Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45b)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45b-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45b/MEKK4 negatively regulates the autophagic process. Macroautophagy (hereafter referred to as autophagy) is a catabolic process, by which the cell degrades cytosolic content to supply metabolic processes with nutrients in order to maintain ATP production and macromolecular synthesis. Thus, autophagy acts as an efficient recycling mechanism in eukaryotic cells.1 Cellular stress, for example, nutrient deprivation, enhances autophagy as a survival mechanism during starvation. In addition, autophagy serves important functions in development, cancer, cell death and immunity in mammals. 1,2 Autophagy is controlled by conserved key regulators known as autophagy-related (Atg) proteins.3 At the onset of the autophagy cascade, Atg6/Beclin-1 forms a complex with the class III phosphatidylinoside kinase Vps34, which induces expansion of the precursor membrane vesicle, the phagophore, via recruitment of additional Atg proteins. During expansion, the double membrane vesicle surrounds cytosolic content, and the completed vesicle, called autophagosome, finally fuses with lysosomes to degrade the autophagosomal content.3 Maturation of autophagosomes is regulated by two ubiquitin-like conjugation systems, namely the Atg8-phosphatidylethanolamine (PE) and the Atg5-12/16L1 conjugation system. The Atg5-Atg12 conjugate interacts with Atg16L1, which tethers the complex to phagophores and autophagosomes. This complex then acts as an E3-like ubiquitin ligase for microtubule-associated protein 1 light chain 3 (LC3) lipidation. The conversion of LC3 to the PE-conjugated LC3-II form and its recruitment to the membrane serves as a wellaccepted marker for autophagy.Although Atg5-independent autophagy has been described, 4 Atg5 is crucial for autophagy under most circumstances and Atg5-deficient mouse embryonic fibroblasts (MEFs) lack LC3 conversion and autophagy. Therefore, Atg5-deficient mice die postnatal owing to their inability to cope with starvation during the neonatal period. 5 In addition, Atg5 see...

The regulatory network for acclimation of the obligate photoautotrophic fresh water cyanobacterium Synechococcus elongatus PCC 7942 to iron (Fe) limitation was studied by transcript profiling with an oligonucleotide whole genome DNA microarray. Six regions on the chromosome with several Fe-regulated genes each were identified. The irpAB and fut region encode putative Fe uptake systems, the suf region participates in [Fe-sulfur] cluster assembly under oxidative stress and Fe limitation, the isiAB region encodes CP43# and flavodoxin, the idiCB region encodes the NuoE-like electron transport associated protein IdiC and the transcriptional activator IdiB, and the ackA/pgam region encodes an acetate kinase and a phosphoglycerate mutase. We also investigated the response of two S. elongatus PCC 7942 mutants to Fe starvation. These were mutant K10, lacking IdiB but containing IdiC, and mutant MuD, representing a idiC-merodiploid mutant with a strongly reduced amount of IdiC as well as IdiB. The absence of IdiB in mutant K10 or the strongly reduced amount of IdiB in mutant MuD allowed for the identification of additional members of the Fe-responsive IdiB regulon. Besides idiA and the irpAB operon somB(1), somA(2), ftr1, ackA, pgam, and nat also seem to be regulated by IdiB. In addition to the reduced amount of IdiB in MuD, the low concentration of IdiC may be responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to the wild type and mutant K10.

Objectives During the current pandemic, antibody testing based on venous serum helps to determine whether the tested person has been previously infected with SARS-CoV-2. Alternatively, capillary blood can be taken via a finger prick (dried blood spots, DBS). In this study, paired DBS and venipuncture samples were tested using two serological assays to evaluate the usability of DBS for the detection of anti-SARS-CoV-2 antibodies. Methods Paired samples of DBS and venous serum were collected from 389 volunteers, of whom 75 had a recent PCR-confirmed SARS-CoV-2 infection, and tested for anti-SARS-CoV-2 IgG antibodies against both viral S1 and nucleocapsid protein (NCP) antigens using two ELISAs. Degree of agreement and correlation coefficients between ELISA results based on the two sampling methods were calculated. Results Results of DBS showed almost perfect agreement and high correlations with results from corresponding serum samples in both the S1-based ELISA and the NCP-based ELISA. Conclusions ELISA results derived from DBS showed very high agreement to those obtained with serum, supposing adequate usability and robustness of DBS as sample material for detection of anti-SARS-CoV-2 antibodies. In the near future, large-scale epidemiological screening for antibodies against SARS-CoV-2 will be carried out. Since DBS reduce the strain on healthcare institutions regarding sample collection, they have a potential to facilitate efficient community- and population-based screening in the current SARS-CoV-2 pandemic.

Autophagy is a catabolic mechanism that is important for many biological processes such as cell homeostasis, development and immunity. Though many molecular components of the autophagy pathway have been identified, the signaling pathways regulating the activity of essential autophagy mediators are still poorly defined. We recently demonstrated that the mitogen-activated protein kinase MAPK14 (p38α), when activated by the GADD45B (Gadd45β)-MAP3K4 (MEKK4) signaling complex (but not other MAPK14 activators), is directed to autophagosomes. Therefore, we demonstrated for the first time that MAPK14 operates at this subcellular compartment. Importantly, activation of MAPK14 impairs autophagosome-lysosome fusion and, thus, autophagy. This was demonstrated by increased autophagic flux in MAPK14-deficient as well as in GADD45B-deficient cells. Moreover, we identified a novel post-translational modification of the crucial autophagy mediator ATG5, since MAPK14 directly phosphorylates ATG5 at threonine 75, which is evolutionarily conserved from yeast to human. Using ATG5-deficient cells, which we reconstituted with either a phosphorylation-defective or a phospho-mimetic mutant of ATG5, we demonstrated that phosphorylation of ATG5 results in impaired autophagy.