Ding Xue

Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans

Interplay among four genes—egl-1, ced-9, ced-4 and ced-3—controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4–CED-9 complex at 2.6 Å resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease

A common feature of all eukaryotic membranes is the non-random distribution of different lipid species in the lipid bilayer (lipid asymmetry). Lipid asymmetry provides the two sides of the plasma membrane with different biophysical properties and influences numerous cellular functions. Alteration of lipid asymmetry plays a prominent role during cell fusion, activation of the coagulation cascade, and recognition and removal of apoptotic cell corpses by macrophages (programmed cell clearance). Here we discuss the origin and maintenance of phospholipid asymmetry, based on recent studies in mammalian systems as well as in Caenhorhabditis elegans and other model organisms, along with emerging evidence for a conserved role of mitochondria in the loss of lipid asymmetry during apoptosis. The functional significance of lipid asymmetry and its disruption during health and disease is also discussed.

Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor

The Caenorhabditis elegans gene ced-9 prevents cells from undergoing programmed cell death and encodes a protein similar to the mammalian cell-death inhibitor Bcl-2 (refs 1,2,3,4,5,6,7). We show here that the CED-9 protein is a substrate for the C. elegans cell-death protease CED-3 (refs 8, 9), which is a member of a family of cysteine proteases first defined by CED-3 and human interleukin-1β converting enzyme (ICE). CED-9 can be cleaved by CED-3 at two sites near its amino terminus, and the presence of at least one of these sites is important for complete protection by CED-9 against cell death. Cleavage of CED-9 by CED-3 generates a carboxy-terminal product that resembles Bcl-2 in sequence and in function. Bcl-2 and the baculovirus protein p35, which inhibits cell death in different species through a mechanism that depends on the presence of its cleavage site for the CED-3/ICE family of proteases,, inhibit cell death additively in C. elegans. Our results indicate that CED-9 prevents programmed cell death in C.elegans through two distinct mechanisms: first, CED-9 may, by analogy with p35 (refs 9, 17), directly inhibit the CED-3 protease by an interaction involving the CED-3 cleavage sites in CED-9; second, CED-9 may directly or indirectly inhibit CED-3 by means of a protective mechanism similar to that used by mammalian Bcl-2.

Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Restarting stalled replication forks partly depends on the break‐induced recombination pathway, in which a DNA double‐stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Single‐stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN‐1), critical in resolving stalled replication fork. In response to replication arrest, FEN‐1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN‐1 interaction with DNA bubble structures. Human FEN‐1, but not the GEN‐deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN‐1 null mutant.

Functional Genomic Analysis of Apoptotic DNA Degradation in C. elegans

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Using an RNAi-based functional genomic approach, we have identified seven additional cell death-related nucleases (crn genes), which along with two known nucleases (CPS-6 and NUC-1) comprise at least two independent pathways that contribute to cell killing, and likely signaling for phagocytosis, by degrading chromosomal DNA. Several crn genes have human homologs that are important for RNA processing, protein folding, DNA replication, and DNA damage repair, suggesting dual roles for CRN nucleases in cell survival and cell death. It should now be possible to systematically decipher the mechanisms of apoptotic DNA degradation.

Role of C. elegans TAT-1 Protein in Maintaining Plasma Membrane Phosphatidylserine Asymmetry

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

Caenorhabditis elegans drp-1 and fis-2 Regulate Distinct Cell-Death Execution Pathways Downstream of ced-3 and Independent of ced-9

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1s proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

Regulation of the mec-3 gene by the C.elegans homeoproteins UNC-86 and MEC-3.

The mec‐3 gene encodes a homeodomain protein with LIM repeats that is required for the specification of touch cell fate in Caenorhabditis elegans. Previous experiments suggested that mec‐3 expression requires the product of the unc‐86 gene, a POU‐type homeoprotein, and mec‐3 itself. We have analyzed the control of mec‐3 expression by identifying potential cis regulatory elements in the mec‐3 gene (by conservation in a related nematode and by DNase I footprinting using unc‐86 and mec‐3 proteins) and testing their importance by transforming C.elegans with mec‐3lacZ fusions in which these sites have been mutagenized in vitro. Both unc‐86 and mec‐3 proteins bind specifically to the promoter of the mec‐3 gene, suggesting that both proteins may be directly involved in the regulation of the mec‐3 gene. In addition, the footprint pattern with mec‐3 protein is altered in the presence of unc‐86 protein. In vivo transformation experiments reveal that some of the binding regions of the two proteins are needed for general positive control and maintenance of mec‐3 expression while others have no detectable, unique function. Interestingly, the unc‐86 gene appears to be required not only to initiate mec‐3 expression but also to maintain it.

CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptotic DNA degradation

Oligonucleosomal fragmentation of chromosomes in dying cells is a hallmark of apoptosis. Little is known about how it is executed or what cellular components are involved. We show that crn‐1, a Caenorhabditis elegans homologue of human flap endonuclease‐1 (FEN‐1) that is normally involved in DNA replication and repair, is also important for apoptosis. Reduction of crn‐1 activity by RNA interference resulted in cell death phenotypes similar to those displayed by a mutant lacking the mitochondrial endonuclease CPS‐6/endonuclease G. CRN‐1 localizes to nuclei and can associate and cooperate with CPS‐6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS‐6 and both the 5′–3′ exonuclease activity and a previously uncharacterized gap‐dependent endonuclease activity of CRN‐1. Our results suggest that CRN‐1/FEN‐1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis.