Mechanisms of apoptotic phosphatidylserine exposure

It has been a long-standing enigma which scramblase causes phosphatidylserine residues to be exposed on the surface of apoptotic cells, thereby facilitating the phagocytic recognition, engulfment and destruction of apoptotic corpses. In a recent paper in Science, Nagata and coworkers reveal that the scramblases Xkr8 and its C. elegans ortholog, CED-8, are activated by caspase cleavage in apoptotic cells.All cells are separated from the extracellular environment by the plasma membrane, a phospholipid bilayer that prevents diffusion of proteins, ions and other essential molecules into the extracellular space and constitutes the structure in which membrane proteins are embedded. In animal cells, the lipid composition of the outer and inner leaflets of the plasma membrane is not symmetrical. Phosphatidylcholine (PC) and sphingomyelin (SM) are mainly present in the outer leaflet of the plasma membrane, whereas phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are restricted to the inner leaflet. This lipid asymmetry is maintained by the combined action of ATP-dependent enzymes called flippases and floppases, which specifically translocate phospholipids and other molecules from the outer to the inner membrane leaflet and from the inner to the outer membrane leaflet, respectively¹. Lipid composition asymmetry not only defines the curvature and electrochemical properties of the plasma membrane, but is also essential for the correct function of determined lipids, as for instance, PI, which only functions as a second messenger if present in the inner leaflet². Nonetheless, several physiologically relevant processes as diverse as platelet activation, neurotransmitter release, sperm capacitation or apoptosis, require dissipation of plasma membrane lipid asymmetry, a process known as scrambling. The enzymes responsible for this activity are called scramblases, and function to randomize the distribution of phospholipids between both membrane leaflets in an ATP-independent manner^2,3,4.Although plasma membrane asymmetry and the existence of flippases, floppases and scramblases have been known for decades, the identity of the specific enzymes involved in these activities has only begun to be revealed during the last few years. Very recently, the group of Shigekazu Nagata identified TMEM16F as the long sought-after calcium-dependent phospholipid scramblase³. However, to date, the identity of the scramblase(s) involved in apoptosis-related (and calcium-independent) PS exposure had remained elusive. Cell surface PS exposure is a classic feature of apoptotic cells and acts as an “eat me” signal allowing phagocytosis of post-apoptotic bodies. In a recent paper in Science, Nagata''s group identified Xk-Related Protein 8 (Xkr8) as the enzyme responsible for this activity and demonstrated an evolutionarily conserved role of this protein in apoptosis-induced lipid scrambling⁵.To identify enzymes involved in membrane lipid scrambling, Nagata''s group took advantage of their previously generated mouse Ba/F3 pro-B cell line³, which presented a high basal level of PS exposure. They then generated a cDNA library from Ba/F3 cells and overexpressed it in the parental cell line. Through sequential enrichment of cells with increased PS exposure, they were able to isolate a cDNA encoding the Xkr8 protein, which enhanced PS scrambling when overexpressed. Xkr8 overexpression (but not that of TMEM16F) was able to increase apoptosis-associated PS exposure. The authors then noticed that both impaired apoptosis-induced PS exposure and deficient post-apoptotic body clearance were correlated with low Xkr8 expression in leukemia and lymphoma cell lines, which was linked to hypermethylation of its promoter. Interestingly, these two alterations were reverted either by overexpressing Xkr8 or by restitution of endogenous Xkr8 expression after treatment with the demethylating agent 5-aza-2′-deoxycytidine (DAC), suggesting that methylation of the Xrk8 promoter may be a mechanism by which tumor cells evade their phagocytosis after apoptotic death, which may result in increased local inflammation, thus favoring tumor progression. Far from being restricted only to PS exposure, Xrk8 overexpression was able to promote scrambling of multiple lipid species during apoptosis, which was demonstrated by incorporation of fluorescent PC and SM analogues. This scrambling activity was restricted to apoptotic events, as Xkr8 overexpression had no effect on Ca²⁺-induced PS exposure. This specificity may be explained by the presence of an evolutionarily conserved caspase recognition site near Xkr8 C-terminal region, whose mutation prevented both Xkr8 cleavage by caspase-3 or -7 and PS exposure during the course of apoptosis (Figure 1). These results from human cell lines were confirmed in Xkr8^−/− mouse embryonic fibroblasts and fetal thymocytes, which were unable to expose PS upon induction of apoptosis, underscoring the broad physiological relevance of Xkr8 in the apoptotic process. Finally, the authors moved to the nematode Caenorhabditis elegans to analyze whether the role of Xpr8 as lipid scramblase is evolutionarily conserved. C. elegans harbors only one ortholog of Xk proteins, CED-8, known to participate in the phagocytic removal of apoptotic corpses⁶. To determine the role of CED-8 in PS exposure, the authors took advantage of the “floater” assay, which is based on the appearance of floating cells (“floaters”) that have detached from developing C. elegans embryos defective for apoptotic cell phagocytosis⁷. Nagata''s group discovered that ced-8 deficiency leads to the accumulation of floaters. Moreover, ced-8 deficiency synergistically enhanced the number of floaters found in other engulfment mutants, which suggests that CED-8 function is not redundant to that developed by previously known engulfment mutants. This enhancing effect of ced-8 deletion was dependent on CED-3, the C. elegans ortholog of caspase-3, confirming the aforementioned results in mammalian cells. The authors then characterized that floaters resulting from ced-8 deletion show a largely deficient PS exposure after developmental apoptosis, confirming the evolutionarily conserved role of Xk-related proteins in apoptosis-induced lipid scrambling. However, they observed that ced-8 deletion does not lead to a total impairment in apoptotic PS presentation, suggesting that additional proteins must be involved in this process. Indeed, apoptosis-inducing factor can induce PS exposure in mammalian cells in a caspase-independent fashion⁸, and the C. elegans AIF ortholog, WAF-1, physically interacts with and activates another scramblase, SCRM-1⁴.Open in a separate windowFigure 1Xrp8 acts as apoptosis-induced lipid scramblase. Under normal conditions, the combined action of multiple mechanisms, including the activity of flippases and floppases, maintains lipid asymmetry between the outer and inner leaflets of the plasma membrane. Once apoptotic program is activated, caspases-3 and -7 are able to cleave and activate Xrp8 protein, which acts as a lipid scramblase and leads to the loss of lipid asymmetry, resulting in PS exposure to the extracellular space. This acts as the “eat-me” signal that will allow phagocytosis of post-apoptotic cell corpses. PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PS, phosphatidylserine.In summary, through a series of elegant manipulations, Nagata''s group has found the long-sought caspase-activated lipid scramblase that mediates the exposure of “eat-me” signals in post-apoptotic cell corpses. Further studies involving Xkr8 protein, including the mechanisms participating in its epigenetic repression may open new roads for the study of autoimmune diseases, such as lupus erythematosus, which is associated with failure in the post-apoptotic corpse clearance system.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms

Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca²⁺-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.     

Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the apoptotic machinery is composed of four basic elements: the caspase CED-3, the Apaf-1 homologue CED-4, and the Bcl-2 family members CED-9 and EGL-1. The ced-9(n1950) gain-of-function mutation prevents most, if not all, somatic cell deaths in C. elegans. It encodes a CED-9 protein with a glycine-to-glutamate substitution at position 169, which is located within the highly conserved Bcl-2 homology 1 domain. We performed biochemical analyses with the CED-9G169E protein to gain insight into the mechanism of programmed cell death. We find that CED-9G169E retains the ability to bind both EGL-1 and CED-4, although its affinity for EGL-1 is reduced. In contrast to the behavior of wild-type CED-9, the interaction between CED-9G169E and CED-4 is not disrupted by expression of EGL-1. Furthermore, CED-4 and CED-9G169E co-localizes with EGL-1 to the mitochondria in mammalian cells, and expression of EGL-1 does not induce translocation of CED-4 to the cytosol. Finally, the ability of EGL-1 to promote apoptosis is impaired by the replacement of wild-type CED-9 with CED-9G169E, and this effect is correlated with the inability of EGL-1 to induce the displacement of CED-4 from the CED-9.CED-4 complex. These studies suggest that the release of CED-4 from the CED-9.CED-4 complex is a necessary step for induction of programmed cell death in C. elegans.     

The Caenorhabditis elegans CED-9 protein does not directly inhibit the caspase CED-3, in vitro nor in yeast

A genetically defined pathway orchestrates the removal of 131 of the 1090 somatic cells generated during the development of the hermaphrodite nematode Caenorhabditis elegans. Regulation of apoptosis is highly evolutionarily conserved and the nematode cell death pathway is a valuable model for studying mammalian apoptotic pathways, the dysregulation of which can contribute to numerous diseases. The nematode caspase CED-3 is ultimately responsible for the destruction of worm cells in response to apoptotic signals, but it must first be activated by CED-4. CED-9 inhibits programmed cell death and considerable data have demonstrated that CED-9 can directly bind and inhibit CED-4. However, it has been suggested that CED-9 may also directly inhibit CED-3. In this study, we used a yeast-based system and biochemical approaches to explore this second potential mechanism of action. While we confirmed the ability of CED-9 to inhibit CED-4, our data argue that CED-9 can not directly inhibit CED-3.     

Failure of Bcl-2 family members to interact with Apaf-1 in normal and apoptotic cells

CED-9 blocks programmed cell death (apoptosis) in the nematode C. elegans by binding to and neutralizing CED-4, an essential activator of the aspartate-directed cysteine protease (caspase) CED-3. In mammals, the CED-9 homologs Bcl-2 and Bcl-xL also block apoptosis by interfering with the activation of CED-3-like caspases. However, it is unknown whether this occurs by binding to the CED-4 homolog Apaf-1. Whilst two groups previously detected an interaction between Bcl-xL and Apaf-1 in immunoprecipitates,1,2 another group found no interaction between Apaf-1 and any of ten individual members of the Bcl-2 family using the same experimental approach.3 In this study, we aimed to resolve this discrepancy by monitoring the binding of Apaf-1 to three Bcl-2 family members within cells. Using immunofluorescence and Western blot analysis, we show that whilst Apaf-1 is a predominantly cytoplasmic protein, Bcl-2, Bcl-xL and Bax mostly reside on nuclear/ER and mitochondrial membranes. This pattern of localization is maintained when the proteins are co-expressed in both normal and apoptotic cells, suggesting that Bcl-2, Bcl-xL or Bax do not significantly sequester cytoplasmic Apaf-1 to intracellular membranes. In addition, we confirm that Apaf-1 does not interact with Bcl-2 and Bcl-xL in immunoprecipitates. Based on these data, we propose that Apaf-1 is not a direct, physiological target of Bcl-2, Bcl-xL or Bax.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Genetic analysis of intracellular aminoglycerophospholipid traffic.

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.     

Chemokines act as phosphatidylserine-bound “find-me” signals in apoptotic cell clearance

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed “find-me” signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other “eat-me” signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein–coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.

Chemokines attract leukocytes by activating chemokine receptors, but many also bind anionic phospholipids. This study shows that phosphatidylserine-binding chemokines endow extracellular apoptotic bodies with “find-me” signals that trigger phagocyte migration for potential apoptotic cell clearance.     

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.     

Structure and Function of the DUF2233 Domain in Bacteria and in the Human Mannose 6-Phosphate Uncovering Enzyme

DUF2233, a domain of unknown function (DUF), is present in many bacterial and several viral proteins and was also identified in the mammalian transmembrane glycoprotein N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” (UCE)). We report the crystal structure of BACOVA_00430, a 315-residue protein from the human gut bacterium Bacteroides ovatus that is the first structural representative of the DUF2233 protein family. A notable feature of this structure is the presence of a surface cavity that is populated by residues that are highly conserved across the entire family. The crystal structure was used to model the luminal portion of human UCE (hUCE), which is involved in targeting of lysosomal enzymes. Mutational analysis of several residues in a highly conserved surface cavity of hUCE revealed that they are essential for function. The bacterial enzyme (BACOVA_00430) has ∼1% of the catalytic activity of hUCE toward the substrate GlcNAc-P-mannose, the precursor of the Man-6-P lysosomal targeting signal. GlcNAc-1-P is a poor substrate for both enzymes. We conclude that, for at least a subset of proteins in this family, DUF2233 functions as a phosphodiester glycosidase.     

Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.     

Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.The removal of apoptotic cells, known as efferocytosis, is a series of arranged events from the recruitment of phagocytes to sites where apoptotic cells are generated to the digestion of apoptotic cells by phagocytes.^{1, 2, 3} One of the key steps during efferocytosis is the recognition of dying cells by phagocytes. Phagocytes can detect apoptotic cells by the direct or indirect association of multiple receptors on phagocytes with ligands on apoptotic cells.^{4, 5, 6, 7, 8, 9} Some receptors on the surface of phagocytic cells not only bind to apoptotic cells but also transduce apoptotic cell recognition signals into phagocytes in order to mediate the ingestion of apoptotic cells. For instance, brain-specific angiogenesis inhibitor 1 (BAI1) and stabilin-2, which are phosphatidylserine (PtdSer) receptors, recognize PtdSer on apoptotic cells and relay signals to the Elmo-Dock-Rac module and Gulp, respectively, via their cytoplasmic tails.^{8, 10, 11} By contrast, it has been suggested that other receptors, called tethering receptors, merely tether apoptotic cells to phagocytes without mediating downstream signal transduction, following which the internalization of apoptotic cells is mediated by the association of these receptors with co-receptors or other engulfment receptors located nearby.^{12, 13, 14, 15, 16} However, it is unclear whether co-receptors for tethering receptors exist in tethering receptor-mediated phagocytosis of apoptotic cells, and, if they do, whether they are indispensable for this process.One intriguing characteristic of tethering receptors is that they have cytoplasmic tails lacking any signaling motifs or are anchored via glycophosphatidylinositol (GPI) to the outer leaflet of the plasma membrane. For example, Tim-4, a PtdSer receptor with a short cytoplasmic tail that promotes the engulfment of apoptotic cells by the binding of its IgV domain to PtdSer on apoptotic cells, lacks signaling motifs in its cytoplasmic tail. It has been known that neither the cytoplasmic tail nor the transmembrane region of Tim-4 is essential for Tim-4-mediated engulfment of apoptotic cells. Accordingly, it functions as a tethering receptor to secure apoptotic cells on phagocytes.^{9, 14} CD14 is located at the exofacial leaflet of the plasma membrane through its GPI anchor, which rules out the possibility that it mediates direct signal transduction into phagocytes after binding to apoptotic cells. Consequently, it is also considered to be a tethering receptor.¹⁵Phospholipids such as PtdSer and phosphatidylcholine (PtdCho) are unequally distributed between the inner and outer leaflets of the plasma membrane in the normal state. For instance, uncharged phospholipids such as PtdCho and sphingomyelin are primarily located in the outer leaflet, whereas positively or negatively charged phospholipids (such as phosphatidylethanolamine or PtdSer, respectively) are restricted to the inner leaflet facing the cytosol.^{17, 18, 19} However, this asymmetric distribution of phospholipids in the plasma membrane is disrupted during apoptosis. In the plasma membrane of apoptotic cells, PtdSer is exposed to the outer leaflet of the plasma membrane by the activity of scramblases and flippases.^{18, 20, 21} Thus, exposed PtdSer is a hallmark of apoptotic cells and is the best characterized ligand on apoptotic cells for efferocytosis. PtdSer on the surface of apoptotic cells can be recognized by various PtdSer-sensing membrane proteins on phagocytes, collectively called PtdSer receptors, including tethering receptors.Besides PtdSer receptors, many PtdSer-binding proteins have been identified. These proteins are involved in various biological processes such as blood coagulation, synaptic vesicle fusion, membrane scaffolding, and signal transduction.²² One of the best known proteins is annexin A5, which has been extensively studied as a PtdSer-binding protein. Annexin A5 belongs to the family of annexins, which are characterized by their Ca²⁺-dependent ability to bind to negatively charged phospholipids and share structural properties. Annexins are considered to be cytosolic proteins because they lack a 5′ leader sequence; however, some annexins, including annexin A5, are also found on the cell surface and in the circulation. This and related properties imply that annexins participate in diverse biological events from membrane dynamics to cell differentiation and migration.^{23, 24, 25} However, the physiological significance of this family is poorly understood. Among annexins, annexin A5 binds to PtdSer with high affinity. Because of this property, annexin A5 has been harnessed as a molecular probe to distinguish apoptotic cells from live cells both in vivo and in vitro for decades.^{25, 26}In this study, annexin A5 was expressed on the cell surface through a GPI anchor to delineate whether a tethering receptor without its co-receptor can promote efferocytosis. GPI-anchored annexin A5 (Anxa5-GPI) should not interact with any plasma membrane or extracellular protein, at least those involved in the engulfment of apoptotic cells. Thus, it is possible to exclude the effects of co-receptors on Anxa5-GPI-mediated phagocytosis of apoptotic cells. The expression of Anxa5-GPI in phagocytes promoted not only the binding but also the internalization of apoptotic cells. By contrast, phagocytosis of carboxylate beads and Escherichia coli was not affected by the expression of Anxa5-GPI in phagocytes. Anxa5-GPI-induced efferocytosis was not only partially dependent on a specific engulfment pathway but also relied on the generally known cytoskeletal engulfment machinery. Our observations suggest that co-receptors are dispensable for tethering receptor-mediated efferocytosis. In addition, tethering receptors could enhance efferocytosis through diverse engulfment machinery located nearby.     

Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans.During development and in tissue homeostasis, multicellular organisms frequently use apoptosis to eliminate cells that are useless or potentially dangerous. Apoptotic cells are readily recognized, internalized and degraded by neighboring or specialized engulfing cells. Rapid clearance of unwanted cells avoids the release of harmful intracellular contents into the surroundings that can lead to inflammation and autoimmune disease.¹The nematode C. elegans serves as a simple yet powerful genetic model organism to study cell corpse clearance in vivo. Many genes involved in recognition, internalization or degradation of apoptotic corpses have been identified through forward and reverse genetic screens in the past two decades.² Loss of engulfment activity not only results in the persistence of cell corpses, but also leads to the survival of some cells destined to die,³ and – in some cases – leads to impaired cell migration.⁴Phenotypic, genetic and biochemical analyses of the early ‘classical'' ced (cell death abnormal) genes led to the identification of three partially redundant signaling cascades that cooperate to regulate cytoskeletal rearrangements and the migration of the engulfing cell around the apoptotic corpse.^{5, 6, 7, 8, 9} In the first pathway, the transmembrane protein CED-1/MEGF10 has been proposed to act as a cell corpse receptor¹⁰ that binds to exposed phosphatidylserine (PS), either directly or indirectly through the action of the bridging molecule TTR-52/TTR.^{11, 12} The lipid transporter homolog CED-7 also plays a role at this stage, at least in part by promoting the exposure of PS in the outer leaflet of the doomed cell.¹³ The adaptor protein CED-6/GULP transduces the signal(s) from CED-1 downstream to CED-10/Rac1 and DYN-1/Dynamin to drive cytoskeletal rearrangements and phagosome maturation.^{8, 14, 15, 16} In the second pathway, activation of CED-10 is promoted by the bipartite GEF (guanine exchange factor) complex composed of CED-12/Elmo–CED-5/Dock180.^{17, 18, 19, 20} This GEF complex in turn is regulated by CED-2/CrkII and the small GTPase MIG-2/RhoG. In the third pathway, the cytoskeletal regulator ABL-1/Abl suppresses corpse clearance by inhibiting ABI-1/Abl-interacting protein.²¹ Active GTP-loaded CED-10 promotes the extensive cytoskeletal rearrangements that are essential for proper cell corpse internalization.⁸ This process is negatively regulated by the GTPase-activating protein (GAP) SRGP-1/srGAP1 that facilitates GTP hydrolysis in CED-10.²²Here we present the identification and characterization of cdc-42 (cell division control protein-42) as an additional mediator of engulfment signaling regulated by SRGP-1 (Slit/Robo GTPase activating protein 1). Our epistatic analyses, performed with cdc-42(lf) mutants and cdc-42 overexpression experiments, suggest that cdc-42 acts downstream or in parallel to the ced-1/6/7 and in parallel to the ced-2/5/12 signaling cascades. Using a functional and rescuing GFP::CDC-42 protein, we show that CDC-42 is recruited to the cell membrane surrounding apoptotic corpses, and that this localization requires the integrin-α PAT-2 but not the canonical ced-1/6/7 or ced-2/5/12 cascades.Taken together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment upstream of the canonical Rac GTPase CED-10, possibly by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling. Our data confirm and significantly expand on recent results published by Hsieh et al.,²³ who independently identified CDC-42 as an engulfment regulator downstream of integrin-α PAT-2.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.     

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A₂ (cPLA₂s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA₂s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA₂β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca²⁺ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca²⁺ on the interfacial binding of mouse cPLA₂β and its C2 domain to vesicles. Ca²⁺-dependent binding of mouse cPLA₂β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca²⁺-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA₂δ also displays Ca²⁺- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A₁, and phospholipase A₂ activities of the full set of mammalian cPLA₂s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA₂δ stands out as having relatively high phospholipase A₁ activity. We also tested the susceptibility of all cPLA₂ family members to a panel of previously reported inhibitors of human cPLA₂α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA₂β. These in vitro studies help determine the regulation and function of the cPLA₂ family members.     

Gln-222 in Transmembrane Domain 4 and Gln-526 in Transmembrane Domain 9 Are Critical for Substrate Recognition in the Yeast High Affinity Glutathione Transporter, Hgt1p

Hgt1p, a member of the oligopeptide transporter family, is a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. We have explored the role of polar or charged residues in the putative transmembrane domains of Hgt1p to obtain insights into the structural features of Hgt1p that govern its substrate specificity. A total of 22 charged and polar residues in the predicted transmembrane domains and other conserved regions were subjected to alanine mutagenesis. Functional characterization of these 22 mutants identified 11 mutants which exhibited significant loss in functional activity. All 11 mutants except T114A had protein expression levels comparable with wild type, and all except E744A were proficient in trafficking to the cell surface. Kinetic analyses revealed differential contributions toward the functional activity of Hgt1p by these residues and identified Asn-124 in transmembrane domain 1 (TMD1), Gln-222 in TMD4, Gln-526 in TMD9, and Glu-544, Arg-554, and Lys-562 in the intracellular loop region 537–568 containing the highly conserved proline-rich motif to be essential for the transport activity of the protein. Furthermore, mutants Q222A and Q526A exhibited a nearly 4- and 8-fold increase in the K_m for glutathione. Interestingly, although Gln-222 is widely conserved among other functionally characterized oligopeptide transporter family members including those having a different substrate specificity, Gln-526 is present only in Hgt1p and Pgt1, the only two known high affinity glutathione transporters. These results provide the first insights into the substrate recognition residues of a high affinity glutathione transporter and on residues/helices involved in substrate translocation in the structurally uncharacterized oligopeptide transporter family.Hgt1p or ScOpt1p, a polytopic membrane protein, from the yeast Saccharomyces cerevisiae, was the first high affinity glutathione transporter to be identified in any system (1). Hgt1p belongs to a relatively novel family of transporters, the oligopeptide transporter (OPT)³ family, that contains a large number of fungal, plant, and prokaryotic members (2). The functional characterizations of a few of the fungal and plants members have demonstrated their ability to transport oligopeptides, glutathione, and metal-secondary amino acid conjugates by harnessing the proton gradient across the plasma membrane (3–7). Furthermore, these studies have also highlighted the physiological significance of this family in assimilation/mobilization of oligopeptides as nutrients in fungi and plants and in maintenance of metal homeostasis in plants. However, the majority of the members are yet uncharacterized and need to be defined with respect to their substrate specificity and physiological role.A complete lack of information on the structural features of the OPT family further limits our understanding of this large, uncharacterized family. Identification of residues or motifs critical for substrate recognition among the functionally characterized members would enable functional characterization of the new members within the family. This has prompted us to initiate a systematic study on the structure-function characterization of Hgt1p as a representative of the OPT family. Not only is Hgt1p the best characterized member of the OPT family in terms of its substrate specificity, being also able to transport some oligopeptides albeit with low affinity (1, 7, 8), its native host S. cerevisiae is a well established model system and easily amendable for mutagenesis-based structure-function studies. We have recently investigated the role of the 12 native cysteine residues in the structural stability and the transporter activity of the protein where 2 of the cysteines were found to be essential for functionality (9). However, no hints on the important motifs or conserved amino acids of Hgt1p (or any other member of the OPT family) that could be involved in substrate recognition have been obtained so far. In the current study we have focused on the polar and charged residues in the transmembrane domains of Hgt1p to explore their role in substrate recognition.Glutathione, the substrate for Hgt1p, is a hydrophilic substrate. Prior studies on structural characterization of transporters of the other hydrophilic substrates using biochemical and genetic strategies, such as site-directed mutagenesis and random mutagenesis, have established the role of polar and charged residues in the transmembrane domains of transporters in recognition, binding, and translocation of substrates (10–18). The availability of the crystal structures of a few transporter proteins have further enabled direct visualization of such interactions between the key residues in the transmembrane domains and the substrate molecule (19–22). In light of these studies we anticipated that few of the charged or polar residues in the predicted transmembrane domains of Hgt1p would be involved in substrate recognition and translocation across the membrane. Hence, a total of 22 polar or charged amino acids spanning the predicted transmembrane domains of Hgt1p were subjected to alanine scanning and functionally characterized. Detailed biochemical characterizations of these mutants revealed that Asn-124, Gln-222, Gln-526, Glu-544, Arg-554, and Lys-562 are key residues for the transport activity of Hgt1p. As replacement of Gln-222 in TMD4 and Gln-526 in TMD9 with alanine resulted in a significant decrease in the affinity of the transporter for glutathione, it suggested that the two residues might directly participate in glutathione recognition as a substrate. These observations provide the first insights into substrate binding residues in Hgt1p, a member of a novel and important transporter family (OPT family).     

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.