SIRT1 regulates DNA damage signaling through the PP4 phosphatase complex.

The Sirtuin family of NAD+-dependent enzymes plays an important role in maintaining genome stability upon stress. Several mammalian Sirtuins have been linked directly or indirectly to the regulation of DNA damage during replication through Homologous recombination (HR). The role of one of them, SIRT1, is intriguing as it seems to have a general regulatory role in the DNA damage response (DDR) that has not yet been addressed. SIRT1-deficient cells show impaired DDR reflected in a decrease in repair capacity, increased genome instability and decreased levels of γH2AX. Here we unveil a close functional antagonism between SIRT1 and the PP4 phosphatase multiprotein complex in the regulation of the DDR. Upon DNA damage, SIRT1 interacts specifically with the catalytical subunit PP4c and promotes its inhibition by deacetylating the WH1 domain of the regulatory subunits PP4R3α/ß. This in turn regulates γH2AX and RPA2 phosphorylation, two key events in the signaling of DNA damage and repair by HR. We propose a mechanism whereby during stress, SIRT1 signaling ensures a global control of DNA damage signaling through PP4.

RhoA/ROCK2 signalling is enhanced by PDGF-AA in fibro-adipogenic progenitor cells: implications for Duchenne muscular dystrophy.

BACKGROUND: The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our aim was to study the molecular role of PDGF-AA in the fibrotic process of DMD. METHODS: Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry results. We evaluated the effects of PDGF-AA on the activation of RhoA pathway using two inhibitors, C3-exoenzyme and fasudil. Cell proliferation and migration were determined by BrdU and migration assay. Actin reorganization and collagen synthesis were measured by phalloidin staining and Sircol assay, respectively. In an in vivo proof of concept study, we treated dba/2J-mdx mice with fasudil for 6 weeks. Muscle strength was assessed with the grip strength. Immunofluorescence and flow cytometry analyses were used to study fibrotic and inflammatory markers in muscle tissue. RESULTS: Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01). CONCLUSIONS: Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.

Evaluation of Tumor Interstitial Fluid-Extraction Methods for Proteome Analysis: Comparison of Biopsy Elution versus Centrifugation.

The analysis of tumor interstitial fluid (TIF) composition is a valuable procedure to identify antimetastatic targets, and different laboratories have set up techniques for TIF isolation and proteomic analyses. However, those methods had never been compared in samples from the same tumor and patient. In this work, we compared the two most used methods, elution and centrifugation, in pieces of the same biopsy samples of cutaneous squamous cell carcinoma (cSCC). First, we established that high G-force (10 000g) was required to obtain TIF from cSCC by centrifugation. Second, we compared the centrifugation method with the elution method in pieces of three different cSCC tumors. We found that the mean protein intensities based in the number of peptide spectrum matches was significantly higher in the centrifuged samples than in the eluted samples. Regarding the robustness of the methods, we observed higher overlapping between both methods (77-80%) than among samples (50%). These results suggest that there exists an elevated consistence of TIF composition independently of the method used. However, we observed a 3-fold increase of extracellular proteins in nonoverlapped proteome obtained by centrifugation. We therefore conclude that centrifugation is the method of choice to study the proteome of TIF from cutaneous biopsies.

SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

Aging-related tau astrogliopathy (ARTAG): not only tau phosphorylation in astrocytes.

Aging-related tau astrogliopathy (ARTAG) is defined by the presence of two types of tau-bearing astrocytes: thorn-shaped astrocytes (TSAs) and granular/fuzzy astrocytes in the brain of old-aged individuals. The present study is focused on TSAs in rare forms of ARTAG with no neuronal tau pathology or restricted to entorhinal and transentorhinal cortices, to avoid bias from associated tauopathies. TSAs show 4Rtau phosphorylation at several specific sites and abnormal tau conformation, but they lack ubiquitin and they are not immunostained with tau-C3 antibodies which recognize truncated tau at Asp421. Astrocytes in ARTAG have atrophic processes, reduced glial fibrillary acidic protein (GFAP) and increased superoxide dismutase 2 (SOD2) immunoreactivity. Gel electrophoresis and western blotting of sarkosyl-insoluble fractions reveal a pattern of phospho-tau in ARTAG characterized by two bands of 68 and 64 kDa, and several middle bands between 35 and 50 kDa which differ from what is seen in AD. Phosphoproteomics of dissected vulnerable regions identifies an increase of phosphorylation marks in a large number of proteins in ARTAG compared with controls. GFAP, aquaporin 4, several serine-threonine kinases, microtubule associated proteins and other neuronal proteins are among the differentially phosphorylated proteins in ARTAG thus suggesting a hyper-phosphorylation background that affects several molecules, including many kinases and proteins from several cell compartments and various cell types. Finally, present results show for the first time that tau seeding is produced in neurons of the hippocampal complex, astrocytes, oligodendroglia and along fibers of the corpus callosum, fimbria and fornix following inoculation into the hippocampus of wild type mice of sarkosyl-insoluble fractions enriched in hyper-phosphorylated tau from selected ARTAG cases. These findings show astrocytes as crucial players of tau seeding in tauopathies.

Disulfide driven folding for a conditionally disordered protein.

Conditionally disordered proteins are either ordered or disordered depending on the environmental context. The substrates of the mitochondrial intermembrane space (IMS) oxidoreductase Mia40 are synthesized on cytosolic ribosomes and diffuse as intrinsically disordered proteins to the IMS, where they fold into their functional conformations; behaving thus as conditionally disordered proteins. It is not clear how the sequences of these polypeptides encode at the same time for their ability to adopt a folded structure and to remain unfolded. Here we characterize the disorder-to-order transition of a Mia40 substrate, the human small copper chaperone Cox17. Using an integrated real-time approach, including chromatography, fluorescence, CD, FTIR, SAXS, NMR, and MS analysis, we demonstrate that in this mitochondrial protein, the conformational switch between disordered and folded states is controlled by the formation of a single disulfide bond, both in the presence and in the absence of Mia40. We provide molecular details on how the folding of a conditionally disordered protein is tightly regulated in time and space, in such a way that the same sequence is competent for protein translocation and activity.

Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC).

UNLABELLED: The human immortalized brain endothelial cell line hCMEC/D3 is considered a simple in-vitro model of the blood-brain-barrier. Our aim was to characterize changes in the secretome of hCMEC/D3 subjected to oxygen and glucose deprivation (OGD) to identify new proteins altered after ischemia and that might trigger blood-brain-barrier disruption and test their potential as blood biomarkers for ischemic stroke. Using a quantitative proteomic approach based on SILAC, 19 proteins were found differentially secreted between OGD and normoxia/normoglycemia conditions. Among the OGD-secreted proteins, protein folding was the main molecular function identified and for the main canonical pathways there was an enrichment in epithelial adherens junctions and aldosterone signaling. Western blot was used to verify the MS results in a set of 9 differentially secreted proteins and 5 of these were analyzed in serum samples of 38 ischemic stroke patients, 18 stroke-mimicking conditions and 18 healthy controls. SIGNIFICANCE: "We characterized changes in the secretome of hCMEC/D3 cells after an ischemic insult by SILAC and identified proteins associated with ischemia that might be involved in the disruption of the blood-brain barrier. Besides we analyzed the putative potential of the candidate proteins to become biomarkers for the diagnosis of ischemic stroke.

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

Human µ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

A mouse model uncovers LKB1 as an UVB-induced DNA damage sensor mediating CDKN1A (p21WAF1/CIP1) degradation.

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.

Relevance of IGFBP2 proteolysis in glioma and contribution of the extracellular protease ADAMTS1.

Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies.

The mitochondrial intermembrane space oxireductase Mia40 funnels the oxidative folding pathway of the cytochrome c oxidase assembly protein Cox19.

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.

Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). BIOLOGICAL SIGNIFICANCE: The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and Quality Control.

Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.

The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.

HER2 carboxyl-terminal fragments regulate cell migration and cortactin phosphorylation.

A group of breast cancer patients with a higher probability of developing metastasis expresses a series of carboxyl-terminal fragments (CTFs) of the tyrosine kinase receptor HER2. One of these fragments, 611-CTF, is a hyperactive form of HER2 that constitutively establishes homodimers maintained by disulfide bonds, making it an excellent model to study overactivation of HER2 during tumor progression and metastasis. Here we show that expression of 611-CTF increases cell motility in a variety of assays. Since cell motility is frequently regulated by phosphorylation/dephosphorylation, we looked for phosphoproteins mediating the effect of 611-CTF using two alternative proteomic approaches, stable isotope labeling with amino acids in cell culture and difference gel electrophoresis, and found that the latter is particularly well suited to detect changes in multiphosphorylated proteins. The difference gel electrophoresis screening identified cortactin, a cytoskeleton-binding protein involved in the regulation of cell migration, as a phosphoprotein probably regulated by 611-CTF. This result was validated by characterizing cortactin in cells expressing this HER2 fragment. Finally, we showed that the knockdown of cortactin impairs 611-CTF-induced cell migration. These results suggest that cortactin is a target of 611-CTF involved in the regulation of cell migration and, thus, in the metastatic behavior of breast tumors expressing this CTF.

Post-transcriptional up-regulation of ADAM17 upon epidermal growth factor receptor activation and in breast tumors.

ADAM17 is a transmembrane metalloprotease involved in the proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Despite its likely participation in tumor progression and its current consideration as a therapeutic target, very little is known about the regulation of the expression of ADAM17. Here we show that long term treatment with epidermal growth factor (EGF) leads to a marked increase in the levels of ADAM17. EGF receptor activation does not affect the levels of the mRNA that encodes for, or the rate of synthesis of, ADAM17 but increases its half-life. The effect of EGF is biologically relevant because it increases the shedding of several substrates of ADAM17, including the desmosomal cadherin Dsg-2. Analysis of protein and mRNA levels in mammary tumor samples shows that in vivo the levels of ADAM17 can also be controlled post-transcriptionally. Finally, we show that both the shed extracellular domains of Dsg-2 and ADAM17 are frequently expressed in tumors, further supporting the participation of the metalloprotease in malignant progression.

Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies.

Recycling of cell surface pro-transforming growth factor-{alpha} regulates epidermal growth factor receptor activation.

Impairments in signal transduction, leading to the regulation of cell proliferation, differentiation, or migration are frequently the cause of cancer. Since the accurate spatial and temporal location of their components is crucial to ensure the correct regulation of these signaling pathways, it could be anticipated that defects in intracellular trafficking are at the base of certain neoplasias. However, the trafficking of many components of pathways frequently up-regulated in cancers, such as the epidermal growth factor receptor (EGFR) pathway, are largely unknown. Here, we show that the pro-transforming growth factor-alpha (pro-TGF-alpha), a prototypical EGFR ligand, is endocytosed from the cell surface via a clathrin-dependent pathway. Internalized pro-TGF-alpha does not progress to the lysosome; instead, it is delivered to the cell surface via recycling endosomes. To analyze the functional meaning of the internalization of pro-TGF-alpha, we used a deletion construct that is normally transported to the cell surface but is deficiently endocytosed. Due to this impairment, the levels of this construct at the cell surface are dramatically augmented. Consequently, the deletion construct displays a higher EGFR-activating ability, revealing a link between the trafficking of pro-TGF-alpha and the signaling by the EGFR and opening the possibility that defects in the trafficking of the growth factor may contribute to the development of tumors.