Monica Cubillos-Rojas

Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-acetate polyacrylamide gels.

To separate and analyze giant and small proteins in the same electrophoresis gel, we have used a 3–15% polyacrylamide gradient gel containing 2.6% of the crosslinker bisacrylamide and 0.2 M of Tris‐acetate buffer (pH 7.0). Samples were prepared in a sample buffer containing lithium dodecyl sulphate and were run in the gel described above using Tris‐Tricine‐SDS‐sodium bisulfite buffer, pH 8.2, as electrophoresis buffer. Here, we show that this system can be successfully used for general applications of SDS‐PAGE such as CBB staining and immunoblot. Thus, by using Tris‐acetate 3–15% polyacrylamide gels, it is possible to simultaneously analyze proteins, in the mass range of 10–500 kDa, such as HERC1 (532 kDa), HERC2 (528 kDa), mTOR (289 kDa), Clathrin heavy chain (192 kDa), RSK (90 kDa), S6K (70 kDa), β‐actin (42 kDa), Ran (24 kDa) and LC3 (18 kDa). This system is highly sensitive since it allows detection from as low as 10 μg of total protein per lane. Moreover, it has a good resolution, low cost, high reproducibility and allows for analysis of proteins in a wide range of weights within a short period of time. All these features together with the use of a standard electrophoresis apparatus make the Tris‐acetate‐PAGE system a very helpful tool for protein analysis.

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.

The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization

Background: HERC2 has been implicated in DNA repair mechanisms and neurological disorders. Results: HERC2 binds p53 and regulates its transcriptional activity, affecting cellular processes modulated by p53 such as cell growth or DNA damage response. Conclusion: HERC2 modulates p53 activity by regulating its oligomerization. Significance: HERC2 is a novel regulator of p53 signaling. 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.

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 that 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.

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

BackgroundSepsis 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.MethodsA 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).ResultsIn 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).ConclusionsThis 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.

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

ABSTRACT 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.

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.

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 p53s signaling.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.

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.