Soon Ji Yoo

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinsons disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinsons disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinsons disease-like pathology. Recently, multiple genes mediating familial forms of Parkinsons disease have been identified, including PTEN-induced kinase 1 (PINK1 ; PARK6 ) and parkin (PARK2 ), which are also associated with sporadic forms of Parkinsons disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1–parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinsons disease pathogenesis.

The Drosophila Caspase Inhibitor DIAP1 Is Essential for Cell Survival and Is Negatively Regulated by HID

Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1s ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.

Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.

The Drosophila inhibitor of apoptosis (IAP) DIAP2 is dispensable for cell survival, required for the innate immune response to gram-negative bacterial infection, and can be negatively regulated by the reaper/hid/grim family of IAP-binding apoptosis inducers.

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-κB homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways.

Reaper is regulated by IAP-mediated ubiquitination

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins inDrosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. In this report, we show that the Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, we also show that regulation of Reaper by ubiquitination is a significant factor in determining its biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each others abundance.

ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli

HslVU is an ATP‐dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase. To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP). HslVU degraded both proteins upon incubation with ATP but not with its non‐hydrolyzable analog, ATPγS, indicating that the degradation of SulA requires ATP hydrolysis. The pulse‐chase experiment using an antibody raised against MBP‐SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon). These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.

Chfr is linked to tumour metastasis through the downregulation of HDAC1

Chfr is a ubiquitin ligase that functions in the mitotic checkpoint by delaying entry into metaphase in response to mitotic stress. It has been suggested that Chfr is a tumour suppressor as Chfr is frequently silenced in human cancers. To better understand how Chfr activity relates to cell-cycle progression and tumorigenesis, we sought to identify Chfr-interacting proteins using affinity purification combined with mass spectrometry. Histone deacetylase 1 (HDAC1), which represses transcription by deacetylating histones, was newly isolated as a Chfr-interacting protein. Chfr binds and downregulates HDAC1 by inducing its polyubiquitylation, both in vitro and in vivo. Ectopic expression of Chfr in cancer cells that normally do not express it results in downregulation of HDAC1, leading to upregulation of the Cdk inhibitor p21CIP1/WAF1 and the metastasis suppressors KAI1 and E-cadherin. Coincident with these changes, cells arrest in the G1 phase of the cell cycle and become less invasive. Collectively, our data suggest that Chfr functions as a tumour suppressor by regulating HDAC1.

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease

HslVU in E. coli is a new type of ATP‐dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain β‐type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site‐directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N‐terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.

Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli

HslU is the ATPase component of the ATP‐dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site‐directed mutagenesis was performed to generate a mutation in the ATP‐binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild‐type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP‐dependent hydrolysis of N‐carbobenzoxy‐Gly‐Gly‐Leu‐7‐amido‐4‐methyl coumarin by HslV. The wild‐type HslU (a mixture of monomer and dimer) formed a multimer containing 6–8 subunits in the presence of either ATP or ADP, indicating that ATP‐binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP‐binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.