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Lysosomal proteins in cell death and autophagy
Michaela Mrschtik and Kevin M. Ryan
Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow, G61 1BD, UK
∗Correspondence: Kevin M. Ryan, Cancer Research UK Beatson Institute, Garscube, Estate, Switchback Road, Glasgow G61 1BD, UK Tel: +441413303655 FAX: +441419426521 E-mail: [email protected]
Abstract Nearly 60 years ago, lysosomes were first described in the laboratory of Christian de Duve, a discovery that significantly contributed to him being awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating “the structural and functional organization of the cell". Initially thought of as a simple waste degradation facility of the cells, these organelles recently emerged as signalling centres with connections to major cellular processes. This minireview will give an overview of the many roles of lysosomal proteins in two of these processes, cell death and autophagy. We will discuss both resident lysosomal proteins as well those that temporarily associate with lysosomes to influence autophagy and cell death pathways. Particular focus will be given to studies in mammalian cells and in vivo systems. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.13253 This article is protected by copyright. All rights reserved.
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Introduction - Lysosome structure and function Lysosomes are single-membrane vesicles that can be found in almost all eukaryotic cells. These organelles are characterized by a low internal pH of 4.5-5, which is generated by the membrane complex v-ATPase[1]. The main function of lysosomes is the degradation of incoming material of all kinds, including organelles, proteins and lipids. These cargoes can be delivered to the lysosomes from outside the cell (via endocytosis) or from inside the cell itself (via autophagy) and monomers generated from the degradation of these materials can either diffuse out of the lysosome or be actively transported to the cytosol by lysosomal proteins for reuse by the cell.
Lysosomes are well equipped for their task as cellular recycling centres - 435 proteins are described to be localized within lysosomes[2], and these include different classes of hydrolytic enzymes, such as proteases, lipases, glycosidases, sulfatases and nucleases, which digest any cargo coming to the lysosomes optimally at the low pH inside the organelle. There are, however, many more proteins that form the structure of lysosomes: the lysosomal membrane contains more than 100 proteins[3], including lysosomal anchoring proteins, transporters, receptors and enzymes. The lysosomal tails of transmembrane proteins need to be heavily glycosylated to avoid degradation in the harsh conditions inside the vesicles, and the inner lysosomal membrane is completely covered by a protective glycocalyx for this purpose. In addition to lysosome resident proteins, there are other factors that can temporarily associate with lysosomes and exert a function at this compartment, such as the mTOR complex[4] or MHC class II molecules[5]. Last but not least, a broad spectrum of proteins, vesicles and even organelles are transported to the lysosomes for degradation, adding to the complex composition of this organelle.
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Given this plethora of proteins that are within or are shuttled to lysosomes, it is not surprising that many of the components of this organelle participate in crucial cellular processes, such as cell growth, survival and death decisions. In the following section, we will describe the role of lysosomal proteins in different cell death processes, followed by a discussion about their involvement in autophagy.
Lysosomes in cell death Ways to die: the different cell death modes Just as there are different reasons for a cell to die, there are different roads that initiate and execute cell death and these modes can have different consequences for the organism. The Nomenclature Committee on Cell Death (NCCD) suggested 12 distinct cell death modes in 2012, defined by biochemical properties[6]. Apoptosis, necrosis, necroptosis and autophagic cell death are the best studied cell death pathways which will be focused on in this review. In this section, the role of lysosomal proteins in apoptosis, necroptosis and necrosis will be discussed while their functions in autophagy and autophagic cell death will be addressed in the second part of the review.
Apoptosis is an evolutionary conserved, regulated form of cellular suicide. The features of apoptotic cell death include nuclear condensation and fragmentation and membrane blebbing, and the main effector molecules of this process are the caspase proteases. There are 2 separate, converging pathways of apoptosis that are triggered by different stimuli.
The intrinsic apoptotic pathway is activated by “internal” signals, such as DNA damage, ER stress or viral infections and is initiated through mitochondrial outer membrane permeabilisation (MOMP), leading to leakage of mitochondrial components including
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cytochrome c that can activate caspases and the apoptotic machinery. Mitochondrial pores are formed by dimerization of the pro-apoptotic Bcl-2 family members Bax and Bak, and their formation can be inhibited by anti-apoptotic Bcl-2 proteins such as Bcl-2, Bcl-XL and MCL1 and promoted by BH3-only proteins like Bim and Bid.
The extrinsic pathway, by contrast, is initiated by death receptor activation, for example by binding of Tumour Necrosis Factor (TNF) or Fas ligand (FasL) to their receptors at the cell surface, which leads to the formation of a death inducing signal complex (DISC). This activates Caspase 8, eventually leading to effector caspase activation. Additionally, the effector mechanism of the intrinsic pathway - MOMP – can be initiated by Caspase 8 through Bid cleavage to enhance the cell death signalling.
Traditionally, necrotic cell death was seen as a passive, immunogenic death mode that can be triggered by catastrophic events such as heat shock or irreparable damage to the cell. This death has been associated with rupture of the lysosomal and cellular membranes and leakage of cellular components to the extracellular matrix. More recently, many of these “unregulated” death events have been found to be regulated and mechanisms have been described[7] such as lysosomal membrane permeabilization (LMP), which is associated with cell death in some, but not all cases. For a more extensive overview of different cell death modalities and their regulation, we suggest [7, 8] for general information, [9] for cell death in cancer and [10, 11] for lysosomal cell death in particular.
Lysosomal components in cell death In the early days after the discovery of lysosomes, these organelles were described as possible “suicide bags” that could cause death in a variety of settings[12]. This idea was,
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however, not followed up thoroughly at the time – in part due to a lack of experimental methods to thoroughly test it. In recent years, an interest in this field has been rediscovered and a multitude of reports on the involvement of lysosomes in different cell death mechanisms has been published. It is now clear that lysosomes are involved in many cell death events. The contribution of lysosomes can be active (leading to cell death) or passive (a consequence of cell death), as well as amplifying the cell death response[13] (Figure 1). Some lysosomal proteins likely to play key roles in different cell death modes are discussed below.
Cathepsins as major effectors of LMP There are many triggers of LMP, ranging from lysosomotropic agents – molecules that accumulate within the lysosomes and cause a destabilisation of the lysosomal membrane - to lysosomal reactive oxygen species (ROS), which are generated by a reaction of H2O2 with lysosomal iron during oxidative stress[11]. Effectors of lysosomal cell death, however, seem clearly defined: the lysosomal protease family consisting of the aspartic cathepsin D and the cysteine cathepsins B, C, F, H, K, L, O, S, V, W and X are commonly attributed to be the effectors of lysosomal cell death downstream of LMP.
Among the cathepsin family, cathepsin D has been predominantly linked to apoptosis in early reports, both following intrinsic pathway stimuli like DNA-damaging agents etoposide and adriamycin in murine embryonic fibroblasts[14] or extrinsic pathway stimuli IFN-γ and Fas in HeLa cells and TNF-α in U937 human lymphoma cells[15]. Strikingly, cathepsin D has been shown to be practically inactive at a neutral pH[16] – a condition it finds inside the cellular lumen, therefore shedding doubt on a major role of this protein downstream of LMP. One report has, however, demonstrated that apoptosis after TNF stimulation can be
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concomitant with cytosolic acidification, which might increase cathepsin activity in the cytosol[17] (Figure 2). Despite some reports to the contrary, a major role in lysosomemediated apoptotic cell death is attributed to cathepsin D to date, and a few molecular apoptotic targets of this protease are described. These include the pro-apoptotic Bcl-2 proteins Bax[18] and Bid in vitro up to a pH of 6.2[19] and the initiator caspase-8 in neutrophil apoptosis[20] (Figure 1).
Several reports indicate that cathepsin B could also be an effector of cell death after LMP, for example after treatment with TNF[21] it has been shown that CtsB -/- mice are protected from TNF-α induced liver cell apoptosis[22]. Nevertheless, cysteine cathepsins have been reported to have no significant role in apoptosis in response to other extrinsic pathway stimuli in cancer cell line models[23]. The cysteine cathepsins retain some activity in pH neutral environments[24], and several major cysteine cathepsin substrates in the apoptotic pathway have been identified, including Bid[25]. Additional substrates include anti-apoptotic Bcl-2 family members Bcl-2, Bcl-Xl and Mcl-1, which prevent apoptosis upstream of MOMP and also XIAP[25], which is an apoptosis inhibitor downstream of MOMP (Figure 1).
In some cellular systems, cathepsin release after LMP is described as an enhancer, but not initiator of apoptosis. Mouse monocytes and fibroblasts deficient in cathepsin B or L display delays in apoptosis in response to some stimuli such as IL-3 withdrawal and etoposide treatment. By contrast, apoptosis after exposure to UV light is not affected by the absence of these cathepsins[26]. Furthermore, this early occurring LMP is shown to be strictly dependent on the presence of Bax/Bak and caspase activation in these cells. This indicates that MOMP and caspase activation precede LMP in this system, with LMP contributing to the extent of apoptosis observed after different apoptotic stimuli.
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Some cathepsins have also been implicated in necrotic forms of cell death. Cathepsin B activity was shown to increase in CA1 neurons after ischaemic shock in monkeys, followed by translocation to the cytoplasm. Administration of the cathepsin B specific inhibitor CA074 directly after the ischaemic insult was sufficient to prevent two thirds of the cell death observed in these neurons[27].
In addition to classic necrosis, cathepsins have also been shown to be involved in regulated or programmed necrosis. One of the first reports on this topic describes a role for cathepsin B in the execution of non-apoptotic cell death following treatment with microtubule stabilising agents in non-small cell lung cancer (NSCLC) cells, where cathepsin B inhibition leads to significant protection from cell death and the morphological changes observed before the cells die[28]. In addition, mouse cathepsin Q, a cathepsin with no known human orthologue, has been shown to contribute to programmed necrosis in DKO Bax/Bak cells after prolonged DNA damage, which displays a late-occurring cell death mode that is independent of caspases and could not be blocked by the necroptosis inhibitor necrostatin[29]. In vivo work also suggests that LMP with subsequent cathepsin release can cause physiological, necrotic death during the involution of mammary gland tissue after weaning[30].
More recently, cathepsins have been shown to play roles in the specific form of programmed necrosis termed necroptosis. In macrophages it has been reported that cathepsins B and S can cleave the Rip1 kinase[31] – a factor central to necroptosis. Conversely, in dendritic cells, cathepsin D has been shown to cleave caspase 8 which serves to cleave Rip1[32]. Hence, in this context, cathepsins promote rather than limit necroptosis.
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Due to the destructive potential of cathepsins, cells have developed natural safeguards to disarm these proteases if they escape lysosomal enclosure – the cystatins. These are potent inhibitors of many cysteine cathepsins, and both intracellular cystatins – often referred to as stefins – and extracellular cystatins exist (Figure 1). Cystatins themselves have been shown to be aberrantly regulated in some diseases, including neurodegerative disorders, cancer and inflammatory diseases[33], adding a further layer of complexity to the contribution of cathepsins in cell death processes.
Lysosomal sphingolipid metabolism and cell death Sphingolipids are cellular lipid components that fulfil various roles in cell death and survival mechanisms. Sphingomyelin, an integral part of cellular membranes, can be metabolised to produce ceramide and sphingosine, which can act as pro-death signalling proteins. The conversion from sphingomyelin to sphingosine via ceramide can be achieved by 2 lysosomal proteins, acid sphingomyelinase (A-SMase) and acid ceramidase (A-CDase) (Figure 2). These two enzymes and their molecular products have been implicated in lysosomal cell death initiation (summarized in[34]). Ceramide produced by A-SMase was found to be an activator of Cathepsin D, leading to its proteolytic activation in the lysosome[35], thereby potentially linking the pro-apoptotic functions of A-SMase to Cathepsin D as an effector molecule. A later report links this Cathepsin D activation by A-SMase to cleavage and activation of Bid by Cathepsin D after TNF stimulation, subsequently leading to caspase activation and apoptosis[19].
Sphingosine, the product of A-CDase has been shown to be a lysosomotropic agent, which accumulates in lysosomes where it becomes trapped by protonation and causes LMP. Low doses of extracellular sphingosine (20μM) caused rapid LMP and necrosis[36]. More recently, it was reported that lysosomal sphingosine causes LMP and cell death in hepatoma cells after treatment with TNF and cycloheximide, which can be inhibited by both knockdown of A-SMase and A-CDase[37].
Lysosomal guardians LMP occurs after a destabilisation of lysosomes, resulting in subsequent spillage of lysosomal components. In contrast to sphingosine, which promotes this process, some lysosomal proteins act as safeguards and stabilise lysosomes to prevent leakage - and their manipulation can therefore modulate cell survival and death events upstream of LMP.
HSP70, a molecular chaperone that is commonly upregulated in cancer[38], is a good potential candidate for the role of lysosomal guardian. It has long been known that HSP70 depletion causes caspase-independent cell death, but only about a decade ago a landmark study showed that it does this through inhibiting LMP[38]. The report demonstrates that HSP70 can localise to endolysosomes and associate with their membranes, which makes these HSP70 containing lysosomes less prone to LMP (Figure 1). The molecular mechanism of this protection from LMP has since been uncovered: HSP70 can bind to an endolysosomal phospholipid called bis(monoacylglycero)phosphate (BMP) in low pH conditions, which is an essential cofactor for A-SMase[39]. This report therefore functionally links the lysosomal sphingolipid metabolism to HSP70-mediated LMP protection.
HSP70 has also been suggested to play a role in neuronal death after ischaemia in monkeys, where it was found to be a potential calpain substrate[40], placing it downstream of calpain activation and upstream of LMP and cathepsin D release in this system. Another study,
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however, found that calpain 1 cleaves the lysosome-associated membrane protein LAMP2 in a cellular system that mimics light-induced retinal degeneration (LIRD)-associated calpainand cathepsin-dependent, caspase-independent cell death. Moreover, LAMP2, but not HSP70 is cleaved in the course of LIRD in vivo[41]. Furthermore, calpain 1 was shown to cleave LAMP2a and the b2 subunit of v-ATPase in vivo in mammary glands undergoing involution in weaned mice, and blocking of calpain 1 can delay the involution process[42]. This study describes a potential mechanism for the LMP-mediated cell death observed in mammary gland involution in vivo[30], suggesting that calpain 1 activation destabilises lysosomes to cause LMP in this setting. Additionally, LAMP1 and 2 protein levels have been found to be decreased by oncogenes such as v-H-Ras, c-srcY527F and K-ras in a cathepsindependent fashion, leading to lysosomal destabilisation and sensitisation to LMP-mediated cell death[43].
Other lysosomal proteins in cell death As discussed previously, the lysosome contains more than 200 different proteins, and many more that temporarily associate with lysosomes, but only a small proportion of these falls into the groups recently discussed. Several of the other lysosomal proteins have, however, been shown to affect cell death mechanisms and the following section discusses a selection of these proteins.
DRAM1, an evolutionary-conserved, p53-induced protein with lysosomal location, has been shown to be required for full apoptotic response after p53 activation[44]. DRAM2, a member of a family of DRAM-related proteins in mammals, which also localizes to lysosomes, does not seem to share this function of DRAM1[45]. DRAM1 has also been shown to be a target of p73, but in contrast to its role in p53-mediated apoptosis DRAM1 expression does not
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affect p73-induced cell death[46]. A direct function of DRAM1 in LMP has recently been described in T cells, which undergo a caspase-independent mode of cell death with LMP, cathepsin release and subsequent MOMP after HIV infection. In this setting, DRAM1 is induced and its ablation leads to decreased LMP and death, but a rise in T cells infected with HIV[47], indicating that DRAM1 might be implicated in the eradication of HIV-infected Tcells.
Another previously undefined lysosome/endoplasmic reticulum-localised protein called TMEM166 has been reported to mediate cell death when overexpressed in HeLa cells. Subsequently, a role for this protein was shown in vivo in rat brains undergoing middle cerebral artery occlusion, where siRNA mediated inhibition of TMEM166 improved the survival rate and decreased the symptoms of the injuries[48]. The mechanism of this action, however, is currently not clear.
Other lysosomal proteins with roles in cell death include the lysosome-associated LAPF, which is suggested to link p53 non-transcriptional pro-death activities to lysosomes[49] and LAPTM5, a protein that is induced in regressing parts of neuroblastoma tumours and which promotes caspase-independent cell death when overexpressed in neuroblastoma tumour cell lines[50]. Additionally, lysosomal storage disorder genes have a variety of impacts on cell death and survival, particularly in neuronal cells[51].
Overall, work from recent years has uncovered a range of proteins that influence cell death through the lysosome, and this exciting field of research is still expanding and constantly yielding new insights into the mechanisms of LMP induction and the role of lysosomal proteins in cell death mechanisms.
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Lysosomes in autophagy Roads to degradation Cellular life involves the generation and degradation of macromolecules. This degradation serves a dual purpose: disposal of unwanted or damaged constituents, thereby protecting the cell, and generation of energy and recycling of “building blocks” in the process. The cellular breakdown of substances can be achieved by different means, depending on the kind of matter degraded and its origin, and several of these waste disposal and recycling roads terminate in the lysosome. Extracellular material can be taken up by endocytosis, while intracellular constituents are often delivered to the lysosome by autophagy. Due to the limitations of this minireview, we will not discuss the role of lysosomal proteins in the late steps of endocytosis (summarised in[52, 53]); instead, we will focus on autophagic processes and the lysosome.
Autophagy is a summarizing term for three distinct cellular processes - macroautophagy, microautophagy and chaperone-mediated autophagy (Figure 3). Their collective purpose is the degradation of intracellular materials with each form of autophagy using different mechanisms for lysosome delivery. They are supported by a second, lysosome-independent intracellular degradation system, the ubiquitin-proteasome pathway. The main difference between the two systems is that the proteasome mainly degrades short-lived proteins while autophagy favours degradation of long-lived and misfolded proteins while additionally being the only way to degrade organelles in a cell.
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During macroautophagy, cargo is engulfed in specialised vesicles called autophagosomes, which terminate in the bulk delivery of proteins and organelles (for example: mitochondria during mitophagy, peroxisomes during pexophagy etc.) to the lysosome via autophagosomelysosome fusion, forming autolysosomes[54] [55].
Macroautophagy has broadly been linked to both cell survival and cell death, with a seemingly paradoxical context-dependent involvement in the promotion of both processes. Cell death with excessive autophagy, or so called autophagic cell death, is commonly described as a distinct cell death mode, but autophagy has also been shown to be a contributing factor for cell death execution by apoptosis and necrosis[56, 57].
Chaperone-mediated autophagy (CMA) is another autophagy pathway that has been identified in mammalian cells. It represents a mode of selective, lysosomal degradation of individual proteins that are recognised through a pentapeptide motif in their sequence. This process is executed one target protein at a time and is critically dependent on the cytosolic chaperone Hsc70 and the lysosomal transmembrane protein LAMP2A which functions as a CMA receptor[58].
The least characterised autophagic process is microautophagy, which is executed through direct invagination of lysosomal vesicles and subsequent uptake of material (both proteins and organelles) in intralysosomal multivesicular bodies[48]. The molecular mechanisms of this pathway(s) remain elusive and equally little is known about the involvement of particular lysosomal proteins in this process.
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In the following sections, we will discuss the involvement of both lysosomal proteins and proteins that temporarily associate with lysosomes in autophagic processes, with particular attention given to macroautophagy.
Autophagy, cell death and the lysosome Lysosomes and autophagy are closely tied, therefore it is not surprising that many diseases display defects in both these processes and that these defects may ultimately lead to cell death. For example, lysosomal storage disorders have been proposed to be autophagic diseases[59]. Niemann-Pick disease type C, caused by mutations in the lysosomal cholesterol transporting system consisting of Npc1 or Npc2, is a prime example of a lysosomal storage disease that involves autophagy deficiencies. About a decade ago, one study described an accumulation of autophagosomes in Purkinje cells of Npc2 Niemann-Pick disease model mice, which undergo massive cell death upon Npc2 loss[60]. More recently, an accumulation of autophagosomes in Npc1 deficient neurons has been shown to be due to a block in the macroautophagy pathway, and autophagy stimulation with the mTORC1 inhibitor rapamycin could partially rescue cell death in this model[61]. Another example is myopathic Danon disease, caused by mutations in LAMP2, which displays a pronounced accumulation of autophagosomes[62, 63].
Some neurological disorders have been shown to have defects in autophagic clearance of specific proteins, leading to the pathologies observed. CMA defects in degrading mutated forms of alpha-synuclein or mutant tau in Parkinson’s disease and Alzheimer’s disease have been causally implicated in these diseases (summarized in[64]).
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In addition to these pathologies, several lysosomal proteins have been shown to both affect autophagy and cell death. The previously described p53 target DRAM1 falls into this category, as it not only contributes to p53 mediated apoptosis, but also induces autophagy[44]. The DRAM1 gene encodes a number of isoforms, and while these localise to different cellular compartments, several are capable of inducing autophagy[65]. Another example is the lysosomal transmembrane protein LAPTM4B. One report indicates that LAPTM4B promotes autophagosome-lysosome fusion and autophagic flux and thereby protects breast cancer cells from starvation-induced cell death[66]. It will also be interesting to investigate the effect of LAPTM4B knockout in vivo to reveal its importance in the autophagososme maturation process. These reports collectively suggest that the lysosome, autophagy and cell death can be closely linked, and future studies will undoubtedly uncover additional lysosomal players in the autophagy-death network.
mTOR: a mediator of lysosome function and macroautophagy The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts as a key regulator of cell growth, which impacts on both lysosomes and autophagy. mTOR exists in 2 distinct complexes, mTORC1 and mTORC2. The former has been researched extensively and is responsible for many of the known mTOR functions in cellular metabolism (for a comprehensive review on this topic, we recommend [67]), while the latter functions mainly in cytoskeleton modulation.
mTORC1 is directly connected to macroautophagy via free amino acids, which are released in lysosomes upon completion of autophagy and sensed by mTORC1 to activate growth and reduce the autophagic process. The molecular players in this process are still not completely established, but recent years have seen a vast improvement in our understanding of how
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mTOR senses the abundance of amino acids. Current evidence points towards a lysosomal amino acid sensing mechanism that leads to mTOR activation, and some lysosomal proteins involved in this process may already be identified (Figure 4).
Rheb has been known to be an activator of mTORC1 in response to amino acids. This interaction was linked to lysosomes in 2010, when a trimeric protein complex termed “Ragulator” was discovered. This complex was found to anchor Rag GTPases at the lysosomal membrane to promote mTOR translocation and activation by the lysosomeresident Rheb in response to amino acid availability[4]. The amino acid sensing mechanism has further been found to be dependent on a lysosomal protein complex: the v-ATPase. Its function in this pathway lies downstream of the accumulation of free amino acids in the lysosomes and upstream of Rag GTPase activation[68].
As previously mentioned, autophagy is known to lead to the lysosomal release of free amino acids. It is therefore not surprising that autophagy – which is commonly induced after deactivation of mTORC1 - can reactivate mTOR after prolonged starvation[69]. This culminates in a process termed autophagic lysosome reformation (ALR), which generates proto-lysosomes from autolysosomes. These vesicles eventually mature into functional lysosomes and replenish the lysosomal population in the cell. mTOR reactivation is essential for ALR, and the lysosomal protein Spinster has been shown to be necessary for mTOR reactivation after prolonged starvation. This effect was shown to be dependent on the sugartransporting capacities of Spinster[70].
Recently, the well-described mTORC1 regulatory complex TSC has been found to be transported to the lysosomes by the Rag GTPases during amino acid starvation where it
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regulates its target Rheb to fully release mTORC1 from the lysosome[71]. The TSC complex is therefore involved in all known mTORC1 metabolic responses.
Lysosome positioning adds another layer of complexity to the mTOR-lysosome-autophagy axis. The clustering of lysosomes in a region of the cell – in the cell periphery or in a perinuclear region – influences mTORC1 signalling and both autophagosome and autolysosome generation[72]. This process is dependent on intracellular pH, which impacts the lysosomal GTPase ARL8B to move lysosomes along microtubules together with the motor protein KIF2.
A network of macroautophagy and lysosome regulation The connection between autophagy and lysosomal function becomes even more apparent through their shared transcription-dependent regulation. In 2009, a network of jointly expressed genes, many of them encoding lysosomal proteins, was discovered[73]. This complex was named the CLEAR (coordinated lysosomal expression and regulation element) network, due to the presence of a palindromic sequence in the promoter of the target genes. The transcription factor responsible for this orchestrated expression of genes is called TFEB, and it was found to directly regulate at least 471 genes – many of them encoding lysosomal proteins[74]. Overexpression of TFEB leads to increased lysosomal mass. Among its targets are also autophagy genes, linking the process of lysosome generation with autophagic functions (Figure 4).
Starvation was found to activate TFEB, which in turn leads to increased expression of autophagy genes and an overall enhanced autophagic flux[75]. This effect was originally attributed to an inhibition of Serine 142 phosphorylation in TFEB by MAPK, thereby
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describing an mTOR-independent, transcriptional regulation of autophagy that is linked to a lysosomal gene regulator. mTORC1 has, however, been found to modulate TFEB phosphorylation in a residue of the serine rich region between amino acids 462-469 to regulate the transcription of the lysosomal target gene v-ATPase by TFEB[76]. Another report finds an mTORC1-dependent regulation of TFEB in Ser211 in full nutrient conditions, leading to scavenging of the transcription factor in the cytosol to render it inactive[77].
TFEB, mTOR, autophagy and lysosomes are connected by several nodes, and it is likely that we have not unravelled their full interaction yet. Future studies will undoubtedly shed more light on the regulation of their interactions and their context-dependent outcomes.
Concluding remarks In this review, we have discussed the role of lysosomes in cell death, autophagy and nutrient sensing. It is important to mention, however, that lysosomes are also critical for other important cellular functions such as iron homeostasis, inflammasome activation, secretion, exocytosis and our innate and adaptive immune responses. Each of these areas of biology is associated with human disease and so it has been a natural progression to ask whether lysosomes can be a target for therapeutic intervention. In this regard, the most promising progress so far is in experimental studies that promote autophagy and as a result lysosomal degradation, to clear protein aggregates associated with neurodegenerative disease or the accumulation of molecules caused by lysosomal storage disorders. This approach appears promising since it seems so far that a moderate up-regulation of lysosomal function can be beneficial in diseased cells without any dramatic detrimental effect on normal tissue.
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Studies have also focused on inhibiting lysosomal function, and this has received a lot of attention recently since it is considered that certain tumours may be autophagy-dependent. The agents used so far have largely been lysosomotropic agents which raise the lysosomal pH and therefore block degradation. While this may be a feasible strategy in the short-term it seems likely that blocking lysosomal function entirely will have consequences for cellular and organismal function long-term. The cell can of course modulate lysosome function in very specific ways and so further studies should yield clues as to how best to either promote or inhibit specific lysosomal functions whether related to cell death, autophagy or beyond.
Acknowledgements We are grateful to members of the Tumour Cell Death Laboratory for advice and for critical reading of the manuscript. Work in the Tumour Cell Death Laboratory is supported by The Association for International Cancer Research and Cancer Research UK.
Conflict of Interest Statement The authors do not have any financial, personal or professional interests to declare that could be construed to influence this paper.
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11. Aits, S. & Jaattela, M. (2013) Lysosomal cell death at a glance, Journal of cell science. 126, 1905-12. 12. de Duve, C. (1983) Lysosomes revisited, European journal of biochemistry / FEBS. 137, 391-7. 13. Johansson, A. C., Appelqvist, H., Nilsson, C., Kagedal, K., Roberg, K. & Ollinger, K. (2010) Regulation of apoptosis-associated lysosomal membrane permeabilization, Apoptosis : an international journal on programmed cell death. 15, 527-40. 14. Wu, G. S., Saftig, P., Peters, C. & El-Deiry, W. S. (1998) Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity, Oncogene. 16, 2177-83. 15. Deiss, L. P., Galinka, H., Berissi, H., Cohen, O. & Kimchi, A. (1996) Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha, The EMBO journal. 15, 3861-70. 16. Capony, F., Morisset, M., Barrett, A. J., Capony, J. P., Broquet, P., Vignon, F., Chambon, M., Louisot, P. & Rochefort, H. (1987) Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells, The Journal of cell biology. 104, 253-62. 17. Nilsson, C., Johansson, U., Johansson, A. C., Kagedal, K. & Ollinger, K. (2006) Cytosolic acidification and lysosomal alkalinization during TNF-alpha induced apoptosis in U937 cells, Apoptosis : an international journal on programmed cell death. 11, 1149-59. 18. Bidere, N., Lorenzo, H. K., Carmona, S., Laforge, M., Harper, F., Dumont, C. & Senik, A. (2003) Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis, The Journal of biological chemistry. 278, 31401-11. 19. Heinrich, M., Neumeyer, J., Jakob, M., Hallas, C., Tchikov, V., Winoto-Morbach, S., Wickel, M., Schneider-Brachert, W., Trauzold, A., Hethke, A. & Schutze, S. (2004)
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Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation, Cell death and differentiation. 11, 550-63. 20. Conus, S., Perozzo, R., Reinheckel, T., Peters, C., Scapozza, L., Yousefi, S. & Simon, H. U. (2008) Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation, The Journal of experimental medicine. 205, 685-98. 21. Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U., Bastholm, L., Boes, M., Elling, F., Leist, M. & Jaattela, M. (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor, The Journal of cell biology. 153, 9991010. 22. Guicciardi, M. E., Miyoshi, H., Bronk, S. F. & Gores, G. J. (2001) Cathepsin B knockout mice are resistant to tumor necrosis factor-alpha-mediated hepatocyte apoptosis and liver injury: implications for therapeutic applications, The American journal of pathology. 159, 2045-54. 23. Spes, A., Sobotic, B., Turk, V. & Turk, B. (2012) Cysteine cathepsins are not critical for TRAIL- and CD95-induced apoptosis in several human cancer cell lines, Biological chemistry. 393, 1417-31. 24. Turk, B., Bieth, J. G., Bjork, I., Dolenc, I., Turk, D., Cimerman, N., Kos, J., Colic, A., Stoka, V. & Turk, V. (1995) Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenous protein inhibitors, cystatins, Biological chemistry Hoppe-Seyler. 376, 225-30. 25. Droga-Mazovec, G., Bojic, L., Petelin, A., Ivanova, S., Romih, R., Repnik, U., Salvesen, G. S., Stoka, V., Turk, V. & Turk, B. (2008) Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues, The Journal of biological chemistry. 283, 19140-50.
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26. Oberle, C., Huai, J., Reinheckel, T., Tacke, M., Rassner, M., Ekert, P. G., Buellesbach, J. & Borner, C. (2010) Lysosomal membrane permeabilization and cathepsin release is a Bax/Bak-dependent, amplifying event of apoptosis in fibroblasts and monocytes, Cell death and differentiation. 17, 1167-78. 27. Yamashima, T., Kohda, Y., Tsuchiya, K., Ueno, T., Yamashita, J., Yoshioka, T. & Kominami, E. (1998) Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpaincathepsin hypothesis', The European journal of neuroscience. 10, 1723-33. 28. Broker, L. E., Huisman, C., Span, S. W., Rodriguez, J. A., Kruyt, F. A. & Giaccone, G. (2004) Cathepsin B mediates caspase-independent cell death induced by microtubule stabilizing agents in non-small cell lung cancer cells, Cancer research. 64, 27-30. 29. Tu, H. C., Ren, D., Wang, G. X., Chen, D. Y., Westergard, T. D., Kim, H., Sasagawa, S., Hsieh, J. J. & Cheng, E. H. (2009) The p53-cathepsin axis cooperates with ROS to activate programmed necrotic death upon DNA damage, Proceedings of the National Academy of Sciences of the United States of America. 106, 1093-8. 30. Kreuzaler, P. A., Staniszewska, A. D., Li, W., Omidvar, N., Kedjouar, B., Turkson, J., Poli, V., Flavell, R. A., Clarkson, R. W. & Watson, C. J. (2011) Stat3 controls lysosomalmediated cell death in vivo, Nature cell biology. 13, 303-9. 31. McComb, S., Shutinoski, B., Thurston, S., Cessford, E., Kumar, K. & Sad, S. (2014) Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase, Journal of immunology. 192, 5671-8. 32. Zou, J., Kawai, T., Tsuchida, T., Kozaki, T., Tanaka, H., Shin, K. S., Kumar, H. & Akira, S. (2013) Poly IC triggers a cathepsin D- and IPS-1-dependent pathway to enhance cytokine production and mediate dendritic cell necroptosis, Immunity. 38, 717-28.
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41. Villalpando Rodriguez, G. E. & Torriglia, A. (2013) Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2, Biochimica et biophysica acta. 1833, 2244-53. 42. Arnandis, T., Ferrer-Vicens, I., Garcia-Trevijano, E. R., Miralles, V. J., Garcia, C., Torres, L., Vina, J. R. & Zaragoza, R. (2012) Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization, Cell death and differentiation. 19, 1536-48. 43. Fehrenbacher, N., Bastholm, L., Kirkegaard-Sorensen, T., Rafn, B., Bottzauw, T., Nielsen, C., Weber, E., Shirasawa, S., Kallunki, T. & Jaattela, M. (2008) Sensitization to the lysosomal cell death pathway by oncogene-induced down-regulation of lysosome-associated membrane proteins 1 and 2, Cancer research. 68, 6623-33. 44. Crighton, D., Wilkinson, S., O'Prey, J., Syed, N., Smith, P., Harrison, P. R., Gasco, M., Garrone, O., Crook, T. & Ryan, K. M. (2006) DRAM, a p53-induced modulator of autophagy, is critical for apoptosis, Cell. 126, 121-34. 45. O'Prey, J., Skommer, J., Wilkinson, S. & Ryan, K. M. (2009) Analysis of DRAM-related proteins reveals evolutionarily conserved and divergent roles in the control of autophagy, Cell cycle. 8, 2260-5. 46. Crighton, D., O'Prey, J., Bell, H. S. & Ryan, K. M. (2007) p73 regulates DRAMindependent autophagy that does not contribute to programmed cell death, Cell death and differentiation. 14, 1071-9. 47. Laforge, M., Limou, S., Harper, F., Casartelli, N., Rodrigues, V., Silvestre, R., Haloui, H., Zagury, J. F., Senik, A. & Estaquier, J. (2013) DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV, PLoS pathogens. 9, e1003328.
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48. Li, W. W., Li, J. & Bao, J. K. (2012) Microautophagy: lesser-known self-eating, Cellular and molecular life sciences : CMLS. 69, 1125-36. 49. Li, N., Zheng, Y., Chen, W., Wang, C., Liu, X., He, W., Xu, H. & Cao, X. (2007) Adaptor protein LAPF recruits phosphorylated p53 to lysosomes and triggers lysosomal destabilization in apoptosis, Cancer research. 67, 11176-85. 50. Inoue, J., Misawa, A., Tanaka, Y., Ichinose, S., Sugino, Y., Hosoi, H., Sugimoto, T., Imoto, I. & Inazawa, J. (2009) Lysosomal-associated protein multispanning transmembrane 5 gene (LAPTM5) is associated with spontaneous regression of neuroblastomas, PloS one. 4, e7099. 51. Boustany, R. M. (2013) Lysosomal storage diseases--the horizon expands, Nature reviews Neurology. 9, 583-98. 52. Luzio, J. P., Gray, S. R. & Bright, N. A. (2010) Endosome-lysosome fusion, Biochemical Society transactions. 38, 1413-6. 53. Luzio, J. P., Parkinson, M. D., Gray, S. R. & Bright, N. A. (2009) The delivery of endocytosed cargo to lysosomes, Biochemical Society transactions. 37, 1019-21. 54. Yang, Z. & Klionsky, D. J. (2010) Mammalian autophagy: core molecular machinery and signaling regulation, Current opinion in cell biology. 22, 124-31. 55. Shen, H. M. & Mizushima, N. (2014) At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy, Trends in biochemical sciences. 39, 6171. 56. Macintosh, R. L. & Ryan, K. M. (2013) Autophagy in tumour cell death, Semin Cancer Biol. 23, 344-351. 57. Ryter, S. W., Mizumura, K. & Choi, A. M. (2014) The impact of autophagy on cell death modalities, International journal of cell biology. 2014, 502676.
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58. Kaushik, S. & Cuervo, A. M. (2012) Chaperone-mediated autophagy: a unique way to enter the lysosome world, Trends Cell Biol. 22, 407-17. 59. Settembre, C., Fraldi, A., Rubinsztein, D. C. & Ballabio, A. (2008) Lysosomal storage diseases as disorders of autophagy, Autophagy. 4, 113-4. 60. Ko, D. C., Milenkovic, L., Beier, S. M., Manuel, H., Buchanan, J. & Scott, M. P. (2005) Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick type C disease, PLoS genetics. 1, 81-95. 61. Sarkar, S., Carroll, B., Buganim, Y., Maetzel, D., Ng, A. H., Cassady, J. P., Cohen, M. A., Chakraborty, S., Wang, H., Spooner, E., Ploegh, H., Gsponer, J., Korolchuk, V. I. & Jaenisch, R. (2013) Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease, Cell reports. 5, 1302-15. 62. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., Sue, C. M., Yamamoto, A., Murakami, N., Shanske, S., Byrne, E., Bonilla, E., Nonaka, I., DiMauro, S. & Hirano, M. (2000) Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease), Nature. 406, 90610. 63. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. L., Hartmann, D., Lullmann-Rauch, R., Janssen, P. M., Blanz, J., von Figura, K. & Saftig, P. (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice, Nature. 406, 902-6. 64. Cuervo, A. M. & Wong, E. (2014) Chaperone-mediated autophagy: roles in disease and aging, Cell research. 24, 92-104. 65. Mah, L. Y., O'Prey, J., Baudot, A. D., Hoekstra, A. & Ryan, K. M. (2012) DRAM-1 encodes multiple isoforms that regulate autophagy, Autophagy. 8, 18-28.
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66. Li, Y., Zhang, Q., Tian, R., Wang, Q., Zhao, J. J., Iglehart, J. D., Wang, Z. C. & Richardson, A. L. (2011) Lysosomal transmembrane protein LAPTM4B promotes autophagy and tolerance to metabolic stress in cancer cells, Cancer research. 71, 7481-9. 67. Shimobayashi, M. & Hall, M. N. (2014) Making new contacts: the mTOR network in metabolism and signalling crosstalk, Nature reviews Molecular cell biology. 15, 155-62. 68. Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y. & Sabatini, D. M. (2011) mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase, Science. 334, 678-83. 69. Yu, L., McPhee, C. K., Zheng, L., Mardones, G. A., Rong, Y., Peng, J., Mi, N., Zhao, Y., Liu, Z., Wan, F., Hailey, D. W., Oorschot, V., Klumperman, J., Baehrecke, E. H. & Lenardo, M. J. (2010) Termination of autophagy and reformation of lysosomes regulated by mTOR, Nature. 465, 942-6. 70. Rong, Y., McPhee, C. K., Deng, S., Huang, L., Chen, L., Liu, M., Tracy, K., Baehrecke, E. H., Yu, L. & Lenardo, M. J. (2011) Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation, Proceedings of the National Academy of Sciences of the United States of America. 108, 7826-31. 71. Demetriades, C., Doumpas, N. & Teleman, A. A. (2014) Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2, Cell. 156, 786-99. 72. Korolchuk, V. I., Saiki, S., Lichtenberg, M., Siddiqi, F. H., Roberts, E. A., Imarisio, S., Jahreiss, L., Sarkar, S., Futter, M., Menzies, F. M., O'Kane, C. J., Deretic, V. & Rubinsztein, D. C. (2011) Lysosomal positioning coordinates cellular nutrient responses, Nature cell biology. 13, 453-60. 73. Sardiello, M., Palmieri, M., di Ronza, A., Medina, D. L., Valenza, M., Gennarino, V. A., Di Malta, C., Donaudy, F., Embrione, V., Polishchuk, R. S., Banfi, S., Parenti, G., Cattaneo,
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E. & Ballabio, A. (2009) A gene network regulating lysosomal biogenesis and function, Science. 325, 473-7. 74. Palmieri, M., Impey, S., Kang, H., di Ronza, A., Pelz, C., Sardiello, M. & Ballabio, A. (2011) Characterization of the CLEAR network reveals an integrated control of cellular clearance pathways, Human molecular genetics. 20, 3852-66. 75. Settembre, C., Di Malta, C., Polito, V. A., Garcia Arencibia, M., Vetrini, F., Erdin, S., Erdin, S. U., Huynh, T., Medina, D., Colella, P., Sardiello, M., Rubinsztein, D. C. & Ballabio, A. (2011) TFEB links autophagy to lysosomal biogenesis, Science. 332, 1429-33. 76. Pena-Llopis, S., Vega-Rubin-de-Celis, S., Schwartz, J. C., Wolff, N. C., Tran, T. A., Zou, L., Xie, X. J., Corey, D. R. & Brugarolas, J. (2011) Regulation of TFEB and V-ATPases by mTORC1, The EMBO journal. 30, 3242-58. 77. Martina, J. A., Chen, Y., Gucek, M. & Puertollano, R. (2012) MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB, Autophagy. 8, 903-14.
Figure Legends
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Figure 1: Lysosomal proteins in cell death mechanisms LMP can culminate in necrosis/necroptosis or apoptosis and it can also amplify the apoptotic response of cells. The executioners of these processes are cathepsins, which are released from the lysosome following LMP. Cathepsin cleavage targets in the apoptotic pathway include Bid, anti-apoptotic Bcl-2 family proteins, Caspase 8 and XIAP. Cystatins, the cellular inhibitors of Cathepsins, can limit cytosolic cathepsin activity. The chaperone HSP70 can prevent LMP by scavenging BMP and thereby inhibiting A-CDase activity. Several other lysosomal proteins, such as DRAM1, LAPTM5 and TMEM166, have been described as cell death effectors that may function independently of LMP. LMP, Lysosomal membrane permeabilisation BMP, Bis(monoacylglycero)phosphate XIAP, X-linked inhibitor of apoptosis DRAM1, DNA-damage-regulated autophagy modulator 1 LAPTM5, lysosomal protein transmembrane 5 A-CDase, acid ceramidase
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Figure 2: Inducers of lysosomal cell death Intrinsic apoptotic stimuli, such as DNA damage by chemotherapeutic drugs, and extrinsic stimuli like TNF-α can cause LMP. Additionally, ROS and lysosomotropic agents are potent LMP inducers. The membrane lipid sphingomyelin can be enzymatically processed in the lysosome to form ceramide – which has been shown to activate Cathepsin B – and sphingosine, which can act as a lysosomotropic agent. LAMP2 and the v-ATPase have been shown to be cleavage targets of Calpain1, leading to lysosomal destabilisation. Several other lysosomal proteins, including DRAM1, LAPTM5 and TMEM166 have been demonstrated to promote cell death. TNF-α, tumour necrosis factor α LMP, lysosomal membrane permeabilisation ROS, Reactive oxygen species LAMP2, lysosomal-associated membrane protein 2 DRAM1, DNA-damage regulated autophagy modulator 1 LAPTM5, lysosomal protein transmembrane 5
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Figure 3: Autophagic pathways The term autophagy describes 3 distinct cellular pathways. Macroautophagy is characterised by the sequestration of cytoplasmic material – including organelles – into double membrane structures that arise from phagophores to produce autophagosomes. These vesicles subsequently fuse with lysosomes to promote bulk degradation of their contents. Chaperonemediated autophagy targets single proteins for lysosomal degradation, a process that depends on LAMP2A as receptor and is aided by several cytoplasmic and lysosomal chaperone proteins. Microautophagy delivers cytoplasmic constituents to lysosomes through a direct lysosomal invagination process. LAMP2A, lysosomal-associated membrane protein 2A
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Figure 4: Macroautophagy and lysosome regulation in response to amino acid availability. When amino acids are abundant (top panel), mTORC1 is tied to the lysosome by Rag GTPases, using the lysosomal complex Ragulator as an anchor. At the lysosome, mTORC1 is activated by TFEB and senses amino acids through an inside out mechanism that involves vATPase. Under these conditions, mTORC1 can promote an inhibitory phosphorylation of the transcription factor RHEB. Altogether, these processes lead to low autophagic flux, high mTORC1 activation and concomitant growth and proliferation. Closely following amino acid withdrawal (middle panel), mTORC1 is inactivated and dissociated from the lysosome, a process that is aided by TSC. mTORC1 inactivation leads to increased autophagic flux. TFEB is activated and translocates to the nucleus, leading to transcription of genes containing CLEAR elements. Overall, these processes culminate in increased autophagic flux and increased lysosomal mass and function. Under prolonged starvation conditions (bottom panel), amino acids are produced by autophagy and mTORC1 can re-associate with lysosomes to be reactivated. This process is positively influenced by the lysosomal protein Spinster. Reactivation of mTORC1 at the lysosome is necessary for autophagic lysosome regeneration (ALR), which regenerates the pool of lysosomes from autolysosomes. The overall outcome of these processes is decreased autophagic flux, reactivation of mTORC1 and regeneration of lysosomes. mTORC1, mechanistic target of rapamycin – complex 1 TFEB, transcription factor EB RHEB, Ras homolog enriched in brain TSC, tuberous sclerosis CLEAR, coordinated lysosomal expression and regulation
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Lysosomal proteins in cell death and autophagy
Michaela Mrschtik and Kevin M. Ryan
Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow, G61 1BD, UK
∗Correspondence: Kevin M. Ryan, Cancer Research UK Beatson Institute, Garscube, Estate, Switchback Road, Glasgow G61 1BD, UK Tel: +441413303655 FAX: +441419426521 E-mail: [email protected]
Abstract Nearly 60 years ago, lysosomes were first described in the laboratory of Christian de Duve, a discovery that significantly contributed to him being awarded a share of the 1974 Nobel Prize in Physiology or Medicine for elucidating “the structural and functional organization of the cell". Initially thought of as a simple waste degradation facility of the cells, these organelles recently emerged as signalling centres with connections to major cellular processes. This minireview will give an overview of the many roles of lysosomal proteins in two of these processes, cell death and autophagy. We will discuss both resident lysosomal proteins as well those that temporarily associate with lysosomes to influence autophagy and cell death pathways. Particular focus will be given to studies in mammalian cells and in vivo systems. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/febs.13253 This article is protected by copyright. All rights reserved.
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Introduction - Lysosome structure and function Lysosomes are single-membrane vesicles that can be found in almost all eukaryotic cells. These organelles are characterized by a low internal pH of 4.5-5, which is generated by the membrane complex v-ATPase[1]. The main function of lysosomes is the degradation of incoming material of all kinds, including organelles, proteins and lipids. These cargoes can be delivered to the lysosomes from outside the cell (via endocytosis) or from inside the cell itself (via autophagy) and monomers generated from the degradation of these materials can either diffuse out of the lysosome or be actively transported to the cytosol by lysosomal proteins for reuse by the cell.
Lysosomes are well equipped for their task as cellular recycling centres - 435 proteins are described to be localized within lysosomes[2], and these include different classes of hydrolytic enzymes, such as proteases, lipases, glycosidases, sulfatases and nucleases, which digest any cargo coming to the lysosomes optimally at the low pH inside the organelle. There are, however, many more proteins that form the structure of lysosomes: the lysosomal membrane contains more than 100 proteins[3], including lysosomal anchoring proteins, transporters, receptors and enzymes. The lysosomal tails of transmembrane proteins need to be heavily glycosylated to avoid degradation in the harsh conditions inside the vesicles, and the inner lysosomal membrane is completely covered by a protective glycocalyx for this purpose. In addition to lysosome resident proteins, there are other factors that can temporarily associate with lysosomes and exert a function at this compartment, such as the mTOR complex[4] or MHC class II molecules[5]. Last but not least, a broad spectrum of proteins, vesicles and even organelles are transported to the lysosomes for degradation, adding to the complex composition of this organelle.
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Given this plethora of proteins that are within or are shuttled to lysosomes, it is not surprising that many of the components of this organelle participate in crucial cellular processes, such as cell growth, survival and death decisions. In the following section, we will describe the role of lysosomal proteins in different cell death processes, followed by a discussion about their involvement in autophagy.
Lysosomes in cell death Ways to die: the different cell death modes Just as there are different reasons for a cell to die, there are different roads that initiate and execute cell death and these modes can have different consequences for the organism. The Nomenclature Committee on Cell Death (NCCD) suggested 12 distinct cell death modes in 2012, defined by biochemical properties[6]. Apoptosis, necrosis, necroptosis and autophagic cell death are the best studied cell death pathways which will be focused on in this review. In this section, the role of lysosomal proteins in apoptosis, necroptosis and necrosis will be discussed while their functions in autophagy and autophagic cell death will be addressed in the second part of the review.
Apoptosis is an evolutionary conserved, regulated form of cellular suicide. The features of apoptotic cell death include nuclear condensation and fragmentation and membrane blebbing, and the main effector molecules of this process are the caspase proteases. There are 2 separate, converging pathways of apoptosis that are triggered by different stimuli.
The intrinsic apoptotic pathway is activated by “internal” signals, such as DNA damage, ER stress or viral infections and is initiated through mitochondrial outer membrane permeabilisation (MOMP), leading to leakage of mitochondrial components including
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cytochrome c that can activate caspases and the apoptotic machinery. Mitochondrial pores are formed by dimerization of the pro-apoptotic Bcl-2 family members Bax and Bak, and their formation can be inhibited by anti-apoptotic Bcl-2 proteins such as Bcl-2, Bcl-XL and MCL1 and promoted by BH3-only proteins like Bim and Bid.
The extrinsic pathway, by contrast, is initiated by death receptor activation, for example by binding of Tumour Necrosis Factor (TNF) or Fas ligand (FasL) to their receptors at the cell surface, which leads to the formation of a death inducing signal complex (DISC). This activates Caspase 8, eventually leading to effector caspase activation. Additionally, the effector mechanism of the intrinsic pathway - MOMP – can be initiated by Caspase 8 through Bid cleavage to enhance the cell death signalling.
Traditionally, necrotic cell death was seen as a passive, immunogenic death mode that can be triggered by catastrophic events such as heat shock or irreparable damage to the cell. This death has been associated with rupture of the lysosomal and cellular membranes and leakage of cellular components to the extracellular matrix. More recently, many of these “unregulated” death events have been found to be regulated and mechanisms have been described[7] such as lysosomal membrane permeabilization (LMP), which is associated with cell death in some, but not all cases. For a more extensive overview of different cell death modalities and their regulation, we suggest [7, 8] for general information, [9] for cell death in cancer and [10, 11] for lysosomal cell death in particular.
Lysosomal components in cell death In the early days after the discovery of lysosomes, these organelles were described as possible “suicide bags” that could cause death in a variety of settings[12]. This idea was,
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however, not followed up thoroughly at the time – in part due to a lack of experimental methods to thoroughly test it. In recent years, an interest in this field has been rediscovered and a multitude of reports on the involvement of lysosomes in different cell death mechanisms has been published. It is now clear that lysosomes are involved in many cell death events. The contribution of lysosomes can be active (leading to cell death) or passive (a consequence of cell death), as well as amplifying the cell death response[13] (Figure 1). Some lysosomal proteins likely to play key roles in different cell death modes are discussed below.
Cathepsins as major effectors of LMP There are many triggers of LMP, ranging from lysosomotropic agents – molecules that accumulate within the lysosomes and cause a destabilisation of the lysosomal membrane - to lysosomal reactive oxygen species (ROS), which are generated by a reaction of H2O2 with lysosomal iron during oxidative stress[11]. Effectors of lysosomal cell death, however, seem clearly defined: the lysosomal protease family consisting of the aspartic cathepsin D and the cysteine cathepsins B, C, F, H, K, L, O, S, V, W and X are commonly attributed to be the effectors of lysosomal cell death downstream of LMP.
Among the cathepsin family, cathepsin D has been predominantly linked to apoptosis in early reports, both following intrinsic pathway stimuli like DNA-damaging agents etoposide and adriamycin in murine embryonic fibroblasts[14] or extrinsic pathway stimuli IFN-γ and Fas in HeLa cells and TNF-α in U937 human lymphoma cells[15]. Strikingly, cathepsin D has been shown to be practically inactive at a neutral pH[16] – a condition it finds inside the cellular lumen, therefore shedding doubt on a major role of this protein downstream of LMP. One report has, however, demonstrated that apoptosis after TNF stimulation can be
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concomitant with cytosolic acidification, which might increase cathepsin activity in the cytosol[17] (Figure 2). Despite some reports to the contrary, a major role in lysosomemediated apoptotic cell death is attributed to cathepsin D to date, and a few molecular apoptotic targets of this protease are described. These include the pro-apoptotic Bcl-2 proteins Bax[18] and Bid in vitro up to a pH of 6.2[19] and the initiator caspase-8 in neutrophil apoptosis[20] (Figure 1).
Several reports indicate that cathepsin B could also be an effector of cell death after LMP, for example after treatment with TNF[21] it has been shown that CtsB -/- mice are protected from TNF-α induced liver cell apoptosis[22]. Nevertheless, cysteine cathepsins have been reported to have no significant role in apoptosis in response to other extrinsic pathway stimuli in cancer cell line models[23]. The cysteine cathepsins retain some activity in pH neutral environments[24], and several major cysteine cathepsin substrates in the apoptotic pathway have been identified, including Bid[25]. Additional substrates include anti-apoptotic Bcl-2 family members Bcl-2, Bcl-Xl and Mcl-1, which prevent apoptosis upstream of MOMP and also XIAP[25], which is an apoptosis inhibitor downstream of MOMP (Figure 1).
In some cellular systems, cathepsin release after LMP is described as an enhancer, but not initiator of apoptosis. Mouse monocytes and fibroblasts deficient in cathepsin B or L display delays in apoptosis in response to some stimuli such as IL-3 withdrawal and etoposide treatment. By contrast, apoptosis after exposure to UV light is not affected by the absence of these cathepsins[26]. Furthermore, this early occurring LMP is shown to be strictly dependent on the presence of Bax/Bak and caspase activation in these cells. This indicates that MOMP and caspase activation precede LMP in this system, with LMP contributing to the extent of apoptosis observed after different apoptotic stimuli.
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Some cathepsins have also been implicated in necrotic forms of cell death. Cathepsin B activity was shown to increase in CA1 neurons after ischaemic shock in monkeys, followed by translocation to the cytoplasm. Administration of the cathepsin B specific inhibitor CA074 directly after the ischaemic insult was sufficient to prevent two thirds of the cell death observed in these neurons[27].
In addition to classic necrosis, cathepsins have also been shown to be involved in regulated or programmed necrosis. One of the first reports on this topic describes a role for cathepsin B in the execution of non-apoptotic cell death following treatment with microtubule stabilising agents in non-small cell lung cancer (NSCLC) cells, where cathepsin B inhibition leads to significant protection from cell death and the morphological changes observed before the cells die[28]. In addition, mouse cathepsin Q, a cathepsin with no known human orthologue, has been shown to contribute to programmed necrosis in DKO Bax/Bak cells after prolonged DNA damage, which displays a late-occurring cell death mode that is independent of caspases and could not be blocked by the necroptosis inhibitor necrostatin[29]. In vivo work also suggests that LMP with subsequent cathepsin release can cause physiological, necrotic death during the involution of mammary gland tissue after weaning[30].
More recently, cathepsins have been shown to play roles in the specific form of programmed necrosis termed necroptosis. In macrophages it has been reported that cathepsins B and S can cleave the Rip1 kinase[31] – a factor central to necroptosis. Conversely, in dendritic cells, cathepsin D has been shown to cleave caspase 8 which serves to cleave Rip1[32]. Hence, in this context, cathepsins promote rather than limit necroptosis.
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Due to the destructive potential of cathepsins, cells have developed natural safeguards to disarm these proteases if they escape lysosomal enclosure – the cystatins. These are potent inhibitors of many cysteine cathepsins, and both intracellular cystatins – often referred to as stefins – and extracellular cystatins exist (Figure 1). Cystatins themselves have been shown to be aberrantly regulated in some diseases, including neurodegerative disorders, cancer and inflammatory diseases[33], adding a further layer of complexity to the contribution of cathepsins in cell death processes.
Lysosomal sphingolipid metabolism and cell death Sphingolipids are cellular lipid components that fulfil various roles in cell death and survival mechanisms. Sphingomyelin, an integral part of cellular membranes, can be metabolised to produce ceramide and sphingosine, which can act as pro-death signalling proteins. The conversion from sphingomyelin to sphingosine via ceramide can be achieved by 2 lysosomal proteins, acid sphingomyelinase (A-SMase) and acid ceramidase (A-CDase) (Figure 2). These two enzymes and their molecular products have been implicated in lysosomal cell death initiation (summarized in[34]). Ceramide produced by A-SMase was found to be an activator of Cathepsin D, leading to its proteolytic activation in the lysosome[35], thereby potentially linking the pro-apoptotic functions of A-SMase to Cathepsin D as an effector molecule. A later report links this Cathepsin D activation by A-SMase to cleavage and activation of Bid by Cathepsin D after TNF stimulation, subsequently leading to caspase activation and apoptosis[19].
Sphingosine, the product of A-CDase has been shown to be a lysosomotropic agent, which accumulates in lysosomes where it becomes trapped by protonation and causes LMP. Low doses of extracellular sphingosine (20μM) caused rapid LMP and necrosis[36]. More recently, it was reported that lysosomal sphingosine causes LMP and cell death in hepatoma cells after treatment with TNF and cycloheximide, which can be inhibited by both knockdown of A-SMase and A-CDase[37].
Lysosomal guardians LMP occurs after a destabilisation of lysosomes, resulting in subsequent spillage of lysosomal components. In contrast to sphingosine, which promotes this process, some lysosomal proteins act as safeguards and stabilise lysosomes to prevent leakage - and their manipulation can therefore modulate cell survival and death events upstream of LMP.
HSP70, a molecular chaperone that is commonly upregulated in cancer[38], is a good potential candidate for the role of lysosomal guardian. It has long been known that HSP70 depletion causes caspase-independent cell death, but only about a decade ago a landmark study showed that it does this through inhibiting LMP[38]. The report demonstrates that HSP70 can localise to endolysosomes and associate with their membranes, which makes these HSP70 containing lysosomes less prone to LMP (Figure 1). The molecular mechanism of this protection from LMP has since been uncovered: HSP70 can bind to an endolysosomal phospholipid called bis(monoacylglycero)phosphate (BMP) in low pH conditions, which is an essential cofactor for A-SMase[39]. This report therefore functionally links the lysosomal sphingolipid metabolism to HSP70-mediated LMP protection.
HSP70 has also been suggested to play a role in neuronal death after ischaemia in monkeys, where it was found to be a potential calpain substrate[40], placing it downstream of calpain activation and upstream of LMP and cathepsin D release in this system. Another study,
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however, found that calpain 1 cleaves the lysosome-associated membrane protein LAMP2 in a cellular system that mimics light-induced retinal degeneration (LIRD)-associated calpainand cathepsin-dependent, caspase-independent cell death. Moreover, LAMP2, but not HSP70 is cleaved in the course of LIRD in vivo[41]. Furthermore, calpain 1 was shown to cleave LAMP2a and the b2 subunit of v-ATPase in vivo in mammary glands undergoing involution in weaned mice, and blocking of calpain 1 can delay the involution process[42]. This study describes a potential mechanism for the LMP-mediated cell death observed in mammary gland involution in vivo[30], suggesting that calpain 1 activation destabilises lysosomes to cause LMP in this setting. Additionally, LAMP1 and 2 protein levels have been found to be decreased by oncogenes such as v-H-Ras, c-srcY527F and K-ras in a cathepsindependent fashion, leading to lysosomal destabilisation and sensitisation to LMP-mediated cell death[43].
Other lysosomal proteins in cell death As discussed previously, the lysosome contains more than 200 different proteins, and many more that temporarily associate with lysosomes, but only a small proportion of these falls into the groups recently discussed. Several of the other lysosomal proteins have, however, been shown to affect cell death mechanisms and the following section discusses a selection of these proteins.
DRAM1, an evolutionary-conserved, p53-induced protein with lysosomal location, has been shown to be required for full apoptotic response after p53 activation[44]. DRAM2, a member of a family of DRAM-related proteins in mammals, which also localizes to lysosomes, does not seem to share this function of DRAM1[45]. DRAM1 has also been shown to be a target of p73, but in contrast to its role in p53-mediated apoptosis DRAM1 expression does not
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affect p73-induced cell death[46]. A direct function of DRAM1 in LMP has recently been described in T cells, which undergo a caspase-independent mode of cell death with LMP, cathepsin release and subsequent MOMP after HIV infection. In this setting, DRAM1 is induced and its ablation leads to decreased LMP and death, but a rise in T cells infected with HIV[47], indicating that DRAM1 might be implicated in the eradication of HIV-infected Tcells.
Another previously undefined lysosome/endoplasmic reticulum-localised protein called TMEM166 has been reported to mediate cell death when overexpressed in HeLa cells. Subsequently, a role for this protein was shown in vivo in rat brains undergoing middle cerebral artery occlusion, where siRNA mediated inhibition of TMEM166 improved the survival rate and decreased the symptoms of the injuries[48]. The mechanism of this action, however, is currently not clear.
Other lysosomal proteins with roles in cell death include the lysosome-associated LAPF, which is suggested to link p53 non-transcriptional pro-death activities to lysosomes[49] and LAPTM5, a protein that is induced in regressing parts of neuroblastoma tumours and which promotes caspase-independent cell death when overexpressed in neuroblastoma tumour cell lines[50]. Additionally, lysosomal storage disorder genes have a variety of impacts on cell death and survival, particularly in neuronal cells[51].
Overall, work from recent years has uncovered a range of proteins that influence cell death through the lysosome, and this exciting field of research is still expanding and constantly yielding new insights into the mechanisms of LMP induction and the role of lysosomal proteins in cell death mechanisms.
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Lysosomes in autophagy Roads to degradation Cellular life involves the generation and degradation of macromolecules. This degradation serves a dual purpose: disposal of unwanted or damaged constituents, thereby protecting the cell, and generation of energy and recycling of “building blocks” in the process. The cellular breakdown of substances can be achieved by different means, depending on the kind of matter degraded and its origin, and several of these waste disposal and recycling roads terminate in the lysosome. Extracellular material can be taken up by endocytosis, while intracellular constituents are often delivered to the lysosome by autophagy. Due to the limitations of this minireview, we will not discuss the role of lysosomal proteins in the late steps of endocytosis (summarised in[52, 53]); instead, we will focus on autophagic processes and the lysosome.
Autophagy is a summarizing term for three distinct cellular processes - macroautophagy, microautophagy and chaperone-mediated autophagy (Figure 3). Their collective purpose is the degradation of intracellular materials with each form of autophagy using different mechanisms for lysosome delivery. They are supported by a second, lysosome-independent intracellular degradation system, the ubiquitin-proteasome pathway. The main difference between the two systems is that the proteasome mainly degrades short-lived proteins while autophagy favours degradation of long-lived and misfolded proteins while additionally being the only way to degrade organelles in a cell.
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During macroautophagy, cargo is engulfed in specialised vesicles called autophagosomes, which terminate in the bulk delivery of proteins and organelles (for example: mitochondria during mitophagy, peroxisomes during pexophagy etc.) to the lysosome via autophagosomelysosome fusion, forming autolysosomes[54] [55].
Macroautophagy has broadly been linked to both cell survival and cell death, with a seemingly paradoxical context-dependent involvement in the promotion of both processes. Cell death with excessive autophagy, or so called autophagic cell death, is commonly described as a distinct cell death mode, but autophagy has also been shown to be a contributing factor for cell death execution by apoptosis and necrosis[56, 57].
Chaperone-mediated autophagy (CMA) is another autophagy pathway that has been identified in mammalian cells. It represents a mode of selective, lysosomal degradation of individual proteins that are recognised through a pentapeptide motif in their sequence. This process is executed one target protein at a time and is critically dependent on the cytosolic chaperone Hsc70 and the lysosomal transmembrane protein LAMP2A which functions as a CMA receptor[58].
The least characterised autophagic process is microautophagy, which is executed through direct invagination of lysosomal vesicles and subsequent uptake of material (both proteins and organelles) in intralysosomal multivesicular bodies[48]. The molecular mechanisms of this pathway(s) remain elusive and equally little is known about the involvement of particular lysosomal proteins in this process.
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In the following sections, we will discuss the involvement of both lysosomal proteins and proteins that temporarily associate with lysosomes in autophagic processes, with particular attention given to macroautophagy.
Autophagy, cell death and the lysosome Lysosomes and autophagy are closely tied, therefore it is not surprising that many diseases display defects in both these processes and that these defects may ultimately lead to cell death. For example, lysosomal storage disorders have been proposed to be autophagic diseases[59]. Niemann-Pick disease type C, caused by mutations in the lysosomal cholesterol transporting system consisting of Npc1 or Npc2, is a prime example of a lysosomal storage disease that involves autophagy deficiencies. About a decade ago, one study described an accumulation of autophagosomes in Purkinje cells of Npc2 Niemann-Pick disease model mice, which undergo massive cell death upon Npc2 loss[60]. More recently, an accumulation of autophagosomes in Npc1 deficient neurons has been shown to be due to a block in the macroautophagy pathway, and autophagy stimulation with the mTORC1 inhibitor rapamycin could partially rescue cell death in this model[61]. Another example is myopathic Danon disease, caused by mutations in LAMP2, which displays a pronounced accumulation of autophagosomes[62, 63].
Some neurological disorders have been shown to have defects in autophagic clearance of specific proteins, leading to the pathologies observed. CMA defects in degrading mutated forms of alpha-synuclein or mutant tau in Parkinson’s disease and Alzheimer’s disease have been causally implicated in these diseases (summarized in[64]).
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In addition to these pathologies, several lysosomal proteins have been shown to both affect autophagy and cell death. The previously described p53 target DRAM1 falls into this category, as it not only contributes to p53 mediated apoptosis, but also induces autophagy[44]. The DRAM1 gene encodes a number of isoforms, and while these localise to different cellular compartments, several are capable of inducing autophagy[65]. Another example is the lysosomal transmembrane protein LAPTM4B. One report indicates that LAPTM4B promotes autophagosome-lysosome fusion and autophagic flux and thereby protects breast cancer cells from starvation-induced cell death[66]. It will also be interesting to investigate the effect of LAPTM4B knockout in vivo to reveal its importance in the autophagososme maturation process. These reports collectively suggest that the lysosome, autophagy and cell death can be closely linked, and future studies will undoubtedly uncover additional lysosomal players in the autophagy-death network.
mTOR: a mediator of lysosome function and macroautophagy The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts as a key regulator of cell growth, which impacts on both lysosomes and autophagy. mTOR exists in 2 distinct complexes, mTORC1 and mTORC2. The former has been researched extensively and is responsible for many of the known mTOR functions in cellular metabolism (for a comprehensive review on this topic, we recommend [67]), while the latter functions mainly in cytoskeleton modulation.
mTORC1 is directly connected to macroautophagy via free amino acids, which are released in lysosomes upon completion of autophagy and sensed by mTORC1 to activate growth and reduce the autophagic process. The molecular players in this process are still not completely established, but recent years have seen a vast improvement in our understanding of how
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mTOR senses the abundance of amino acids. Current evidence points towards a lysosomal amino acid sensing mechanism that leads to mTOR activation, and some lysosomal proteins involved in this process may already be identified (Figure 4).
Rheb has been known to be an activator of mTORC1 in response to amino acids. This interaction was linked to lysosomes in 2010, when a trimeric protein complex termed “Ragulator” was discovered. This complex was found to anchor Rag GTPases at the lysosomal membrane to promote mTOR translocation and activation by the lysosomeresident Rheb in response to amino acid availability[4]. The amino acid sensing mechanism has further been found to be dependent on a lysosomal protein complex: the v-ATPase. Its function in this pathway lies downstream of the accumulation of free amino acids in the lysosomes and upstream of Rag GTPase activation[68].
As previously mentioned, autophagy is known to lead to the lysosomal release of free amino acids. It is therefore not surprising that autophagy – which is commonly induced after deactivation of mTORC1 - can reactivate mTOR after prolonged starvation[69]. This culminates in a process termed autophagic lysosome reformation (ALR), which generates proto-lysosomes from autolysosomes. These vesicles eventually mature into functional lysosomes and replenish the lysosomal population in the cell. mTOR reactivation is essential for ALR, and the lysosomal protein Spinster has been shown to be necessary for mTOR reactivation after prolonged starvation. This effect was shown to be dependent on the sugartransporting capacities of Spinster[70].
Recently, the well-described mTORC1 regulatory complex TSC has been found to be transported to the lysosomes by the Rag GTPases during amino acid starvation where it
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regulates its target Rheb to fully release mTORC1 from the lysosome[71]. The TSC complex is therefore involved in all known mTORC1 metabolic responses.
Lysosome positioning adds another layer of complexity to the mTOR-lysosome-autophagy axis. The clustering of lysosomes in a region of the cell – in the cell periphery or in a perinuclear region – influences mTORC1 signalling and both autophagosome and autolysosome generation[72]. This process is dependent on intracellular pH, which impacts the lysosomal GTPase ARL8B to move lysosomes along microtubules together with the motor protein KIF2.
A network of macroautophagy and lysosome regulation The connection between autophagy and lysosomal function becomes even more apparent through their shared transcription-dependent regulation. In 2009, a network of jointly expressed genes, many of them encoding lysosomal proteins, was discovered[73]. This complex was named the CLEAR (coordinated lysosomal expression and regulation element) network, due to the presence of a palindromic sequence in the promoter of the target genes. The transcription factor responsible for this orchestrated expression of genes is called TFEB, and it was found to directly regulate at least 471 genes – many of them encoding lysosomal proteins[74]. Overexpression of TFEB leads to increased lysosomal mass. Among its targets are also autophagy genes, linking the process of lysosome generation with autophagic functions (Figure 4).
Starvation was found to activate TFEB, which in turn leads to increased expression of autophagy genes and an overall enhanced autophagic flux[75]. This effect was originally attributed to an inhibition of Serine 142 phosphorylation in TFEB by MAPK, thereby
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describing an mTOR-independent, transcriptional regulation of autophagy that is linked to a lysosomal gene regulator. mTORC1 has, however, been found to modulate TFEB phosphorylation in a residue of the serine rich region between amino acids 462-469 to regulate the transcription of the lysosomal target gene v-ATPase by TFEB[76]. Another report finds an mTORC1-dependent regulation of TFEB in Ser211 in full nutrient conditions, leading to scavenging of the transcription factor in the cytosol to render it inactive[77].
TFEB, mTOR, autophagy and lysosomes are connected by several nodes, and it is likely that we have not unravelled their full interaction yet. Future studies will undoubtedly shed more light on the regulation of their interactions and their context-dependent outcomes.
Concluding remarks In this review, we have discussed the role of lysosomes in cell death, autophagy and nutrient sensing. It is important to mention, however, that lysosomes are also critical for other important cellular functions such as iron homeostasis, inflammasome activation, secretion, exocytosis and our innate and adaptive immune responses. Each of these areas of biology is associated with human disease and so it has been a natural progression to ask whether lysosomes can be a target for therapeutic intervention. In this regard, the most promising progress so far is in experimental studies that promote autophagy and as a result lysosomal degradation, to clear protein aggregates associated with neurodegenerative disease or the accumulation of molecules caused by lysosomal storage disorders. This approach appears promising since it seems so far that a moderate up-regulation of lysosomal function can be beneficial in diseased cells without any dramatic detrimental effect on normal tissue.
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Studies have also focused on inhibiting lysosomal function, and this has received a lot of attention recently since it is considered that certain tumours may be autophagy-dependent. The agents used so far have largely been lysosomotropic agents which raise the lysosomal pH and therefore block degradation. While this may be a feasible strategy in the short-term it seems likely that blocking lysosomal function entirely will have consequences for cellular and organismal function long-term. The cell can of course modulate lysosome function in very specific ways and so further studies should yield clues as to how best to either promote or inhibit specific lysosomal functions whether related to cell death, autophagy or beyond.
Acknowledgements We are grateful to members of the Tumour Cell Death Laboratory for advice and for critical reading of the manuscript. Work in the Tumour Cell Death Laboratory is supported by The Association for International Cancer Research and Cancer Research UK.
Conflict of Interest Statement The authors do not have any financial, personal or professional interests to declare that could be construed to influence this paper.
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Figure Legends
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Figure 1: Lysosomal proteins in cell death mechanisms LMP can culminate in necrosis/necroptosis or apoptosis and it can also amplify the apoptotic response of cells. The executioners of these processes are cathepsins, which are released from the lysosome following LMP. Cathepsin cleavage targets in the apoptotic pathway include Bid, anti-apoptotic Bcl-2 family proteins, Caspase 8 and XIAP. Cystatins, the cellular inhibitors of Cathepsins, can limit cytosolic cathepsin activity. The chaperone HSP70 can prevent LMP by scavenging BMP and thereby inhibiting A-CDase activity. Several other lysosomal proteins, such as DRAM1, LAPTM5 and TMEM166, have been described as cell death effectors that may function independently of LMP. LMP, Lysosomal membrane permeabilisation BMP, Bis(monoacylglycero)phosphate XIAP, X-linked inhibitor of apoptosis DRAM1, DNA-damage-regulated autophagy modulator 1 LAPTM5, lysosomal protein transmembrane 5 A-CDase, acid ceramidase
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Figure 2: Inducers of lysosomal cell death Intrinsic apoptotic stimuli, such as DNA damage by chemotherapeutic drugs, and extrinsic stimuli like TNF-α can cause LMP. Additionally, ROS and lysosomotropic agents are potent LMP inducers. The membrane lipid sphingomyelin can be enzymatically processed in the lysosome to form ceramide – which has been shown to activate Cathepsin B – and sphingosine, which can act as a lysosomotropic agent. LAMP2 and the v-ATPase have been shown to be cleavage targets of Calpain1, leading to lysosomal destabilisation. Several other lysosomal proteins, including DRAM1, LAPTM5 and TMEM166 have been demonstrated to promote cell death. TNF-α, tumour necrosis factor α LMP, lysosomal membrane permeabilisation ROS, Reactive oxygen species LAMP2, lysosomal-associated membrane protein 2 DRAM1, DNA-damage regulated autophagy modulator 1 LAPTM5, lysosomal protein transmembrane 5
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Figure 3: Autophagic pathways The term autophagy describes 3 distinct cellular pathways. Macroautophagy is characterised by the sequestration of cytoplasmic material – including organelles – into double membrane structures that arise from phagophores to produce autophagosomes. These vesicles subsequently fuse with lysosomes to promote bulk degradation of their contents. Chaperonemediated autophagy targets single proteins for lysosomal degradation, a process that depends on LAMP2A as receptor and is aided by several cytoplasmic and lysosomal chaperone proteins. Microautophagy delivers cytoplasmic constituents to lysosomes through a direct lysosomal invagination process. LAMP2A, lysosomal-associated membrane protein 2A
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Figure 4: Macroautophagy and lysosome regulation in response to amino acid availability. When amino acids are abundant (top panel), mTORC1 is tied to the lysosome by Rag GTPases, using the lysosomal complex Ragulator as an anchor. At the lysosome, mTORC1 is activated by TFEB and senses amino acids through an inside out mechanism that involves vATPase. Under these conditions, mTORC1 can promote an inhibitory phosphorylation of the transcription factor RHEB. Altogether, these processes lead to low autophagic flux, high mTORC1 activation and concomitant growth and proliferation. Closely following amino acid withdrawal (middle panel), mTORC1 is inactivated and dissociated from the lysosome, a process that is aided by TSC. mTORC1 inactivation leads to increased autophagic flux. TFEB is activated and translocates to the nucleus, leading to transcription of genes containing CLEAR elements. Overall, these processes culminate in increased autophagic flux and increased lysosomal mass and function. Under prolonged starvation conditions (bottom panel), amino acids are produced by autophagy and mTORC1 can re-associate with lysosomes to be reactivated. This process is positively influenced by the lysosomal protein Spinster. Reactivation of mTORC1 at the lysosome is necessary for autophagic lysosome regeneration (ALR), which regenerates the pool of lysosomes from autolysosomes. The overall outcome of these processes is decreased autophagic flux, reactivation of mTORC1 and regeneration of lysosomes. mTORC1, mechanistic target of rapamycin – complex 1 TFEB, transcription factor EB RHEB, Ras homolog enriched in brain TSC, tuberous sclerosis CLEAR, coordinated lysosomal expression and regulation
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