility, and it can be used for general applications of PAGE such as Coomassie Brilliant Blue staining and immunoblotting. Moreover, we describe how to generate mini Tris-acetate polyacrylamide gels to use them in miniprotein electrophoresis systems. These economical gels are easy to generate and to manipulate and allow a rapid analysis of proteins. All these features make the Tris-acetate-PAGE system a very helpful tool for protein analysis.

The E3 ubiquitin ligase HERC1 controls the ERK signaling pathway targeting C-RAF for degradation.

The RAF/MEK/ERK cascade is a conserved intracellular signaling pathway that controls fundamental cellular processes including growth, proliferation, differentiation, survival and migration. Aberrant regulation of this signaling pathway has long been associated with human cancers. A major point of regulation of this pathway occurs at the level of the serine/threonine protein kinase C-RAF. Here, we show how the E3 ubiquitin ligase HERC1 regulates ERK signaling. HERC1 knockdown induced cellular proliferation, which is associated with an increase in ERK phosphorylation and in C-RAF protein levels. We demonstrate that overexpression of wild-type C-RAF is sufficient to increase ERK phosphorylation. Experiments with pharmacological inhibitors of RAF activity, or with interference RNA, show that the regulation of ERK phosphorylation by HERC1 is RAF-dependent. Immunoprecipitation, pull-down and confocal fluorescence microscopy experiments demonstrate an interaction between HERC1 and C-RAF proteins. Mechanistically, HERC1 controls C-RAF stability by regulating its polyubiquitylation in a lysine 48-linked chain. In vitro ubiquitylation assays indicate that C-RAF is a substrate of the E3 ubiquitin ligase HERC1. Altogether, we show how HERC1 can regulate cell proliferation through the activation of ERK signaling by a mechanism that affects C-RAF's stability.

Myeloid p38α signaling promotes intestinal IGF-1 production and inflammation-associated tumorigenesis.

The protein kinase p38α plays a key role in cell homeostasis, and p38α signaling in intestinal epithelial cells protects against colitis-induced tumorigenesis. However, little is known on the contribution of p38α signaling in intestinal stromal cells. Here, we show that myeloid cell-specific downregulation of p38α protects mice against inflammation-associated colon tumorigenesis. The reduced tumorigenesis correlates with impaired detection in the colon of crucial chemokines for immune cell recruitment. We identify insulin-like growth factor-1 (IGF-1) as a novel mediator of the p38α pathway in macrophages. Moreover, using genetic and pharmacological approaches, we confirm the implication of IGF-1 produced by myeloid cells in colon inflammation and tumorigenesis. We also show a correlation between IGF-1 pathway activation and the infiltration of myeloid cells with active p38α in colon samples from patients with ulcerative colitis or colon cancer. Altogether, our results uncover an important role for myeloid IGF-1 downstream of p38α in colitis-associated tumorigenesis and suggest the interest in evaluating IGF-1 therapies for inflammation-associated intestinal diseases, taking into consideration IGF-1 signaling and immune cell infiltration in patient biopsies.

Mesenchymal stem cells decrease lung inflammation during sepsis, acting through inhibition of the MAPK pathway.

BACKGROUND: Sepsis is a severe medical condition that ranks among the top 10 causes of death worldwide and which has permanently high incidence rates. Mesenchymal stem cells (MSCs) have been found to be potent modulators of immune responses. More importantly, there is evidence that MSCs have a beneficial effect on preclinical models of polymicrobial sepsis. However, the changes caused by the MSCs in the effector cells of the host immune system remain unclear. METHODS: A mouse model of sepsis (male C57BL/6 mice) with three experimental groups was used for experiments in vivo: a control group, an untreated septic group, and a septic group treated with MSCs. In vitro experiments were performed using a cell line of pulmonary macrophages (RAW 264.7) co-cultured with MSCs and stimulated with lipopolysaccharide (LPS). RESULTS: In vivo we demonstrated that treatment with MSCs was able to reduce the expression of cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB), and thereby decrease the production of inflammatory cytokines. In vitro experiments using a co-culture of macrophages with MSCs showed a decrease in COX-2 and NF-κB, and showed that this reduction was directly related to the ability of MSCs to inhibit phosphorylation of ERK, RSK, and p38, enzymes that belong to the family of mitogen-activated protein kinases (MAPKs). CONCLUSIONS: This study demonstrated that MSCs are able to inhibit the MAPK pathway activation, modulating the inflammatory response during sepsis. This understanding that MSCs can remodel the response of host cells and improve the course of sepsis is essential for developing new treatments for this pathology.

NEURL4 regulates the transcriptional activity of tumor suppressor protein p53 by modulating its oligomerization.

p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53's signaling.

p53 inhibits SP7/Osterix activity in the transcriptional program of osteoblast differentiation.

Osteoblast differentiation is achieved by activating a transcriptional network in which Dlx5, Runx2 and Osx/SP7 have fundamental roles. The tumour suppressor p53 exerts a repressive effect on bone development and remodelling through an unknown mechanism that inhibits the osteoblast differentiation programme. Here we report a physical and functional interaction between Osx and p53 gene products. Physical interaction was found between overexpressed proteins and involved a region adjacent to the OSX zinc fingers and the DNA-binding domain of p53. This interaction results in a p53-mediated repression of OSX transcriptional activity leading to a downregulation of the osteogenic programme. Moreover, we show that p53 is also able to repress key osteoblastic genes in Runx2-deficient osteoblasts. The ability of p53 to suppress osteogenesis is independent of its DNA recognition ability but requires a native conformation of p53, as a conformational missense mutant failed to inhibit OSX. Our data further demonstrates that p53 inhibits OSX binding to their responsive Sp1/GC-rich sites in the promoters of their osteogenic target genes, such as IBSP or COL1A1. Moreover, p53 interaction to OSX sequesters OSX from binding to DLX5. This competition blocks the ability of OSX to act as a cofactor of DLX5 to activate homeodomain-containing promoters. Altogether, our data support a model wherein p53 represses OSX-DNA binding and DLX5-OSX interaction, and thereby deregulates the osteogenic transcriptional network. This mechanism might have relevant roles in bone pathologies associated to osteosarcomas and ageing.

Guaraná, a Highly Caffeinated Food, Presents in vitro Antitumor Activity in Colorectal and Breast Cancer Cell Lines by Inhibiting AKT/mTOR/S6K and MAPKs Pathways.

The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinases (MAPKs) pathways are frequently upregulated in cancer. Some authors have reported that some antioxidant molecules could be potential inhibitors of these pathways. Therefore, we investigated the in vitro antitumor effect of guaraná by inhibiting the AKT/mTOR/S6K and MAPKs pathways. Colorectal and breast cancer cell lineages, HT-29 and MCF-7 cells, respectively, were exposed to different guaraná concentrations (0.1, 1, 10, and 100 µg/mL) as well as its main bioactive molecule, caffeine, in proportional concentrations to those found in the extract. Western blot, clonogenic assay, and growth curve were performed. Moreover, we investigated the potential cytotoxic effect of guaraná in normal cells. The results revealed that guaraná and caffeine inhibited some MAPKs proteins (p-p38 and p-HSP27) in MCF-7 cells. However, they did not affect this pathway in HT-29 cells. Furthermore, guaraná inhibited mTORC1 (p-S6K) and mTORC2 (p-AKT) in MCF-7 cells, but only mTORC1 in HT-29 cells. Caffeine only inhibited the mTOR pathway in MCF-7 cells. Guaraná decreased the colony formation and cell growth in MCF-7 and HT-29 cells. Guaraná did not affect normal cells. In conclusion, guaraná could be an important agent in antitumor pharmacologic therapies by inhibiting the mTOR and MAPKs pathways.

Analysis of Protein Oligomerization by Electrophoresis.

A polypeptide chain can interact with other polypeptide chains and form stable and functional complexes called "oligomers." Frequently, biochemical analysis of these complexes is made difficult by their great size. Traditionally, size exclusion chromatography, immunoaffinity chromatography, or immunoprecipitation techniques have been used to isolate oligomers. Components of these oligomers are then further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by immunoblotting with specific antibodies. Although they are sensitive, these techniques are not easy to perform and reproduce. The use of Tris-acetate polyacrylamide gradient gel electrophoresis allows the simultaneous analysis of proteins in the mass range of 10-500 kDa. We have used this characteristic together with cross-linking reagents to analyze the oligomerization of endogenous proteins with a single electrophoretic gel. We demonstrate how the oligomerization of p53, the pyruvate kinase isoform M2, or the heat shock protein 27 can be studied with this system. We also show how this system is useful for studying the oligomerization of large proteins such as clathrin heavy chain or the tuberous sclerosis complex. Oligomerization analysis is dependent on the cross-linker used and its concentration. All of these features make this system a very helpful tool for the analysis of protein oligomerization.

The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

Fenton Reaction-Generated Advanced Oxidation Protein Products Induces Inflammation in Human Embryonic Kidney Cells.

Fenton reaction is a new mechanism able to generate advanced oxidation protein products (AOPPs) by exposing the human serum albumin to the Fenton system. Here, we characterized the effects of Fenton reaction-generated advanced oxidation protein products (AOPP-FR) on the gene transcription of the nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) in human embryonic kidney cells (HEK 293). To investigate the effects of AOPP-FR and AOPP-HOCl on transcription of inflammatory genes, the NF-κB, COX-2, and IL-6 luciferase promoter activities were analyzed. AOPP-FR and AOPP-HOCl were able to induce the activation of the gene transcription of NF-κB, COX-2, and IL-6 in HEK 293 cells. However, the effects of AOPP-FR were significantly higher than the effects of AOPP-HOCl in relation to COX-2 and IL-6. AOPP-FR induces the activation of the gene transcription of NF-κB, COX-2, and IL-6 and may represent a novel pathogenic mediator of inflammation in kidney.

Tris-acetate polyacrylamide gradient gel electrophoresis for the analysis of protein oligomerization.

Here we report a new approach for studying protein oligomerization in cells using a single electrophoresis gel. We combined the use of a crosslinking reagent for sample preparation, such as glutaraldehyde, with the analysis of oligomers by Tris-acetate polyacrylamide gel electrophoresis. The use of a 3-15% Tris-acetate polyacrylamide gradient gel allows for the simultaneous analysis of proteins of masses ranging from 10 to 500 kDa. We showed the usefulness of this method for analyzing endogenous p53 oligomerization with high resolution and sensitivity in human cells. Oligomerization analysis was dependent on the crosslinker concentration used. We also showed that this method could be used to study the regulation of oligomerization. In all experiments, Tris-acetate polyacrylamide gel electrophoresis proved to be a robust, manageable, and cost- and time-efficient method that provided excellent results using a single gel. This approach can be easily extrapolated to the study of other oligomers. All of these features make this method a highly useful tool for the analysis of protein oligomerization.

Functional and pathological relevance of HERC family proteins: a decade later.

The HERC gene family encodes proteins with two characteristic domains in their sequence: the HECT domain and the RCC1-like domain (RLD). In humans, the HERC family comprises six members that can be divided into two groups based on their molecular mass and domain structure. Whereas large HERCs (HERC1 and HERC2) contain one HECT and more than one RLD, small HERCs (HERC3-6) possess single HECT and RLD domains. Accumulating evidence shows the HERC family proteins to be key components of a wide range of cellular functions, including neurodevelopment, DNA damage repair, cell growth and immune response. Considering the significant recent advances made regarding HERC functionality, an updated review summarizing the progress is greatly needed at 10 years since the last HERC review. We provide an integrated view of HERC function and go into detail about its implications for several human diseases such as cancer and neurological disorders.

The E3 ubiquitin protein ligase HERC2 modulates the activity of tumor protein p53 by regulating its oligomerization.

The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292-2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling.

Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide.

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals' knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

Mutation of HERC2 causes developmental delay with Angelman-like features.

BACKGROUND: Deregulation of the activity of the ubiquitin ligase E6AP (UBE3A) is well recognised to contribute to the development of Angelman syndrome (AS). The ubiquitin ligase HERC2, encoded by the HERC2 gene is thought to be a key regulator of E6AP. METHODS AND RESULTS: Using a combination of autozygosity mapping and linkage analysis, we studied an autosomal-recessive neurodevelopmental disorder with some phenotypic similarities to AS, found among the Old Order Amish. Our molecular investigation identified a mutation in HERC2 associated with the disease phenotype. We establish that the encoded mutant HERC2 protein has a reduced half-life compared with its wild-type counterpart, which is associated with a significant reduction in HERC2 levels in affected individuals. CONCLUSIONS: Our data implicate a model in which disruption of HERC2 function relates to a reduction in E6AP activity resulting in neurodevelopmental delay, suggesting a previously unrecognised role of HERC2 in the pathogenesis of AS.