Inducing autophagy Harder, Lea M; Bunkenborg, Jakob; Andersen, Jens S
Autophagy,
02/2014, Letnik:
10, Številka:
2
Journal Article
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Autophagy is a lysosomal-mediated catabolic process, which through degradation of different cytoplasmic components aids in maintaining cellular homeostasis and survival during exposure to extra- or ...intracellular stresses. Ammonia is a potential toxic and stress-inducing byproduct of glutamine catabolism, which has recently been found to induce autophagy in an MTOR independent way and support cancer cell survival. In this study, quantitative phosphoproteomics was applied to investigate the initial signaling events linking ammonia to the induction of autophagy. The MTOR inhibitor rapamycin was used as a reference treatment to emphasize the differences between an MTOR-dependent and -independent autophagy-induction. By this means 5901 phosphosites were identified of which 626 were treatment-specific regulated and 175 were coregulated. Investigation of the ammonia-specific regulated sites supported that MTOR activity was not affected, but indicated increased MAPK3 activity, regulation of proteins involved in Rho signal transduction, and a novel phosphorylation motif, serine-proline-threonine (SPT), which could be linked to cytoskeleton-associated proteins. MAPK3 could not be identified as the primary driver of ammonia-induced autophagy but instead the data suggested an upregulation of AMPK and the unfolded protein response (UPR), which might link ammonia to autophagy induction. Support of UPR induction was further obtained from the finding of increased protein levels of the ER stress markers DDIT3/CHOP and HSPA5 during ammonia treatment. The large-scale data set presented here comprises extensive high-quality quantitative information on phosphoprotein regulation in response to 2 very different autophagy inducers and should therefore be considered a general resource for the community.
Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism ...in the lake, comprising more than half of the bacterial population, and its biomass increases 3.8-fold over the summer. Cells from four positions in the water column were used for comparative analysis of the Chl. clathratiforme proteome in order to investigate changes in protein composition in response to the chemical and physical gradient in their environment, with special focus on how the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC-MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively compared 621 of these in the four samples. Our results showed that compared with cells obtained from the photic zone, cells collected from the dark part of the water column had the same expression level of key enzymes involved in carbon metabolism and photosynthetic light harvesting. However, most proteins participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N₂ and the complete oxidation of sulfide to sulfate while these processes are probably not active in the dark. Instead we propose that Chl. clathratiforme cells in the dark part of the water column obtain energy for maintenance from the fermentation of polyglucose. Based on the observed protein compositions we have constructed possible pathways for C, N and S metabolism in Chl. clathratiforme.
Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5–15% FBS. Contamination by bovine proteins is difficult to ...avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC‐MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.
The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 ...enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
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•ARS2 transcription termination activity is independent of the INT and CPA pathways•The ZC3H4 restrictor factor interacts with ARS2 via a short linear motif (SLiM)•ARS2 is required for ZC3H4 recruitment to chromatin at a number of loci•ARS2-ZC3H4 triggers transcription termination and decay of the cognate RNA
Rouviere et al. show that the RNA-binding protein ARS2, previously shown to act in early transcription termination, does so by recruiting the ZC3H4 “restrictor” factor to chromatin. The ARS2-ZC3H4 axis commits terminated transcripts for degradation by the nuclear exosome via a direct recruitment of the nuclear exosome targeting (NEXT) complex.
Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far ...depended on purification methods based on either density gradient centrifugations or magnetic purification of iron‐loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar‐type H+‐ATPase. Quantitative MS‐based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal membrane proteome was significantly altered by the ectopic expression of an active form of the ErbB2 oncogene, which renders the cells highly metastatic. The furthermost ErbB2‐associated changes included increased levels of CD63, S100A11 and ferritin heavy chain. Overall, our data introduce the antibody‐based purification of lysosomes as a suitable method for the characterization of lysosomes from a variety of pathological conditions with altered lysosomal density and stability.
Phosphoproteomic experiments are routinely conducted in laboratories worldwide, and because of the fast development of mass spectrometric techniques and efficient phosphopeptide enrichment methods, ...researchers frequently end up having lists with tens of thousands of phosphorylation sites for further interrogation. To answer biologically relevant questions from these complex data sets, it becomes essential to apply computational, statistical, and predictive analytical methods. Here we provide an advanced bioinformatic platform termed “PhosphoSiteAnalyzer” to explore large phosphoproteomic data sets that have been subjected to kinase prediction using the previously published NetworKIN algorithm. NetworKIN applies sophisticated linear motif analysis and contextual network modeling to obtain kinase–substrate associations with high accuracy and sensitivity. PhosphoSiteAnalyzer provides an algorithm to retrieve kinase predictions from the public NetworKIN webpage in a semiautomated way and applies hereafter advanced statistics to facilitate a user-tailored in-depth analysis of the phosphoproteomic data sets. The interface of the software provides a high degree of analytical flexibility and is designed to be intuitive for most users. PhosphoSiteAnalyzer is a freeware program available at http://phosphosite.sourceforge.net.
The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and ...participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.
Are meta-analysis–based comparisons solid evidence? Calderon, Moises A., MD; Andersen, Jens S., PhD; Nelson, Harold S., MD
Journal of allergy and clinical immunology,
08/2013, Letnik:
132, Številka:
2
Journal Article
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Because small single-center trials commonly have less variation, their SMDs will be large, not because of a larger effect but because of less variation. ...the random-effects model favors ...single-center trials and not the guideline-compliant, multinational, multisite confirmatory trials used in modern development of specific immunotherapy. ...moving toward evidence-based medicine implies the integration of individual clinical expertise with the best available external clinical evidence from systematic research.
During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome ...segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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