Autophagy--alias self-eating--appetite and ageing

Two articles—one published online in January and in the March issue EMBO reports—implicate autophagy in the control of appetite by regulating neuropeptide production in hypothalamic neurons. Autophagy decline with age in POMC neurons induces obesity and metabolic syndrome.Kaushik et al. EMBO reports, this issue doi:10.1038/embor.2011.260Macroautophagy, which I will call autophagy, is a critical process that degrades bulk cytoplasm, including organelles, protein oligomers and a range of selective substrates. It has been linked with diverse physiological and disease-associated functions, including the removal of certain bacteria, protein oligomers associated with neurodegenerative diseases and dysfunctional mitochondria [1]. However, the primordial role of autophagy—conserved from yeast to mammals—appears to be its ability to provide nutrients to starving cells by releasing building blocks, such as amino acids and free fatty acids, obtained from macromolecular degradation. In yeast, autophagy deficiency enhances death in starvation conditions [2], and in mice it causes death from starvation in the early neonatal period [3,4]. Two recent articles from the Singh group—one of them in this issue of EMBO reports—also implicate autophagy in central appetite regulation [5,6].Autophagy seems to decline with age in the liver [7], and it has thus been assumed that autophagy declines with age in all tissues, but this has not been tested rigorously in organs such as the brain. Conversely, specific autophagy upregulation in Caenorhabditis elegans and Drosophila extends lifespan, and drugs that induce autophagy—but also perturb unrelated processes, such as rapamycin—promote longevity in rodents [8].Autophagy literally means self-eating, and it is therefore interesting to see that this cellular ‘self-eating'' has systemic roles in mammalian appetite control. The control of appetite is influenced by central regulators, including various hormones and neurotransmitters, and peripheral regulators, including hormones, glucose and free fatty acids [9]. Autophagy probably has peripheral roles in appetite and energy balance, as it regulates lipolysis and free fatty acid release [10]. Furthermore, Singh and colleagues have recently implicated autophagy in central appetite regulation [5,6].The arcuate nucleus in the hypothalamus has received extensive attention as an integrator and regulator of energy homeostasis and appetite. Through its proximity to the median eminence, which is characterized by an incomplete blood–brain barrier, these neurons rapidly sense metabolic fluctuations in the blood. There are two different neuronal populations in the arcuate nucleus, which appear to have complementary effects on appetite (Fig 1). The proopiomelanocortin (POMC) neurons produce the neuropeptide precursor POMC, which is cleaved to form α-melanocyte stimulating hormone (α-MSH), among several other products. The α-MSH secreted from these neurons activates melanocortin 4 receptors on target neurons in the paraventricular nucleus of the hypothalamus, which ultimately reduce food intake. The second group of neurons contain neuropeptide Y (NPY) and Agouti-related peptide (AgRP). Secreted NPY binds to downstream neuronal receptors and stimulates appetite. AgRP blocks the ability of α-MSH to activate melanocortin 4 receptors [11]. Furthermore, AgRP neurons inhibit POMC neurons [9].Open in a separate windowFigure 1Schematic diagram illustrating the complementary roles of POMC and NPY/AgRP neurons in appetite control. AgRP, Agouti-related peptide; MC4R, melanocortin 4 receptor; α-MSH, α-melanocyte stimulating hormone; NPY, neuropeptide Y; POMC, proopiomelanocortin.The first study from Singh''s group started by showing that starvation induces autophagy in the hypothalamus [5]. This finding alone merits some comment. Autophagy is frequently assessed by using phosphatidylethanolamine-conjugated Atg8/LC3 (LC3-II), which is specifically associated with autophagosomes and autolysosomes. LC3-II levels on western blot and the number of LC3-positive vesicles strongly correlate with the number of autophagosomes [1]. To assess whether LC3-II formation is altered by a perturbation, its level can be assessed in the presence of lysosomal inhibitors, which inhibit LC3-II degradation by blocking autophagosome–lysosome fusion [12]. Therefore, differences in LC3-II levels in response to a particular perturbation in the presence of lysosomal inhibitors reflect changes in autophagosome synthesis. An earlier study using GFP-LC3 suggested that autophagy was not upregulated in the brains of starved mice, compared with other tissues where this did occur [13]. However, this study only measured steady state levels of autophagosomes and was performed before the need for lysosomal inhibitors was appreciated. Subsequent work has shown rapid flux of autophagosomes to lysosomes in primary neurons, which might confound analyses without lysosomal inhibitors [14]. Thus, the data of the Singh group—showing that autophagy is upregulated in the brain by a range of methods including lysosomal inhibitors [5]—address an important issue in the field and corroborate another recent study that examined this question by using sophisticated imaging methods [15].“…decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with age”Singh and colleagues then analysed mice that have a specific knockout of the autophagy gene Atg7 in AgRP neurons [5]. Although fasting increases AgRP mRNA and protein levels in normal mice, these changes were not seen in the knockout mice. AgRP neurons provide inhibitory signals to POMC neurons, and Kaushik and colleagues found that the AgRP-specific Atg7 knockout mice had higher levels of POMC and α-MSH, compared with the normal mice. This indicated that starvation regulates appetite in a manner that is partly dependent on autophagy. The authors suggested that the peripheral free fatty acids released during starvation induce autophagy by activating AMP-activated protein kinase (AMPK), a known positive regulator of autophagy. This, in turn, enhances degradation of hypothalamic lipids and increases endogenous intracellular free fatty acid concentrations. The increased intracellular free fatty acids upregulate AgRP mRNA and protein expression. As AgRP normally inhibits POMC/α-MSH production in target neurons, a defect in AgRP responses in the autophagy-null AgRP neurons results in higher α-MSH levels, which could account for the decreased mouse bodyweight.In follow-up work, Singh''s group have now studied the effects of inhibiting autophagy in POMC neurons, again using Atg7 deletion [6]. These mice, in contrast to the AgRP autophagy knockouts, are obese. This might be accounted for, in part, by an increase in POMC preprotein levels and its cleavage product adrenocorticotropic hormone in the knockout POMC neurons, which is associated with a failure to generate α-MSH. Interestingly, these POMC autophagy knockout mice have impaired peripheral lipolysis in response to starvation, which the authors suggest might be due to reduced central sympathetic tone to the periphery from the POMC neurons. In addition, POMC-neuron-specific Atg7 knockout mice have impaired glucose tolerance.This new study raises several interesting issues. How does the autophagy defect in the POMC neurons alter the cleavage pattern of POMC? Is this modulated within the physiological range of autophagy activity fluctuations in response to diet and starvation? Importantly, in vivo, autophagy might fluctuate similarly (or possibly differently) in POMC and AgRP neurons in response to diet and/or starvation. Given the tight interrelation of these neurons, how does this affect their overall response to appetite regulation in wild-type animals?Finally, the study also shows that hypothalamic autophagosome formation is decreased in older mice. To my knowledge, this is the first such demonstration of this phenomenon in the brain. The older mice phenocopied aspects of the POMC-neuron autophagy null mice—increased hypothalamic POMC preprotein and ACTH and decreased α-MSH, along with similar adiposity and lipolytic defects, compared with young mice. These data are provocative from several perspectives. In the context of metabolism, it is tantalizing to consider that decreasing autophagy with ageing in POMC neurons could contribute to the metabolic problems associated with ageing. Again, this model considers the POMC neurons in isolation, and it would be important to understand how reduced autophagy in aged AgRP neurons counterbalances this situation. In a more general sense, the data strongly support the concept that neuronal autophagy might decline with age.Autophagy is a major clearance route for many mutant, aggregate-prone intracytoplasmic proteins that cause neurodegenerative disease, such as tau (Alzheimer disease), α-synuclein (Parkinson disease), and huntingtin (Huntington disease), and the risk of these diseases is age-dependent [1]. Thus, it is tempting to suggest that the dramatic age-related risks for these diseases could be largely due to decreased neuronal capacity of degrading these toxic proteins. Neurodegenerative pathology and age-related metabolic abonormalities might be related—some of the metabolic disturbances that occur in humans with age could be due to the accumulation of such toxic proteins. High levels of these proteins are seen in many people who do not have, or who have not yet developed, neurodegenerative diseases, as many of them start to accumulate decades before any sign of disease. These proteins might alter metabolism and appetite either directly by affecting target neurons, or by influencing hormonal and neurotransmitter inputs into such neurons.     

Sirtuins and ageing—new findings

Sirtuins are a promising avenue for orally administered drugs that might deliver the anti-aging benefits normally provided by calorie restriction.Calorie (or dietary) restriction was first shown to extend rodent lifespan almost 80 years ago, and remains the most robust longevity-promoting intervention in mammals, genetic or dietary. Sirtuins are NAD-dependent deacylases homologous to yeast Sir2p and were first shown to extend replicative lifespan in budding yeast [1]. Because of their NAD requirement, sirtuins were proposed as mediators of the anti-ageing effects of calorie restriction [1]. Indeed, many studies in yeast, Caenorhabditis elegans, Drosophila melanogaster and mice have supported these ideas [2]. However, a 2011 paper posed a challenge: transgenic strains of C. elegans and Drosophila that overexpress SIR2 were found not to be long-lived [3].Rather than review the extensive sirtuin literature previous to that paper, I focus on a few key studies that have followed it, which underscore a conserved role of sirtuins in slowing ageing. In the first study, two highly divergent budding yeast strains—a lab strain and a clinical isolate—were crossed. A genome-wide quantitative trait locus analysis was then performed to map genes that determine differences in replicative lifespan [4]. The top hit was SIR2, explaining more than one-half of the difference in replicative lifespan between the two strains (due to five codon differences between the SIR2 alleles). In Drosophila, overexpression of dSIR2 in the fat body extended the lifespan of flies on the normal diet, whereas deletion of dSIR2 in the fat body abolished the extension of lifespan by a calorie-restriction-like protocol [5]. This example illustrates the key role of dSIR2 in lifespan determination and its central role in mediating dietary effects on longevity, discussed further below. Another study showed that two transgenic mouse lines that overexpress the mammalian SIRT6—mammals have seven sirtuins—had significantly extended lifespans [6]. Finally, a recent study clearly showed that worm sir2.1 could extend lifespan by regulating two distinct longevity pathways involving insulin-like signalling and the mitochondrial unfolded protein response [7]. All told, this body of work supports the original proposal that sirtuins are conserved mediators of longevity.Many other studies also illustrate that sirtuins can mediate the effects of diet. As an example, calorie restriction completely protected against ageing-induced hearing loss in wild type but not SIRT3^−/− mice [8]. The mitochondrial sirtuin SIRT3 thus helps to protect the neurons of the inner ear against oxidative damage during calorie restriction. Of course, these studies do not imply that sirtuins are the only mediators of calorie restriction effects, but they do indicate that they must be central components.Finally, what about the translational potential of this research, namely using putative SIRT1-activating compounds—resveratrol and newer, synthetic STACs? Two new studies provide strong evidence that the effects of these compounds really do occur through SIRT1. First, acute deletion of SIRT1 in adult mice prevented many of the physiological effects of resveratrol and other STACs [9]. Second, a single mutation adjacent to the SIRT1 catalytic domain abolished the ability of STACs to activate the enzyme in vitro, or to promote the canonical physiological changes in vivo [10].In summary, sirtuins seem to represent a promising avenue by which orally available drugs might deliver anti-ageing benefits normally triggered by calorie restriction. Indeed, the biology of sirtuins is complex and diverse, but this is an indication of their deep reach into key disease processes. Connections between sirtuins and cancer metabolism are but one new example of this. The future path of discovery promises to be exciting and might lead to new drugs that maintain robust health.     

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing,Low-Alcohol Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1_221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3°.A large number of quality wines produced by modern winemaking practices, which favor harvesting fully ripened grapes, frequently contain an excessive ethanol content. This tendency is observed in the majority of the world''s wine-producing areas, and reducing the alcohol levels in wines has become a major concern of the wine industry. Consequently, numerous attempts have been made to engineer Saccharomyces cerevisiae yeast strains with reduced ethanol yields, which would offer faster and less expensive biological alternatives to the current physical processes available for the production of low- and reduced-alcohol wines (29). The biological approaches used so far are all based on diverting sugar metabolism toward by-products other than ethanol by metabolic engineering strategies (7, 8, 18, 19, 21, 22, 30). However, these strategies have so far not satisfied the need to obtain a significant reduction in the ethanol yield without causing the accumulation of undesirable secondary products and/or without affecting yeast physiology. Among these various advances, an efficient strategy is based on the rerouting of the carbon flux toward the production of glycerol. This polyol is a relatively neutral compound from an olfactory perspective, and it is has been demonstrated previously to contribute positively to wine quality through enhanced sweetness and viscosity (27). In yeast, glycerol plays a major role as an osmolyte under osmotic stress conditions and also functions as an essential redox sink in the absence of oxygen, when the reoxidation of excess cytosolic NADH is required (1, 2, 42, 44, 46). This compound is formed by the reduction of dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (encoded by GPD1 and GPD2), followed by dephosphorylation by glycerol-3-phosphatase, which exists as two isoforms: Gpp1 and Gpp2p (see Fig. ​Fig.1).1). By overexpressing GPD1 or GPD2, the production of glycerol has been greatly enhanced, making it possible to decrease the ethanol yield as the result of carbon diversion and reduced NADH availability for the alcohol dehydrogenase reaction. This strategy has been used previously to reduce the ethanol yields in wine and brewer''s yeast (4, 7, 23, 25, 26, 31). For wine, it was shown that these glycerol-overproducing strains have the potential to reduce the ethanol content by 1 to 2°. Nevertheless, major modifications in the production levels of other metabolites, in particular acetate and acetoin (23, 31), which are undesirable at high concentrations in wine, are generated. The production of acetate in glycerol-overproducing wine yeasts has been reduced to a normal level by the deletion of ALD6, coding for an acetaldehyde dehydrogenase (4, 30). The major problem of the accumulation of acetoin, which was shown to accumulate at several grams per liter in commercial GPD1 ald6 wine yeast strains (4), remains to be overcome. At usual concentrations in wine, which vary from undetectable levels to 80 mg/liter (32, 33, 40), this compound has no negative organoleptic influence. However, at concentrations higher than its threshold level (around 150 mg/liter [11]), acetoin can confer an unpleasant buttery flavor on wines. In contrast, the reduced form of acetoin, 2,3-butanediol (2,3-BD), has neutral sensory qualities (data not shown). It is found in wines at concentrations ranging from 0.2 to 3 g/liter (14, 41).Open in a separate windowFIG. 1.Schematic representation of metabolic pathways implicated in our design strategy for a low-alcohol yeast. GP, glycerol phosphatase, encoded by GPP1 and GPP2; GPDH, glycerol phosphate dehydrogenase, encoded by GPD1 and GPD2; PDC, pyruvate decarboxylase, encoded by PDC1, PDC5, and PDC6; ACDH, acetaldehyde dehydrogenase, encoded by ALD4, ALD5, and ALD6; ADH, alcohol dehydrogenase, encoded by ADH1; BDH, Bdh1, encoded by BDH1 (other BDHs exist; however, no other identified gene has been associated with BDH activity); ALS, acetolactate synthase, encoded by ILV2; DS, diacetyl synthetase; DR, diacetyl reductase; G3P, glycerol-3-phosphatase; DHAP, dihydroxyacetone phosphate; acetyl CoA, acetyl coenzyme A; TPP, thiamine PP_i.Bdh1, encoded by BDH1, is the only identified enzyme in yeast catalyzing the reduction of acetoin into 2,3-BD (12). This enzyme has strict stereospecificity for the OHs of carbons in R configuration and acts preferentially as a reductase rather than as a dehydrogenase (11, 12). It is essentially responsible for the formation of (2R,3R)-2,3-BD and part of meso-2,3-BD from (3R)-acetoin and (3S)-acetoin, respectively.The accumulation of acetoin in strains engineered for glycerol overproduction has been attributed to several factors (4). On one hand, it was assumed that the amount of Bdh1 is a rate-limiting factor in the conversion of acetoin into 2,3-BD. On the other hand, it is possible that the Bdh1 reaction is limited by the low level of available NADH since this coenzyme is preferentially used for glycerol synthesis in these strains.The aim of the present study was to investigate in detail the metabolic prerequisites for reducing accumulated acetoin in S. cerevisiae overproducing glycerol and exhibiting reduced acetate formation by promoting the conversion of acetoin into the compound 2,3-BD, which has neutral sensory characteristics. In this study, we first determined the role of Bdh1 in the reduction of acetoin into 2,3-BD during wine fermentation. Next, we studied the impact of the overproduction of NADH-dependent Bdh1 or an engineered NADPH-dependent form of Bdh1 in a model wine yeast, V5, overexpressing GPD1 and lacking ALD6 during fermentation in synthetic must with various sugar concentrations. The NADPH-dependent Bdh1 has been obtained previously by the replacement of three amino acids involved in the NADH binding domain, resulting in the complete reversal of the coenzyme specificity from NADH to NADPH (6). The effects on the growth and fermentation properties of the engineered strains and the levels of by-products and key aromatic compounds formed by the strains were determined.     

Poring over exosome structure

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.164Almost all RNA molecules are processed by RNases to form mature RNAs. In addition, many RNAs are degraded, either because they are no longer needed or because they are aberrant. All of these functions—RNA processing, normal RNA degradation and RNA quality control—are carried out by the eukaryotic RNA exosome complex. In this issue of EMBO reports, the Lorentzen group provide structural insight into the eukaryotic exosome and the mechanism by which it degrades RNA from 3′ to 5′ (Malet et al, 2010).The crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006; Bonneau et al, 2009) and the related bacterial PNPase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these RNA-degrading machines from the three domains of life have a similar structure (Fig 1). They are all composed of a ring of six RNase PH domains, one side of which has a cap that contains putative RNA-binding domains. Although this overall structure is conserved, the way that it is formed is not. Bacterial PNPase is a homotrimer of which each monomer contains two RNase PH domains, an S1 domain and a KH domain. The archaeal PH ring consists of three copies of two proteins and the cap is made of three copies of either one of two proteins. Finally, the eukaryotic exosome core is composed of nine proteins: six with one RNase PH domain each and three cap proteins.Open in a separate windowFigure 1Exosome structures. The bacterial PNPase (left), the archaeal exosome (middle) and eukaryotic core exosome (right) have a common overall structure. The top panels are schematic views from above, showing the cap proteins. The bottom panels show a view from the side, with one-third of the exosome cut away to reveal the RNA in the central channel.In PNPase and the archaeal exosome, substrates enter the PH ring from the cap-side. The putative RNA-binding domains of the cap are therefore probably important for controlling entry to the PH ring. In both archaea and bacteria, the active sites are on the inner side of the PH ring and thus the ribonucleic catalysis occurs inside the central channel. However, in humans and yeast each of the RNase PH domains have point mutations that make the exosome ring catalytically inactive (Dziembowski et al, 2007). Instead, catalysis is carried out by a tenth subunit—Rrp44/Dis3—which binds to the PH ring on the opposite side to the cap proteins (Bonneau et al, 2009; Wang et al, 2007). This organization made it unclear whether RNA also enters the central channel of the exosome in eukaryotes (Fig 1), or whether substrate RNAs directly access the catalytic subunit.Malet and colleagues now provide structural information that resolves this by reconstituting the ten-subunit yeast exosome and analysing its structure with electron microscopy, in the presence and absence of RNA. This analysis suggests that the RNase PH ring of the exosome is stable, but that the cap and catalytic subunits are more flexible than previously appreciated. It is the first structural evidence that in eukaryotes RNA is threaded through the central channel before being degraded by Rrp44.     

Chaperoning the synapse—NMNAT protects Bruchpilot from crashing

Maintaining active zone structure is crucial for synaptic function. In this issue of EMBO reports, NMNAT is shown to act as a chaperone that protects the active zone structural protein Bruchpilot from degradation.EMBO reports (2013) 14 1, 87–94 doi:10.1038/embor.2012.181Synapses perform several tasks independently from the cell body of the neuron, including synaptic vesicle recycling through endocytosis or local protein maturation and degradation. Failure to regulate protein function locally is detrimental to the nervous system as evidenced by neuronal dysfunctions that arise as a consequence of synaptic ageing. This relative synaptic autonomy comes with a need for mechanisms that ensure correct protein (re)folding, and there is accumulating evidence that key chap-erones have a central role in the regulation and maintenance of synaptic structural integrity and function [1]. Work by Grace Zhai''s group, published in this issue of EMBO reports, demonstrates a key role of the Drosophila nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degeneration (Fig 1; [2]).Open in a separate windowFigure 1Results reported by Zang and colleagues [2] reveal a specific role of nicotinamide mononucleotide adenylyltransferase (NMNAT) in preserving active zone structure against use-dependent decline. This protection is exerted by direct interaction with BRP and protection of this key structural protein against ubiquitination and subsequent degradation. BRP, Bruchpilot; Ub, ubiquitin.Active zones, the specialized sites for neurotransmitter release at presynaptic terminals, are characterized by a dense protein network called the cytomatrix at the active zone (CAZ). The protein machinery of the CAZ is responsible for efficient synaptic vesicle tethering, docking and fusion with the presynaptic membrane and, thus, for reliable signal transmission from the neuron to the postsynaptic cell. Clearly, proteins in the CAZ are tightly regulated, especially in response to external cues such as synaptic activity [3,4]. Yet, this particularly crowded protein environment might be favourable for the formation of non-functional—and sometimes toxic—protein aggregates. Chaperones that act at the synapse reduce the probability of crucial protein aggregation by preventing and reverting these inappropriate interactions, which happen as a result of environmental stress.One of these chaperones, the Drosophila neuroprotective NMNAT, was identified in a genetic screen for factors involved in synapse function [5]. Its chaperone activity was later confirmed by using in vitro and in vivo protein folding assays [6]. NMNAT null mutants show severe and early onset neurodegeneration, whereas neurodevelopment does not seem to be strongly affected. Interestingly, degeneration of photoreceptors lacking NMNAT can be significantly attenuated by limiting synaptic activity, either by rearing flies in the dark or by introducing the no receptor potential A (norpA) mutation that blocks phototransduction [5]. These results indicate that NMNAT protects adult neurons from activity-induced degeneration.In this issue of EMBO reports, Zang and colleagues report a role for NMNAT at the synapse. They observed that loss or reduced levels of NMNAT leads to a concomitant loss of several synaptic markers including cysteine-string protein (CSP), synaptotagmin and the active zone structural protein Bruchpilot (BRP). Remarkably, BRP was the only one of these proteins found to co-immunoprecipitate with NMNAT from brain lysates. Both proteins show approximately 50% co-localization at the neuromuscular junction when imaged by 3D-SIM^™ super-resolution microscopy, suggesting that NMNAT might act directly as a chaperone for maintaining a functional BRP conformation.Consistent with a protective role of NMNAT against BRP degradation, RNA interference-mediated NMNAT knockdown leads to BRP ubiquitination, whereas this modification was not detected in control brain lysates. Given the involvement of the ubiquitin proteasome pathway in regulating synaptic development and function [1], the authors tested the effect of the proteasome inhibitor MG-132 on BRP ubiquitination. They observed an increased level of BRP ubiquitination in wild-type flies fed with this drug, suggesting a role for the proteasome in the clearance of ubiquitinated BRP. By contrast, overexpression of NMNAT reduces the level of BRP ubiquitination both in the absence and the presence of MG-132, providing further evidence for the protective role of this chaperone against ubiquitination of BRP (Fig 1).a key role of the […] nicotinamide mononucleotide adenylyltransferase (NMNAT) chaperone in the protection of active zone components against activity-induced degenerationBRP is a cytoskeletal-like protein that is an integral component of T-bars—electron-dense structures that project from the presynaptic membrane and around which synaptic vesicles cluster. In agreement with a protective role of NMNAT against BRP ubiquitination, reduced levels of this chaperone give rise to a marked decrease in T-bar size in an age-dependent manner (Fig 1). Active zones are known to show dynamic changes in response to synaptic activity, and NMNAT was previously reported to protect photoreceptors against activity-induced degeneration [5]. The authors thus tested the effect of minimizing photoreceptor activity on active zone structure by keeping flies in the dark or inhibiting phototransduction by means of the norpA mutation. Both manipulations largely reversed the effect of NMNAT knockdown on T-bar size. Absence of light exposure also significantly reduced the amount of BRP that co-immunoprecipitates with NMNAT, indicating that neuronal activity regulates NMNAT–BRP interaction. Further experiments are needed to examine whether there is a positive correlation between synaptic activity and BRP ubiquitination levels, and whether NMNAT can indeed keep T-bar structure intact by protecting BRP against this modification under conditions of high synaptic activity.Finally, the study shows that reduced NMNAT levels not only caused a loss of BRP from the synapse but also a specific mislocalization of this protein to the cell body, where it accumulates in clusters together with the remaining NMNAT protein. Under these conditions BRP co-immunoprecipitated with the stress-induced Hsp70, a chaperone classically used as a marker for protein aggregation. It is still unclear whether these BRP clusters form as a result of defective anterograde trafficking and/or of enhanced retrograde transport of BRP. In the absence of light stimulation T-bars are properly assembled in nmnat null photoreceptors, but at this stage a role of NMNAT in regulating the axonal transport of BRP under conditions of normal synaptic activity cannot be excluded. Noticeably, two independent recent reports show involvement of NMNAT in mitochondrial mobility [7,8].As BRP and NMNAT co-localize and interact with one another, the simplest model that accounts for all the observations by Zang et al is that NMNAT directly prevents activity-induced ubiquitination of BRP and subsequent degradation. Yet, as its name indicates, this chaperone is an essential enzyme in NAD synthesis. It was previously shown by the Bellen lab that mutant versions of NMNAT, impaired for NAD production, rescue photoreceptor degeneration caused by loss of NMNAT [5]. This strongly suggests that NAD production is not required for stabilization of BRP but this might need further scrutiny [9].…reduced levels of this chaperone [NMNAT] give rise to a marked decrease in T-bar sizeWhile providing further insights into the role of NMNAT at the active zone in Drosophila, the paper by Zang et al might also have important implications for neurodegeneration in mammals. When ectopically expressed in mice, Nmnat has a protective role against Wallerian degeneration, that is, synapse and axon degeneration that rapidly occurs distal from an axonal wound in wild-type animals. This process is significantly delayed in mice overexpressing a chimaeric protein consisting of the amino-terminal 70 residues of the ubiquitination factor E4B (Ube4b) fused through a linker to Nmnat1, known as the Wallerian degeneration slow (Wld^s) protein. Conversely, mutations in the human NMNAT1 gene were characterized in several families with Leber congenital amaurosis—a severe, early-onset neurodegenerative disease of the retina [10,11,12,13]. As Wld^s or Nmnat1 overexpression protects axons from degeneration in various disease models [9], Nmnat1 emerges as a promising candidate for developing protective strategies against axonal degeneration in peripheral neuropathies such as amyotrophic lateral sclerosis but also in glaucoma, AIDS and other diseases [9].     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.     

Autophagy plays a WASHing game

EMBO J (2013) 32: 2685–2696 doi:10.1038/emboj.2013.189; published online August232013Beclin 1 is a crucial regulator of autophagy. It forms a complex with ATG14L, VPS34 or the class III phosphatidylinositol 3-kinase AMBRA1 to control autophagosome formation. A study in this issue of The EMBO Journal (Xia et al, 2013) reports that WASH, a protein known to regulate endosomal sorting, hampers autophagy by inhibiting the AMBRA1-dependent polyubiquitylation of Beclin 1. This modification is required to promote VPS34 activity and to initiate autophagy during starvation.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components. Autophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome that sequesters the autophagic cargo and eventually fuses with the lysosomal compartment, leading to cargo degradation. Autophagy plays a major role in cytoplasm homeostasis by removing protein aggregates and controlling organelle quality, and it is stimulated to promote cell survival during stressful situations, such as nutrient starvation and microbial infection. Autophagosome formation is dependent on evolutionarily conserved Atg (Autophagy-related) proteins initially identified in yeast (Mizushima et al, 2011). They function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via several stages (initiation, elongation and sealing). Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homologue of yeast Atg6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34 (Cecconi and Levine, 2008; Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P) to recruit Atg18 homologues (WIPIs), has to be tightly regulated to keep autophagy under control.Open in a separate windowFigure 1Role of WASH in autophagosome formation. WASH interacts with Beclin 1 via a sequence (121–221) that is not involved in endosomal sorting. In the absence of WASH, when autophagy is stimulated by nutrient deprivation, Beclin 1 interacts with VPS34 (and its adaptor VPS15), ATG14L and AMBRA1. In this complex, Beclin 1 is polyubiquitylated by AMBRA1, which acts as an E3 ligase. The activation of VPS34 produces PI3P at the phagophore to recruit WIPI proteins, and then triggers the machinery to elongate and seal the membrane, thus forming an autophagosome. The VAC domain of WASH involved in endosomal sorting is not required for autophagy regulation.In this issue of The EMBO Journal, Xia et al (2013) demonstrate that WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitylation of Beclin 1. WASH forms a complex with four other proteins and plays an essential role in endosomal sorting by promoting actin polymerization to facilitate segregation of endosomal proteins (Seaman et al, 2013). Using WASH−/− mice, Xia et al (2013) observe that WASH deficiency causes embryonic lethality (E7.5−E9.5) with massive apoptosis-independent cell death and the accumulation of autophagic structures. However, whether autophagy is instrumental in the cell death observed in WASH−/− embryos remains to be investigated. From a series of classical readouts, the authors conclude that WASH downregulates starvation-induced autophagy. Interestingly, WASH is detected on the autophagosomal membrane before and after closure of the autophagosome, but not on the autolysosomes (organelles formed when the autophagosome fuses with the lysosome). The authors first show that WASH has distinct roles in autophagy and in endosomal sorting, because deletion of the VCA domain of WASH that is required for its endosomal function and the knockdown of FAM21 (a protein that directs WASH to the endosomal membrane) do not influence autophagy. They then show that WASH interacts with the coil-coiled domain of Beclin 1. WASH depletion induces more VPS34 to interact with Beclin 1, leading to augmented formation of PtdIns3P and increased recruitment of WIPI-1 to the autophagosomal membrane. However, WASH does not influence the stability of Beclin 1, although it does regulate its K63-linked polyubiquitylation at position K437 during starvation (K63-linked polyubiquitylation suggests a regulatory role, whereas K48-linked polyubiquitylation is frequently associated with protein degradation). Overexpression of WASH reduces the ubiquitylation of Beclin 1, and the K437R Beclin 1 mutant that cannot be ubiquitylated has a low level of interaction with VPS34, which prevents the stimulation of autophagy by starvation (Figure 1). Recently, Beclin 1 has been shown to be ubiquitylated at position K117 during TLR4-induced autophagy (Shi and Kehrl, 2010). Interestingly, Xia et al (2013) show that the K117R Beclin 1 mutant is still ubiquitylated in response to nutrient starvation. Finally, the authors identify the substrate-specific E3 ubiquitin ligase for Beclin 1 in this context. They exclude TRAF6 and NEDD4, two E3 ligases reported to ubiquitylate Beclin 1 (Kuang et al, 2013). AMBRA1, which regulates autophagy in the PI3K complex I, is also known as DCAF3 and interacts with the Cullin 4–DDB1 E3 ligase complex (Jin et al, 2006). Following in vitro E3 ligase assays, Xia et al (2013) conclude that AMBRA1 is the substrate receptor for Beclin 1 ubiquitylation at position K437, adding Cullin4/DDB1 and AMBRA1 to the list of E3 ligases involved in autophagy (Kuang et al, 2013). The finding that WASH and AMBRA1 compete to regulate starvation-induced autophagy raises several questions: how do WASH and AMBRA1 influence one another''s binding to Beclin 1? WASH is evolutionarily conserved, although it is not present in yeast (Linardopoulou et al, 2007)—does it also reduce starvation-induced autophagy in non-mammalian cells? WASH has recently been reported to be required for the lysosomal digestion of autophagy cargo in Dictyostelium during starvation (King et al, 2013). Does this mean that the function of WASH in autophagy has evolved from controlling cargo degradation to forming autophagosomes? Xia et al (2013) do not report any association between WASH and the lysosomal compartment during autophagy; the role of WASH in autophagosome maturation calls for further investigation. Finally, a recent report demonstrates that AMBRA1 interacts with the E3 ligase TRAF6 to ubiquitylate ULK1 (the mammalian orthologue of the yeast Atg1) and to ensure its subsequent stabilization and function (Nazio et al, 2013). ULK1 is part of a complex that acts with PI3K complex 1 to regulate the initiation of autophagy (Mizushima et al, 2011). How are these two AMBRA1-dependent ubiquitylation processes coordinated to control autophagy? In conclusion, both the study reported here (Xia et al, 2013) and the recent study of Nazio et al (2013) clearly demonstrate that AMBRA1 plays a central role in regulating the initiation of autophagy.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Up for grabs; trashing peroxisomes

EMBO J 31 13, 2852–2868 (2012); published online May292012Together with the proteasome, autophagy is one of the major catabolic pathways of the cell. In response to cellular needs or environmental cues, this transport route targets specific structures for degradation into the mammalian lysosomes or the yeast and plant vacuoles. The mechanisms allowing exclusive autophagic elimination of unwanted structures are currently the object of intensive investigations. The emerging picture is that there is a series of autophagy receptors that determines the specificity of the different selective types of autophagy. How cargo binding and recognition is regulated by these receptors, however, is largely unknown. In their study, Motley et al (2012) have shed light into the molecular principles underlying the turnover of excess peroxisomes in the budding yeast Saccharomyces cerevisiae.Peroxisomes perform a series of crucial functions and their number is regulated in response to the metabolic demands of the cell. After proliferation and when no more required, a selective type of autophagy called pexophagy degrades superfluous peroxisomes (Manjithaya et al, 2010). This turnover allows the cell to save the energy required for the maintenance of excess organelles and to generate metabolites that can be used to carry out other functions. Like all selective types of autophagy, pexophagy relies on the conserved core of the autophagy-related (Atg) machinery, but also requires additional proteins that confer specificity of the pathway such as cargo selection and membrane dynamics (Manjithaya et al, 2010). It is still unclear, however, which peroxisomal protein allows the recognition of peroxisomes by the autophagosomes. Although Pex3 and Pex14 have previously been indicated as possible suspects (Bellu et al, 2001, 2002; Farre et al, 2008), their specific contribution to pexophagy was difficult to establish. Deletion of either PEX3 or PEX14, as well as most other PEX genes, leads to defects in peroxisome biogenesis, which makes the dissection of their contribution to peroxisome degradation very difficult to assess. Motley et al (2012) have elegantly exploited S. cerevisiae genetics to isolate pex3 alleles specifically impaired in pexophagy and could thus demonstrate that Pex3 (and not Pex14) mediates the selective engulfment of peroxisomes by autophagosomes. In support to this result, the authors have also identified a new protein, Atg36, which binds Pex3 (Figure 1). Importantly, Atg36 interacts with Atg11, an autophagy adaptor involved in numerous selective types of autophagy in yeast, thereby bringing peroxisomes to the site where autophagosomes will be generated and coordinating the activation of the Atg machinery at this location (Kim et al, 2001; Reggiori et al, 2005; Monastyrska et al, 2008). Atg36, however, is only present in S. cerevisiae and related yeasts. Methylotrophic yeasts, in contrast, appear to have a different protein with the same properties, Atg30 (Farre et al, 2008). It is unclear, however, whether Atg30 is the functional counterpart of Atg36 because these two proteins do not display similarities in their amino-acid sequence.Open in a separate windowFigure 1Schematic representation for a putative Pex3 checkpoint. The peroxisomal integral membrane protein Pex3 acts as a master regulator to determine peroxisome fate. Organelle abundance is regulated by formation of new organelles, and their subsequent segregation (inheritance) and degradation. A new paradigm has been uncovered, whereby Pex3 controls peroxisome abundance through the regulated binding to specific co-factors. At the endoplasmic reticulum (ER), together with Pex19, it initiates biogenesis of new peroxisomes. At the peroxisomal membrane, it ensures that both mother and daughter cells obtain the correct number of peroxisomes, whereas when the organelles become dispensable, Pex3 can initiate their selective degradation. To keep peroxisomes in the mother cell during cell division, Pex3 associates with Inp1 and tether peroxisomes to cortical actin patches. Under pexophagy-inducing conditions, Pex3 binds the newly identified pexophagy factor Atg36 and delivers peroxisomes to the site of autophagosome formation for subsequent degradation into the vacuole.While it is unmistakable that Atg36 (and Atg30) is essential for pexophagy, it remains unclear whether this protein is an autophagy receptor. This class of molecules has four characteristics (Kraft et al, 2010). First, each autophagy receptor binds a specific cargo. Second, they often interact with adaptor proteins, which function as scaffolds that bring the cargo–receptor complex in contact with the core Atg machinery to allow the specific sequestration of the cargo. Third, they possess at least one LC3-interacting region (LIR) motif that enables them to interact with the LC3/Atg8 pool present in the interior autophagomes and assures the hermetic enwrapping of the cargo into these vesicles. Fourth, autophagy receptors are degraded in the lysosome/vacuole together with the cargo that they bind to. While Atg36 (and Atg30) binds both the cargo and the adaptor protein Atg11, this protein does not appear to be turned over in the vacuole during pexophagy and a LIR motif has not been pinpointed yet. Consequently, it is unclear whether Atg36 is a new type of autophagy receptor or acts together with a not yet identified autophagy receptor involved in pexophagy.A very interesting concept emerging from the work of Motley et al (2012) is that a single protein, that is, Pex3, could be the central regulator of peroxisome homoeostasis (Figure 1). Pex3 is involved in peroxisome biogenesis, segregation and degradation (Bellu et al, 2002; Hoepfner et al, 2005; Farre et al, 2008; Munck et al, 2009; Ma et al, 2011). As a result, the cell could regulate peroxisome abundance by modulating Pex3 function and/or its array of interactions. In this context, it would be particularly interesting to determine whether Pex3 is also the decision maker of a quality control mechanism that eliminates peroxisomes when not correctly assembled and thus dysfunctional, or when not accurately distributed during cell division. Clearly, additional experiments are needed to understand how Pex3 regulates peroxisome homoeostasis, but this protein and this organelle could represent a convenient system to unveil the principles that regulate the steady-state level of other subcellular compartments.     

Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase

EMBO J 32 15, 2099–2112 doi:10.1038/emboj.2013.125; published online May312013Mutations in Parkin represent ∼50% of disease-causing defects in autosomal recessive-juvenile onset Parkinson''s disease (AR-JP). Recently, there have been four structural reports of autoinhibited forms of this RING-IBR-RING (RBR) ubiquitin ligase (E3) by the Gehring, Komander, Johnston and Shaw groups. The important advances from these studies set the stage for the next steps in understanding the molecular basis for Parkinson''s disease (PD).Regulated protein degradation requires that E3s and their access to substrates be exquisitely controlled. RBR family E3s provide striking examples of this regulation. The complex and compact structures of Parkin (Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) as well as another RBR E3, human homologue of Ariadne (HHARI) (Duda et al, 2013), demonstrate extraordinarily intricate inter-domain arrangements. These autoinhibited structures ensure that their functions are restricted until activated.Until recently, RBR E3s were believed to be a subclass of RING E3s, which allosterically activate E2 conjugated with ubiquitin (E2∼Ub). However, Wenzel et al (2011) determined that they are actually hybrid E3s, containing an E2 binding site in RING1 and a catalytic cysteine residue in the domain designated as RING2. The catalytic cysteine is an acceptor for an ubiquitin from RING1-bound E2∼Ub forming an intermediate (E3∼Ub) that leads to substrate or autoubiquitination. In this way, RBRs resemble HECT E3s, which also form catalytic intermediates in ubiquitination. There are 13 human RBR family E3s. Besides Parkin, two notable RBRs are HOIL-1 and HOIP, which form part of a complex integral to NF-κB activation (Wenzel and Klevit, 2012).In addition to causal roles in AR-JP, single allele mutations of Parkin are found in some sporadic cases of PD (references in Wauer and Komander, 2013). Mutations in the Parkin-associated kinase PINK1, which is upstream of Parkin, also account for a significant number of AR-JP cases (Hardy et al, 2009; Narendra et al, 2012; Lazarou et al, 2013). A number of diverse Parkin substrates have been postulated to be associated with PD. There is substantial evidence that one role for Parkin is at mitochondria. Once activated and recruited to damaged/depolarized mitochondria by PINK1, it ubiquitinates exposed mitochondrial proteins leading to both proteasomal degradation and mitophagy (Narendra et al, 2012; Sarraf et al, 2013). Parkin has also been implicated in cell surface signalling and as a tumour suppressor (see references in Wauer and Komander, 2013).Parkin encodes five structured domains, beginning with an N-terminal ubiquitin-like domain (UbLD) and followed by four domains that each bind two zinc (Zn) atoms (Figure 1A). The most N-terminal of the Zn-binding domains is RING0. C-terminal to this is the RBR, consisting of RING1, the IBR and RING2. The crystal structures of inactive Parkin from Riley et al (2013), Trempe et al (2013) and Wauer and Komander (2013) show remarkable congruity. Spatially, the IBR is at the complete opposite end of the molecule from RING2, to which it is connected by a partially unstructured ∼37 residue linker. This linker includes a two-turn helix, referred to as the repressor element of Parkin (REP) or tether, which binds and occludes the E2 binding face of RING1. RING1 occupies the central position in these structures, and RING0 separates RING1 from RING2 (Figure 1B and C). The latter contains the residue identified by Wenzel et al (2011), and confirmed by all three groups, to be the catalytic cysteine, C431. A lower resolution structure also includes the UbLD and places this domain adjacent to RING1 (Trempe et al, 2013). A second unstructured linker connects the UbLD and RING0. UbLDs are involved in a number of protein–protein interactions and small angle X-ray scattering confirms that this domain is integral to the core structure of Parkin (Spratt et al, 2013; Trempe et al, 2013). Biophysical characterization of Parkin and HHARI suggests that each is a monomer in solution.Open in a separate windowFigure 1Schematic and spatial representation of Parkin. (A) Primary structure and domain designations of Parkin, including the REP sequence within the otherwise unstructured IBR-RING2 linker. (B) Structural representation of full-length Parkin (PDB 4K95) highlighting the complex domain interactions in the three-dimensional structure, the catalytic C431 residue, and residue W403 within the REP, which plays a role in stabilizing the autoinhibited form of Parkin. (C) A model of Parkin with the E2 UbcH5B/Ube2D2 bound (devised using PDB 4K95 and PDB 4AP4 to mimic the position of an E2 bound to RING1) to illustrate the required displacement of UbLD and REP and the large distance between the E2∼Ub attachment site of the E2 and the catalytic active site of Parkin. Note that in this conformation the catalytic Cys within RING2 (C431) remains buried by RING0.RING1 is the only bona fide RING domain. All NMR and crystal structures of IBR domains from Parkin, HHARI and HOIP (PDB ID: 2CT7) are in good agreement. The Parkin and HHARI RING2s are structurally highly homologous and share a common Zn-coordinating arrangement with IBR domains. In contrast to the IBR and RING2, RING0 has a distinct arrangement of Zn-coordinating residues (Beasley et al, 2007; Duda et al, 2013; Riley et al, 2013; Spratt et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013) (see Figure 1F of Trempe et al (2013) for the various Zn coordination arrangements).All of the Parkin crystal structures represent inactive forms of the E3. This is imposed by the quaternary positioning of the domains, which precludes activity in multiple ways. RING0 plays two obvious roles to maintain Parkin in an inactive state. RING0 shares an interface with RING2 and buries C431, making it unavailable as an ubiquitin acceptor. Moreover, RING0 intervenes between RING1 and RING2, creating an insurmountable separation of >50 Å between the active site Cys of an E2 bound to RING1 and C431 (Figure 1B and C). Thus, RING0 must be displaced for ubiquitin transfer to occur. Accordingly, deletion of RING0 results in a marked increase in Parkin autoubiquitination and in C431 reactivity (Riley et al, 2013; Trempe et al, 2013; Wauer and Komander, 2013). In HHARI, these two inhibitory functions are fulfilled by the C-terminal Ariadne domain, which similarly interposes between RING1 and RING2 (Duda et al, 2013).Additional inhibition is provided by the REP, which binds to RING1 at the canonical RING-E2 binding site and prevents E2 binding. This provides at least a partial explanation for the impaired ability of Parkin to bind E2 when compared to HHARI, which lacks this element (Duda et al, 2013). A disease-associated REP mutant (A398T) at the RING1 interface increases autoubiquitination (Wauer and Komander, 2013). The significance of inhibition by REP-RING1 binding was verified by mutating a critical RING1-interacting REP residue (W403A). This increased autoubiquitination and E2 binding (Trempe et al, 2013). Consistent with the requirement for charging C431 with ubiquitin in mitochondrial translocation (Lazarou et al, 2013), Parkin association with depolarized mitochondria is accelerated with this mutation (Trempe et al, 2013). Interestingly, W403 also interacts with the C-terminal Val of Parkin within RING2, and could therefore potentially further stabilize the autoinhibited form of the protein (Riley et al, 2013), consistent with previous observations (Henn et al, 2005).The quaternary structure of full-length Parkin also suggests that displacement of its N-terminal UbLD must occur for full activation (Trempe et al, 2013). The positioning of the UbLD adjacent to RING1 indicates that it would provide a steric impediment to E2∼Ub binding (Figure 1B and C). Additionally, displacement of the UbLD could be important to relieve interactions with the IBR-RING2 linker, which, as suggested in a previous study (Chaugule et al, 2011), might help to maintain Parkin in an inactive state. Finally, the crystal structure of the full-length Parkin indicates that the UbLD is not available for interactions with other proteins. This would limit Parkin''s range of intermolecular interactions.RBR E3s have at least two domains critical for sequential ubiquitin transfer and full activity, RING1 and RING2. The RING1 of Parkin, as well as all other RBR E3s, is notable in lacking the basic residue in the second Zn coordinating loop (or its equivalent in U-box proteins), which has recently been implicated in RING-mediated transfer of Ub from E2∼Ub (Metzger et al, 2013). This suggests that other factors play compensatory roles in positioning ubiquitin for transfer from E2∼Ub to C431. A non-mutually exclusive possibility is that the lack of this basic residue in RING1 limits unwanted attack on the E2∼Ub linkage, thereby minimizing the unregulated ubiquitination. Turning to RING2, the area surrounding the active site C431 of Parkin is notable in that it includes a sequence recognizable as a catalytic triad, similar to that in deubiquitinating enzymes. The Cys-His-Glu grouping, found in Parkin and other RBR E3s, contributes to in vitro activity (Riley et al, 2013; Wauer and Komander, 2013). Interestingly, however, the Glu was dispensable in a cellular assay (Riley et al, 2013). This triad is conserved in HHARI, where an Asn between the Cys and His residues (found in a number of RBRs but not conserved in Parkin), was found to be important for catalysis (Duda et al, 2013).The advances made in these studies impart significant information about an important and clinically relevant E3. However, Parkin, as well as HHARI, has been captured in their inactive, unmodified forms. One obvious question is how does Parkin transition between inactive and active states. PINK1 is implicated in phosphorylating Parkin on its UbLD and potentially other sites, with evidence that phosphorylation contributes to Parkin activation (Narendra et al, 2012). How phosphorylation could contribute to protein interactions that might facilitate Parkin activation, potentially including Parkin oligomerization (Lazarou et al, 2013), is unknown. Regardless, it is evident that considerable unwinding of its quaternary structure must take place.While there is much work ahead to understand these processes, one important interface that must be disrupted for activation is that between the REP and RING1. It is intriguing to consider that such interruption might be associated with other alterations in the IBR-RING2 linker, potentially facilitating the movement of the UbLD from RING1 and contributing to activation. Related to activation is the all-important question of how Parkin recognizes and targets specific substrates. While the UbLD represents a potential site of interaction, most purported substrates are not known to have UbLD-interaction domains. Although interactions involving the UbLD could occur indirectly, through bridging molecules, there is also evidence that other regions of Parkin, including the RBR region, might recognize substrates either directly or indirectly (Tsai et al, 2003) and that some substrates may be phosphorylated by PINK1 (Narendra et al, 2012). Conformational changes induced by substrate interactions, particularly in the IBR RING2 linker, could, as above, represent an important aspect of activation.There are over 75 missense mutations of Parkin associated with AR-JP, most of these inactivate the protein, but there are also some that are activating (Wauer and Komander, 2013). Activating mutations presumably result in pathology at least partially as a consequence of increased autoubiquitination and degradation (e.g., A398T). The current studies help to provide a classification of missense mutations into those that affect (i) folding or stability, (ii) catalytic mechanism, and (iii) interactions between domains. Interdomain mutations might inactivate or contribute to constitutive activation leading to autoubiquitination and degradation.Finally, we know little about how the autosomal recessive and the much more prevalent sporadic forms of PD overlap in their molecular pathology. However, mitochondrial dysfunction is increasingly a common theme. Thus, with the structure of the inactive protein in hand, there is hope that we can begin to consider ways in which domain interactions might be altered in a controlled manner to activate, but not hyperactivate, this critical E3 and lessen the progression of PD.     

Cilia born out of shock and stress

EMBO J (2013) 32 23, 3029–3040 10.1038/emboj.2013.223; published online October112013Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins.Centrioles are barrel-shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule-organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle-dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule-dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM-1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet-Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: First, in cycling cells they may serve to sequester essential ciliary proteins (Stowe et al, 2012). Second, upon initiation of the ciliogenesis programme, centriolar satellite components seem to promote the recruitment of specific ciliary proteins to basal bodies (Ferrante et al, 2006; Lopes et al, 2011; Stowe et al, 2012).In a new study in The EMBO Journal, Villumsen et al (2013) now describe how stress–response pathways conspire to control ciliogenesis. The authors observed that specific environmental stresses, such as ultraviolet light radiation (UV) or heat shock, but not ionizing radiation (IR), trigger rapid displacement of PCM-1, AZI1 and CEP290 from centriolar satellites. However, OFD1 remained associated with satellites, indicating that centriolar satellites persist despite UV-induced removal of PCM-1. This might come as some surprise, since PCM-1 depletion by RNA interference (RNAi) is thought to disrupt satellite integrity (Kim et al, 2008; Lopes et al, 2011); however, satellite loss upon PCM-1 RNAi may be a consequence of prolonged depletion of PCM-1, while acute PCM-1 displacement by stress might only ‘remodel'' centriolar satellites. It is also possible that not all satellites are created equal, and they do vary in protein composition (Kim et al, 2008; Staples et al, 2012). If so, UV-induced PCM-1 removal may disrupt some, but not all satellites.A good candidate regulator of centriolar satellite remodelling was the stress-activated MAP kinase p38, and indeed, Villumsen et al (2013) found p38 MAPK activity to be stimulated by both UV and heat shock but not IR in U2OS cells, mirroring those very stress pathways that also cause displacement of AZI1 and PCM-1 from satellites. Furthermore, p38 MAPK was essential for UV-induced dispersal of PCM-1 and AZI1. The authors then tested the hypothesis that stress-induced centriolar satellite remodelling could involve changes in the interactome of AZI1, and—consistent with an earlier proteomics study (Akimov et al, 2011)—identified PCM-1, CEP290 and the mindbomb E3 ubiquitin protein ligase 1 (MIB1) as the main AZI1 binding partners. GFP-MIB1 localized to centriolar satellites and mono-ubiquitylated AZI1, PCM-1 and CEP290 in cycling cells. In response to UV, both ubiquitylation of these proteins and MIB1 activity were reduced; notably, UV-induced MIB1 inactivation was independent of p38 MAPK activity, indicating that these two enzymes may act via distinct pathways (Figure 1A).Open in a separate windowFigure 1(A) Regulation of centriolar satellite remodelling. (B) Schematic summary of how centriolar satellite remodelling might facilitate ciliogenesis. See text for details.What could be the purpose of MIB1-dependent ubiquitylation of these satellite proteins? It certainly does not seem to regulate subcellular targeting, as in MIB1-depleted cells, AZI1 and PCM-1 both localised normally to centriolar satellites and could still be displaced by UV. Instead, ubiquitylation seems to suppress the interaction between AZI1 and PCM-1, consistent with the observation that UV, a condition that also reduces their ubiquitylation, enhances the binding of AZI1 to PCM-1.PCM-1, CEP290 and AZI1 all participate in ciliogenesis (Kim et al, 2008; Wilkinson et al, 2009; Stowe et al, 2012), raising the possibility that MIB1 might also affect this process. Indeed, serum starvation, which is known to promote cilia formation, attenuated MIB1 activity. Furthermore, MIB1 overexpression reduced the ciliogenesis observed in serum-starved cells, while MIB1 depletion in proliferating cells triggered a marked increase in the proportion of cells that formed cilia; this seems to reflect a direct effect of MIB1 on ciliogenesis, since neither MIB1 depletion nor overexpression altered cell cycle progression. Taken together, downregulation of MIB1 enzymatic activity appears to be a pre-requisite for efficient ciliogenesis, regardless of whether it is triggered by physiological ciliogenesis-promoting signals or by environmental stresses, making MIB1 a novel negative regulator of cilia formation.The recent discovery of ciliopathy-associated mutations in constituents of the DNA damage response signalling pathway pointed to a connection between DNA damage and ciliogenesis (Chaki et al, 2012). With the new link between UV and centriolar satellites, the authors next asked if UV radiation might affect ciliogenesis. Remarkably, UV and heat shock both triggered cilia assembly in RPE-1 cells in a p38 MAPK-dependent manner. MIB1 depletion further enhanced ciliogenesis after UV radiation, again implying an additive effect of p38 MAPK signalling and MIB1 suppression (Figure 1A).While finer details on the precise role of centriolar satellite components in cilia formation are still lacking, a more coherent picture is finally starting to emerge. In cycling cells, ubiquitination by MIB1 could serve to limit the interaction between AZI1 and PCM-1 on centriolar satellites (Figure 1B). Under these conditions PCM-1 may bind and sequester CEP290, an essential ciliogenic protein, thereby precluding untimely cilia formation (Stowe et al, 2012). Both during normal and stress-induced ciliogenesis programs, remodelling of centriolar satellites creates a permissive environment for cilia formation, and a key step in this process is downregulation of MIB1 activity. While it remains to be established how the latter is achieved, it is clear that MIB1 inactivation causes loss of ubiquitylation and increased binding between AZI1 and PCM-1. Preferential interaction of PCM-1 with AZI1 could in turn facilitate release of CEP290 from centriolar satellites and its subsequent accumulation at the centrosome. Once CEP290 reaches the optimum concentration at the centriole/basal body, it could serve to tether AZI1–PCM-1 complexes. PCM-1 could then concentrate Rab8 GTPase near centrosomes, allowing CEP290 to recruit Rab8 into the cilium, where it acts to extend the ciliary membrane (Kim et al, 2008).Collectively, the findings reported here provide strong experimental support to the notion that centriolar satellites are negative regulators of ciliogenesis in proliferating cells. Their role is central to limit untimely formation of cilia in cells. Environmental strains elicit stress–response pathways that converge to relieve the ciliogenesis block imposed by satellites. It is tempting to speculate that stress-induced cilia might serve as signalling platforms and contribute to checkpoint activation or perhaps initiation of repair mechanisms, but more work is needed to establish the true purpose of ciliogenesis in this context. It is of considerable interest that a recent study reports that autophagy, another stress-induced pathway, selectively removes OFD1 from satellites to promote ciliogenesis (Tang et al, 2013). Therefore stress-mediated centriolar satellite remodelling seems to be an evolving theme in the control of ciliogenesis.