Prosurvival AMBRA1 turns into a proapoptotic BH3-like protein during mitochondrial apoptosis

Autophagy and apoptosis are 2 stress-response mechanisms that are closely interconnected. However, the molecular interplays between these 2 pathways remain to be clarified. Here we report that the crucial proautophagic factor AMBRA1 can act as a positive mediator of mitochondrial apoptosis. Indeed, we show that, in a proapoptotic positive feedback loop, the C-terminal part of AMBRA1, generated by CASP/CASPASE cleavage upon apoptosis induction, inhibits the antiapoptotic factor BCL2 by a direct binding through its BH3-like domain. The mitochondrial AMBRA1-BCL2 complex is thus at the crossroad between autophagy and cell death and may represent a novel target in development of therapeutic approaches in clinical diseases.     

AMBRA1 is able to induce mitophagy via LC3 binding,regardless of PARKIN and p62/SQSTM1

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general'' autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.Autophagy is an important eukaryotic process involved in the lysosomal degradation of cytosolic components in both physiological and pathological conditions. During autophagy, the autophagosomes − specific double-membraned vesicles − engulf a number of different cargoes and then fuse with the lysosomes for subsequent recycling of their content. Several key proteins are involved in autophagosome formation, such as BECLIN 1 and its positive regulator AMBRA1;^{1, 2} a pool of AMBRA1 is localized at the mitochondria, where its pro-autophagic activity is inhibited by mitochondrial resident Bcl-2.³ Interestingly, mitochondria have been described as a source for autophagosome biogenesis;⁴ they play a key role in the cross-talk between autophagy and apoptosis regulation and they are involved in the cell death versus survival decision (reviewed in Strappazzon et al.³).Another mechanistic link exists between autophagy and mitochondria in mammals. Indeed, mitochondria damaged by the uncoupler CCCP (carbonyl cyanide m-chlorophenyl hydrazone) − because of a loss of their mitochondrial membrane potential (ΔΨ_m) − are subjected to a form of selective autophagy, termed mitophagy.^{5, 6, 7} During this process, depolarized mitochondria are ubiquitylated; they then recruit p62 (a protein involved in linking polyubiquitinated protein aggregates to the autophagic machinery) and next they are transported along microtubules to the perinuclear region, where they form rough aggregate structures termed ‘mito-aggresomes'',^{8, 9, 10} a step preceding their lysosomal degradation.Although mitophagy has been described in a number of tissues and in various physiological or pathological conditions (reviewed in Andreux et al.¹¹), very few are the known molecular mechanisms that regulate mitophagy; this is despite the fact that its manipulation may represent a forefront strategy in several human diseases. Thus, rather scarse is yet the availability of chemicals and drug candidates to modulate the process. The autophagy receptor NIX and the kinase Ulk1 mediate developmental removal of mitochondria during retyculocyte differentiation.^{6, 12, 13} Smurf1 has been defined as a new recognized mediator of both viral autophagy and mitophagy.¹⁴ In contrast, the E3 ubiquitin ligase PARKIN and the Ser/Thr kinase PINK1, both found to be mutated in autosomal recessive forms of Parkinson''s disease (PD), regulate mitophagy after mitochondrial damage.⁵ In more detail, PINK1 recruits PARKIN to depolarized mitochondria in order to remove damaged mitochondria. This mitochondrial quality control, driven by PINK1/PARKIN proteins, has recently been better characterized by RNAi screens.¹⁵ In fact, new proteins such as HSPA1L, BAG4 and SIAH3 have been found to modulate translocation of PARKIN to damaged mitochondria, whereas TOMM7 stabilizes PINK1 on the mitochondria. Interestingly, it has been demonstrated that after mitochondrial depolarization, the cytosolic pool of AMBRA1 interacts with PARKIN to enhance mitochondrial clearance.¹⁶In this study, we investigate the molecular mechanism(s) responsible for the AMBRA1-dependent enhancement of PARKIN-mediated mitophagy. We describe for the first time AMBRA1 as a new LIR (LC3 interacting region)-containing protein, and we demonstrate that this motif is essential for the binding between AMBRA1 and LC3, following mitophagy induction. Furthermore, we show that this interaction is crucial in a number of cell systems in order to both amplify PARKIN-mediated mitochondrial clearance and regulate PARKIN-independent mitophagy. In addition, to better understand the role of AMBRA1 at the mitochondria and as AMBRA1 does not possess a clear mitochondrial targeting sequence, we generated and expressed an organelle-targeted mutant of AMBRA1 in two different cell systems. Our data indicate that mitochondrial AMBRA1 induces (1) relocalization of the mitochondrial network around the nucleus, (2) depolarization and ubiquitylation of mitochondria and (3) recruitment of the molecular platform necessary to induce functional mitophagy through a PARKIN/p62-independent pathway.This work thus places AMBRA1 as a central player of mitophagy: we suggest that this molecule facilitates mitochondrial clearance by bringing damaged mitochondria onto autophagosomes via its LIR-mediated LC3 interaction. In addition, we show that high levels of mitochondrial AMBRA1 trigger mitophagy, a finding that could herald new therapies to fight important human disorders, ranging from muscle dystrophy to neurodegeneration.     

Small artery elasticity is decreased in patients with systemic lupus erythematosus without increased intima media thickness

Introduction  

The objectives of this study were to determine small arterial elasticity (SAE) in systemic lupus erythematosus (SLE) and to investigate its relationship with intima media thickness (IMT), accumulation of advanced glycation end products (AGEs), endothelial activation and inflammation.     

The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy

Autophagy is an evolutionary conserved catabolic process involved in several physiological and pathological processes such as cancer and neurodegeneration. Autophagy initiation signaling requires both the ULK1 kinase and the BECLIN 1-VPS34 core complex to generate autophagosomes, double-membraned vesicles that transfer cellular contents to lysosomes. In this study, we show that the BECLIN 1-VPS34 complex is tethered to the cytoskeleton through an interaction between the BECLIN 1-interacting protein AMBRA1 and dynein light chains 1/2. When autophagy is induced, ULK1 phosphorylates AMBRA1, releasing the autophagy core complex from dynein. Its subsequent relocalization to the endoplasmic reticulum enables autophagosome nucleation. Therefore, AMBRA1 constitutes a direct regulatory link between ULK1 and BECLIN 1-VPS34, which is required for core complex positioning and activity within the cell. Moreover, our results demonstrate that in addition to a function for microtubules in mediating autophagosome transport, there is a strict and regulatory relationship between cytoskeleton dynamics and autophagosome formation.     

Regulation of the adaptation to zinc deficiency in plants

The molecular mechanisms by which plants sense their micronutrient status, and adapt to their environment in order to ensure a sufficient micronutrient supply, are poorly understood. Zinc is an essential micronutrient for all living organisms. when facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation were recently identified. in this mini-review, we highlight recent progress in understanding the adaptation to zinc deficiency in plants and discuss the future challenges to fully unravel its molecular basis.Key words: adaptation, zinc deficiency, biofortification, molecular regulators, plant nutritionIn an increasingly populated world, agricultural production is an essential element of social development. Agriculture is the primary source of all nutrients required for human life, and nutrient sufficiency is the basis for good health and welfare of the human population.¹ Soils with zinc deficiency are widespread in the world, affecting large areas of cultivated soils in India, Turkey, China, Brazil and Australia,^2,3 making zinc the most common crop micronutrient deficiency.⁴ In addition, risk of inadequate zinc diet and zinc malnutrition are estimated to affect one-third of the global human population, i.e., around two billion people.⁵ Most affected are people living in developing countries, where diets are rich in cereal-based foods. Cereal grains are rich in phytate, which is a potent anti-nutrient, limiting micronutrient bioavailability.⁶ Zinc deficiency in crop production can be easily ameliorated through zinc fertilization, making agronomic biofortification an important strategy,³ however in the poorer regions, the required infrastructure to provide a reliable supply of zinc fertilizers of sufficient quality, is often not available. In those situations, biofortified crops, in which the zinc status of crops is genetically improved by selective breeding or via biotechnology, offer a rural-based intervention that will more likely reach the population.⁷ Different traits can be targeted to developing such improved crops, such as plant zinc deficiency tolerance, zinc use efficiency and the accumulation of zinc in edible parts. However, insufficient knowledge on the molecular mechanisms and the regulation of the zinc homeostasis network in plants is a serious bottleneck when pursuing zinc biofortification.     

A first insight into the genetic diversity of Mycobacterium tuberculosis in Dar es Salaam, Tanzania, assessed by spoligotyping

Background  

Tanzania has a high tuberculosis incidence, and genotyping studies of Mycobacterium tuberculosis in the country are necessary in order to improve our understanding of the epidemic. Spoligotyping is a potentially powerful genotyping method due to fast generation of genotyping results, high reproducibility and low operation costs. The recently constructed SpolDB4 database and the model-based program 'spotclust' can be used to assign isolates to families, subfamilies and variants. The results of a study can thus be analyzed in a global context.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.