Autophagy is important in the basal or stress-induced clearance of bulk cytosol, damaged organelles, pathogens and selected proteins by specific vesicles, the autophagosomes. Following mTOR ...(mammalian target of rapamycin) inhibition, autophagosome formation is primed by the ULK1 and the beclin-1-Vps34-AMBRA1 complexes, which are linked together by a scaffold platform, the exocyst. Although several regulative steps have been described along this pathway, few targets of mTOR are known, and the cross-talk between ULK1 and beclin 1 complexes is still not fully understood. We show that under non-autophagic conditions, mTOR inhibits AMBRA1 by phosphorylation, whereas on autophagy induction, AMBRA1 is dephosphorylated. In this condition, AMBRA1, interacting with the E3-ligase TRAF6, supports ULK1 ubiquitylation by LYS-63-linked chains, and its subsequent stabilization, self-association and function. As ULK1 has been shown to activate AMBRA1 by phosphorylation, the proposed pathway may act as a positive regulation loop, which may be targeted in human disorders linked to impaired autophagy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these ...two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per ...experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO2-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO2 beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.
Protein phosphorylation is an important mechanism of cellular signaling, and many proteins are precisely regulated through the interplay of stimulatory and inhibitory phosphorylation sites. ...Phosphoproteomics offers great opportunities to unravel this complex interplay, generating a mechanistic understanding of vital cellular processes. However, protein phosphorylation is substoichiometric and, in particular, peptides carrying multiple phosphorylation sites are extremely difficult to detect in a highly complex mixture of abundant nonphosphorylated peptides. Chromatographic methods are employed to reduce sample complexity and thereby significantly increase the number of phosphopeptide identifications. We previously demonstrated that combinatorial strong cation exchange–electrostatic repulsion–hydrophilic interaction chromatography yields a surplus in overall identifications of phosphopeptides compared with single chromatographic approaches. Here we present a simple and rapid strategy implemented as solid-phase extraction not requiring specific instrumentation such as off-line HPLC systems. It is inexpensive, adaptable for high and low amounts of starting material, and saves time by allowing multiplexed sample preparation without any carry-over problem.
In large-scale phosphoproteomics studies, fractionation by strong cation exchange (SCX) or electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is commonly used to reduce sample ...complexity, fractionate phosphopeptides from their unmodified counterparts, and increase the dynamic range for phosphopeptide identification. However, these procedures do not succeed to separate, both singly and multiply phosphorylated peptides due to their inverse physicochemical characteristics. Hence, depending on the chosen method only one of the two peptide classes can be efficiently separated. Here, we present a novel strategy based on the combinatorial separation of singly and multiply phosphorylated peptides by SCX and ERLIC for in-depth phosphoproteome analysis. In SCX, mostly singly phosphorylated peptides are retained and fractionated while not-retained multiply phosphorylated peptides are fractionated in a subsequent ERLIC approach (SCX–ERLIC). In ERLIC, multiply phosphorylated peptides are fractionated, while not-retained singly phosphorylated peptides are separated by SCX (ERLIC–SCX). Compared to single step fractionations by SCX, the combinatorial strategies, SCX–ERLIC and ERLIC–SCX, yield up to 48% more phosphopeptide identifications as well as a strong increase in the number of detected multiphosphorylated peptides. Phosphopeptides identified in two subsequent, complementary fractionations had little overlap (5%) indicating that ERLIC and SCX are orthogonal methods ideally suited for in-depth phosphoproteome studies.
Recessive dystrophic epidermolysis bullosa (RDEB), a genetic skin blistering disease, is a paradigmatic condition of tissue fragility‐driven multi‐organ fibrosis. Here, longitudinal analyses of the ...tissue proteome through the course of naturally developing disease in RDEB mice revealed that increased pro‐inflammatory immunity associates with fibrosis evolution. Mechanistically, this fibrosis is a consequence of altered extracellular matrix organization rather than that of increased abundance of major structural proteins. In a humanized system of disease progression, we targeted inflammatory cell fibroblast communication with Ang‐(1‐7)—an anti‐inflammatory heptapeptide of the renin‐angiotensin system, which reduced the fibrosis‐evoking aptitude of RDEB cells. In vivo, systemic administration of Ang‐(1‐7) efficiently attenuated progression of multi‐organ fibrosis and increased survival of RDEB mice. Collectively, our study shows that selective down‐modulation of pro‐inflammatory immunity may mitigate injury‐induced fibrosis. Furthermore, together with published data, our data highlight molecular diversity among fibrotic conditions. Both findings have direct implications for the design of therapies addressing skin fragility and fibrosis.
SYNOPSIS
Injury‐evoked fibrosis in recessive dystrophic epidermolysis bullosa (RDEB) associates with inflammatory immunity. This fibrosis is a consequence of altered extracellular matrix (ECM) organization. Targeting fibro‐inflammatory communication with Ang‐(1‐7) effectively attenuates fibrosis in RDEB.
Inflammatory immunity with seemingly little involvement of tissue‐repair immunity supports fibrosis in RDEB.
Established fibrotic lesions in RDEB display modest changes of major structural proteins including fibrillar collagens, but show changed abundance of minor ECM organizer and altered ECM architecture.
Selective targeting of dysregulated fibro‐inflammatory communication in injured RDEB tissue with Ang‐(1‐7) effectively attenuates progression of multi‐organ fibrosis.
Injury‐evoked fibrosis in recessive dystrophic epidermolysis bullosa (RDEB) associates with inflammatory immunity. This fibrosis is a consequence of altered extracellular matrix (ECM) organization. Targeting fibro‐inflammatory communication with Ang‐(1‐7) effectively attenuates fibrosis in RDEB.
Heterozygous missense mutations in the human COL7A1 gene - coding for collagen VII - lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB) that is ...characterized by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of EB which frequently show early lethality and severe disease, these mouse models which to our knowledge are the first for DDEB show no reduction in growth and survival and - together with a relatively mild phenotype - represent a practically and ethically tractable tool for better understanding and treating EB.
Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays ...using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.
High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ...ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) – a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.
•High sequence conservation of murine and human collagen VII makes development of species-specific antibodies challenging.•Divergence in the immune epitope of a conserved peptide allowed for generation of species-specific collagen VII antibodies.•The antibodies allow strong, simultaneous visualization of human and murine collagen VII in immunocompetent hosts.
Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin ...efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux. To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis revealing regulated phosphorylation sites on proteins involved in a wide range of cellular processes and an impact of the treatments on the kinome. To approach the potential function of the identified phosphorylation sites we performed a screen for MAP1LC3-interacting proteins and identified a group of binding partners exhibiting dynamic phosphorylation patterns. The data presented here provide a valuable resource on phosphorylation events underlying early autophagy induction.
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