Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4

Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy. We show that starvation stimulates formation of ROS, specifically H(2)O(2). These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H(2)O(2), and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.     

Autophagy mediates nonselective RNA degradation in starving yeast

During nitrogen starvation, a nonselective bulk degradation of cytosolic proteins and organelles including ribosomes, termed macro‐autophagy (hereafter termed autophagy), is induced. The precise mechanism of RNA degradation by this pathway has not been yet elucidated. In this issue of the The EMBO Journal, Huang et al characterize an autophagy‐dependent RNA catabolism in yeast and identify the enzymes responsible for the degradation process.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

Role of the pulvinar-lateralis posterior nucleus complex (P-LP) in the experimental epilepsy of the cat

The ability of the pulvinar-lateralis posterior nucleus complex (P-LP) to evoke epileptic activity when stimulated, was studied in 20 adult cats. Twelve animals were analyzed after they recovered from the surgical procedure (chronic model). In seven of them a cannula with electrodes was implanted in the P-LP and one twisted bipolar electrode was placed ipsilaterally in the following structures: hippocampus, superior colliculus, caudate nucleus and cerebral cortex. Through the cannula Na penicillin was injected. The electrodes allowed both to stimulate and to record the electrical activity. In the remaining five cats, the cannula was implanted in hippocampus in order to compare its sensitivity to generate epileptic activity to that of P-LP. Another group of eight cats were surgically implanted and studied in the same day (acute model). In four of them the cannula was placed in the P-LP through the temporal pathway, to avoid crossing the hippocampus and the ventricle. In another four, penicillin was injected in the P-LP after suctioning the cerebral cortex and the hippocampus overlying the former structure. Epileptic activity could be induced in P-LP and it spread rapidly to hippocampus and after a while to the other implanted structures. This was observed both with penicillin and electrical stimulation. The sensitivity of P-LP to generate epileptic activity was lower than that of the hippocampus. In particular, it was necessary to use two to ten times more penicillin and three times the electrical current intensity in the P-LP as compared to the values needed in the hippocampus. These results are discussed in view of the controversial problem about the ability of the thalamus to generate and spread epileptic activity.     

TECPR2

Autophagy dysfunction has been implicated in a group of progressive neurodegenerative diseases, and has been reported to play a major role in the pathogenesis of these disorders. We have recently reported a recessive mutation in TECPR2, an autophagy-implicated WD repeat-containing protein, in five individuals with a novel form of monogenic hereditary spastic paraparesis (HSP). We found that diseased skin fibroblasts had a decreased accumulation of the autophagy-initiation protein MAP1LC3B/LC3B, and an attenuated delivery of both LC3B and the cargo-recruiting protein SQSTM1/p62 to the lysosome where they are subject to degradation. The discovered TECPR2 mutation reveals for the first time a role for aberrant autophagy in a major class of Mendelian neurodegenerative diseases, and suggests mechanisms by which impaired autophagy may impinge on a broader scope of neurodegeneration.     

The intracellular nucleotide‐binding leucine‐rich repeat receptor (SlNRC4a) enhances immune signalling elicited by extracellular perception

Plant recognition and defence against pathogens employs a two‐tiered perception system. Surface‐localized pattern recognition receptors (PRRs) act to recognize microbial features, whereas intracellular nucleotide‐binding leucine‐rich repeat receptors (NLRs) directly or indirectly recognize pathogen effectors inside host cells. Employing the tomato PRR LeEIX2/EIX model system, we explored the molecular mechanism of signalling pathways. We identified an NLR that can associate with LeEIX2, termed SlNRC4a (NB‐LRR required for hypersensitive response‐associated cell death‐4). Co‐immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR‐mediated responses. SlNRC4a overexpression enhances defence responses, whereas silencing SlNRC4 reduces plant immunity. Moreover, the coiled‐coil domain of SlNRC4a is able to associate with LeEIX2 and is sufficient to enhance responses upon EIX perception. On the basis of these findings, we propose that SlNRC4a acts as a noncanonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perceptions.     

Purification of the D-2 dopamine receptor from bovine striatum

The D-2 dopamine receptor has been purified 21500 fold from bovine striatal membranes. Solubilized receptor preparation was partially purified by affinity chromatography on a haloperidol adsorbent followed by gel filtration on a Sephacryl S-300 column. The fractions eluted from this column which contained the ligand binding activity were further chromatographed on wheat germ agglutinin conjugated to Sepharose. The resulting receptor preparation displays a major polypeptide band of an apparent molecular weight of 92 kDa, and exhibits a specific binding activity of 2490 pmol spiperone per mg protein. This purified receptor preparation can reabsorb specifically to the haloperidol affinity column indicating that the 92 kDa polypeptide represents the ligand binding unit of the D-2 dopamine receptor.