Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

The Mei5-Sae3 Protein Complex Mediates Dmc1 Activity in Saccharomyces cerevisiae

During homologous recombination, a number of proteins cooperate to catalyze the loading of recombinases onto single-stranded DNA. Single-stranded DNA-binding proteins stimulate recombination by coating single-stranded DNA and keeping it free of secondary structure; however, in order for recombinases to load on single-stranded-DNA-binding protein-coated DNA, the activity of a class of proteins known as recombination mediators is required. Mediator proteins coordinate the handoff of single-stranded DNA from single-stranded DNA-binding protein to recombinase. Here we show that a complex of Mei5 and Sae3 from Saccharomyces cerevisiae preferentially binds single-stranded DNA and relieves the inhibition of the strand assimilation and DNA binding abilities of the meiotic recombinase Dmc1 imposed by the single-stranded DNA-binding protein replication protein A. Additionally, we demonstrate the physical interaction of Mei5-Sae3 with replication protein A. Our results, together with previous in vivo studies, indicate that Mei5-Sae3 is a mediator of Dmc1 assembly during meiotic recombination in S. cerevisiae.During meiosis, recombination between homologous chromosomes ensures proper segregation into haploid products. Recombination events are initiated by the formation of double strand breaks (DSBs)² in DNA (1). This is followed by resection of free DNA ends to yield 3′ single-stranded tails, upon which recombinase assembles to form nucleoprotein filaments. Following recombinase assembly, the nucleoprotein filament engages a donor chromatid, searches for homologous DNA sequences on that chromatid, and promotes strand exchange to yield a heteroduplex DNA intermediate often referred to as a joint molecule. Although recombinase alone is capable of promoting homology search and strand exchange in vitro, genetic and biochemical studies have demonstrated that normal recombinase function in vivo requires the activity of a number of accessory factors (2). These factors enhance the assembly of nucleoprotein filaments, target capture, homology search, and dissociation of recombinase from duplex DNA.Most eukaryotes possess two recombinases, both homologues of the Escherichia coli recombinase RecA: Rad51, which is the major recombinase in mitotic cells and is also important during meiotic recombination, and Dmc1, which functions only in meiosis. Dmc1 and Rad51 have been shown to assemble at DSBs by immunofluorescence and chromatin immunoprecipitation (3–6), and both proteins oligomerize on single-stranded DNA (ssDNA) to form nucleofilaments that catalyze strand invasion (7–9).A number of biochemical studies have defined the role of accessory factors in stimulating the activity of Rad51 (10–12). Replication protein A (RPA), the yeast ssDNA-binding protein (SSB), removes secondary structure in ssDNA that otherwise prevents formation of fully functional nucleoprotein filaments (13). Both Rad52 protein (11, 12) and the heterodimeric protein Rad55/Rad57 (14) can overcome the inhibitory effect of RPA on Rad51 nucleoprotein filament formation in purified systems, mediating a handoff between RPA and Rad51. It is thought that the mechanism for the mediator activity of Rad52 involves Rad52 recognizing and binding to RPA-coated ssDNA, where it provides nucleation sites for the recruitment of free molecules of Rad51 (15). The tumor suppressor protein BRCA2 also serves as an assembly factor for Rad51 during mitosis in a variety of species that encode orthologues of this protein, including mice (16), corn smut (17), and humans (18).The meiosis-specific recombinase Dmc1 is stimulated by a distinct set of accessory factors. Immunostaining studies suggest that the Rad51 mediators Rad52 and Rad55/Rad57 are not required for assembly of Dmc1 foci in vivo, although Rad51 itself promotes Dmc1 foci (19–21). More recently, immunostaining and chromatin immunoprecipitation experiments demonstrated a role for the Mei5 and Sae3 proteins of Saccharomyces cerevisiae in assembly of Dmc1 at sites of DSBs in vivo (22, 23). Consistent with these observations, mei5 and sae3 mutants display markedly similar meiotic defects as compared with dmc1 mutants, including defects in sporulation, spore viability, crossing over, DSB repair, progression through meiosis, and synaptonemal complex formation (19, 22–24). Finally, the three proteins have been shown to physically interact; Mei5 and Sae3 have been co-purified and co-immunoprecipitated, and an N-terminal portion of Mei5 has been shown to interact with Dmc1 in a two-hybrid assay (22).The fission yeast Schizosaccharomyces pombe encodes two proteins, Swi5 and Sfr1, which share sequence homology with Sae3 and Mei5, respectively (22). Swi5 and Sfr1 have been shown to stimulate the strand exchange activity of Rhp51 (the S. pombe Rad51 homologue) and Dmc1 (25). Although some results indicate functional similarity of Swi5-Sfr1 and Mei5-Sae3, there are also clear differences. The Mei5-Sae3 complex of budding yeast is expressed solely during meiosis, and no mitotic phenotypes have been reported for mei5 or sae3 mutants (22, 24, 26). In contrast, the Swi5-Sfr1 complex of fission yeast is expressed in mitotic and meiotic cells, and mutations in SWI5 have been shown to cause defects in mitotic recombination (27). Furthermore, although mei5 and sae3 mutants are phenotypically similar to dmc1 mutants, swi5 and sfr1 mutants display more severe meiotic defects during fission yeast meiosis than do dmc1 mutants (27–29). These data suggest that although Swi5-Sfr1 clearly contributes to Rad51 activity in fission yeast, it is possible that the activity of Mei5-Sae3 is restricted to stimulating Dmc1 in budding yeast.In this study, a biochemical approach is used to test the budding yeast Mei5-Sae3 complex for properties expected of a recombinase assembly mediator. We show that Mei5-Sae3 binds both ssDNA and double-stranded DNA (dsDNA) but binds ssDNA preferentially. We also show that Mei5-Sae3 can overcome the inhibitory effects of RPA on the ssDNA binding and strand assimilation activities of Dmc1. Finally, we show that Mei5-Sae3 and RPA bind one another directly. These results indicate that Mei5-Sae3 acts directly as a mediator protein for assembly of Dmc1.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed “P4M” (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the α-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila ΔsidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative agent of Legionnaires disease, but it evolved as a parasite of various species of environmental predatory protozoa, including the social amoeba Dictyostelium discoideum (1, 2). The human disease is linked to the inhalation of contaminated aerosols, followed by replication in alveolar macrophages. To accommodate the transfer between host cells, L. pneumophila alternates between replicative and transmissive phases, the regulation of which includes an apparent quorum-sensing system (3–5).In macrophages and amoebae, L. pneumophila forms a replicative compartment, the Legionella-containing vacuole (LCV).³ LCVs avoid fusion with lysosomes (6), intercept vesicular traffic at endoplasmic reticulum (ER) exit sites (7), and fuse with the ER (8–10). The uptake of L. pneumophila and formation of LCVs in macrophages and amoebae depends on the Icm/Dot type IV secretion system (T4SS) (11–14). Although more than 100 Icm/Dot substrates (“effector” proteins) have been identified to date, only few are functionally characterized, including effectors that interfere with host cell signal transduction, vesicle trafficking, or apoptotic pathways (15–18).Two Icm/Dot-translocated substrates, SidM/DrrA (19, 20) and RalF (21), have been characterized as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small GTPases. These bacterial GEFs are recruited to and activate their targets on LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic signal transduction pathways and in actin cytoskeleton regulation (22). Inactive Rho GTPases bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases are activated by removal of the GDI and the exchange of GDP with GTP by GEFs, which promotes the interaction with downstream effector proteins, such as protein or lipid kinases and various adaptor proteins. The cycle is closed by hydrolysis of the bound GTP, which is mediated by GTPase-activating proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle transport, and additionally, SidM acts as a GDI displacement factor (GDF) to activate Rab1 (23, 24). The function of SidM is assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA preferentially binds to activated Rab1, thus supporting the recruitment of early secretory vesicles by SidM (19, 20, 23, 25, 26). Another Icm/Dot substrate, LepB (27), contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its GTPase-activating protein function, thus acting as an antagonist of SidM (24).The Icm/Dot substrate RalF recruits and activates the small GTPase ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle transport from Golgi to ER (21). Dominant negative Arf1 (7, 28) or knockdown of Arf1 by RNA interference (29) impairs the formation of LCVs, as well as the recruitment of the Icm/Dot substrate SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane (31), where the proteins specifically bind to the host cell lipid phosphatidylinositol 4-phosphate (PtdIns(4)P) (32, 33). Phosphoinositides (PIs) regulate eukaryotic receptor-mediated signal transduction, actin remodeling, and membrane dynamics (34, 35). PtdIns(4)P is present on the cytoplasmic membrane, but localizes preferentially to the trans-Golgi network (TGN), where this PI is produced by an Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ) (36) to promote trafficking along the secretory pathway. Recently, PtdIns(4)P was found to also mediate the export of early secretory vesicles from ER exit sites (37). At present, the L. pneumophila effector proteins that mediate exploitation of host PI signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using different PIs coupled to agarose beads, we identified SidM as a major PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs, including the P4M domain, were found to be translocated and bind the LCV membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ.     

Analysis of a Critical Interaction within the Archaeal Box C/D Small Ribonucleoprotein Complex

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2′-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full functionality in the cell (1). Currently there are over 100 known RNA modification types ranging from small functional group substitutions to the addition of large multi-cyclic ring structures (2). Transfer RNA, one of many functional RNAs targeted for modification (3-6), possesses the greatest modification type diversity, many of which are important for proper biological function (7). Ribosomal RNA, on the other hand, contains predominantly two types of modified nucleotides: pseudouridine and 2′-O-methylribose (8). The crystal structures of the ribosome suggest that these modifications are important for proper folding (9, 10) and structural stabilization (11) in vivo as evidenced by their strong tendency to localize to regions associated with function (8, 12, 13). These roles have been verified biochemically in a number of cases (14), whereas newly emerging functional modifications are continually being investigated.Box C/D ribonucleoprotein (RNP)³ complexes serve as RNA-guided site-specific 2′-O-methyltransferases in both archaea and eukaryotes (15, 16) where they are referred to as small RNP complexes and small nucleolar RNPs, respectively. Target RNA pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl group of the nucleotide five bases upstream of either the D or D′ box motif of the sRNA (Fig. 1, star) (17, 18). In archaea, the internal C′ and D′ motifs generally conform to a box C/D consensus sequence (19), and each sRNA contains two guide regions ∼12 nucleotides in length (20). The bipartite architecture of the RNP potentially enables the complex to methylate two distinct RNA targets (21) and has been shown to be essential for site-specific methylation (22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D, C′, and D′ boxes are labeled. The target RNA binds the sRNA through Watson-Crick pairing and is methylated by fibrillarin at the fifth nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three proteins for activity (23): the ribosomal protein L7Ae (24, 25), fibrillarin, and the Nop56/Nop58 homolog Nop5p (Fig. 1). L7Ae binds to both box C/D and the C′/D′ motifs (26), which respectively comprise kink-turn (27) or k-loop structures (28), to initiate the assembly of the RNP (29, 30). Fibrillarin performs the methyl group transfer from the cofactor S-adenosylmethionine to the target RNA (31-33). For this to occur, the active site of fibrillarin must be positioned precisely over the specific 2′-hydroxyl group to be methylated. Although fibrillarin methylates this functional group in the context of a Watson-Crick base-paired helix (guide/target), it has little to no binding affinity for double-stranded RNA or for the L7Ae-sRNA complex (22, 26, 33, 34). Nop5p serves as an intermediary protein bringing fibrillarin to the complex through its association with both the L7Ae-sRNA complex and fibrillarin (22). Along with its role as an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses other functions not yet fully understood. For example, Nop5p self-dimerizes through a coiled-coil domain (35) that in most archaea and eukaryotic homologs includes a small insertion sequence of unknown function (36, 37). However, dimerization and fibrillarin binding have been shown to be mutually exclusive in Methanocaldococcus jannaschii Nop5p, potentially because of the presence of this insertion sequence (36). Thus, whether Nop5p is a monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate its interaction with a L7Ae box C/D RNA complex because both the fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal structures (29, 35, 38). Individual residues on the surface of a monomeric form of Nop5p (referred to as mNop5p) (22) were mutated to alanine, and the effect on binding affinity for a L7Ae box C/D motif RNA complex was assessed through the use of electrophoretic mobility shift assays. These data reveal that residues important for binding cluster within the highly conserved NOP domain (39, 40). To demonstrate that this domain is solely responsible for the affinity of Nop5p for the preassembled L7Ae box C/D RNA complex, we expressed and purified it in isolation from the full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA complex with nearly wild type affinity, demonstrating that the Nop-RBD is truly an autonomously folding and functional module. Comparison of our data with the crystal structure of the homologous spliceosomal hPrp31-15.5K protein-U4 snRNA complex (41) suggests the adoption of a similar mode of binding, further supporting a crucial role for the NOP domain in RNP complex assembly.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase implicated in diverse cell signaling processes (1–3), plays a critical role in cellular function. Homozygous deletion of Exon 2 (4) or Exon 3 (5) of the SHP-2 gene (PTPN11) in mice leads to early embryonic lethality prior to and at midgestation, respectively. SHP-2 null mutant mice die much earlier, at peri-implantation (4). Exon 3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic stem cells both in vitro and in vivo (6–8), whereas SHP-2 null mutation causes inner cell mass death and diminished trophoblast stem cell survival (4). Recent studies on SHP-2 conditional knock-out or tissue-specific knock-out mice have further revealed an array of important functions of this phosphatase in various physiological processes (9–12). The phenotypes demonstrated by loss of SHP-2 function are apparently attributed to the role of SHP-2 in the cell signaling pathways induced by growth factors/cytokines. SHP-2 generally promotes signal transmission in growth factor/cytokine signaling in both catalytic-dependent and -independent fashion (1–3). The positive role of SHP-2 in the intracellular signaling processes, in particular, the ERK³ and PI3K/Akt kinase pathways, has been well established, although the underlying mechanism remains elusive, in particular, the signaling function of the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation, the phenotypes induced by loss of SHP-2 function may be associated with its role in cell migration. Indeed, dominant negative SHP-2 disrupts Xenopus gastrulation, causing tail truncations (13, 14). Targeted Exon 3 deletion mutation in SHP-2 results in decreased cell spreading, migration (15, 16), and impaired limb development in the chimeric mice (7). The role of SHP-2 in cell adhesion and migration has also been demonstrated by catalytically inactive mutant SHP-2-overexpressing cells (17–20). The molecular mechanisms by which SHP-2 regulates these cellular processes, however, have not been well defined. For example, the role of SHP-2 in the activation of the Rho family small GTPases that is critical for cell motility is still controversial. Both positive (19, 21, 22) and negative roles (18, 23) for SHP-2 in this context have been reported. Part of the reason for this discrepancy might be due to the difference in the cell models used. Catalytically inactive mutant SHP-2 was often used to determine the role of SHP-2 in cell signaling. In the catalytically inactive mutant SHP-2-overexpressing cells, the catalytic activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions independent of its catalytic activity, overexpression of catalytically deficient SHP-2 may also increase its scaffolding function, generating complex effects.The critical role of SHP-2 in cellular function is further underscored by the identification of SHP-2 mutations in human diseases. Genetic lesions in PTPN11 that cause hyperactivation of SHP-2 catalytic activity have been identified in the developmental disorder Noonan syndrome (24) and various childhood leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute lymphoblastic leukemia, and acute myeloid leukemia (25, 26). In addition, activating mutations in SHP-2 have been identified in sporadic solid tumors (27). The SHP-2 mutations appear to play a causal role in the development of these diseases as SHP-2 mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are mutually exclusive in the patients (24–27). Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor cells, and JMML-like myeloproliferative disease in mice (28–32). Gain-of-function cell models derived from the newly available SHP-2 GOF mutation (D61G) knock-in mice (28) now provide us with a good opportunity to clarify the role of SHP-2 in cell motility. Unlike the dominant negative approach in which overexpression of mutant forms of SHP-2 generates complex effects, the SHP-2 D61G knock-in model eliminates this possibility as the mutant SHP-2 is expressed at the physiological level (28). Additionally, defining signaling functions of GOF mutant SHP-2 in cell movement can also help elucidate the molecular mechanisms by which SHP-2 mutations contribute to the relevant diseases.     

Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.     

VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.Frontotemporal lobar degeneration (FTLD)² accounts for 10% of all late onset dementias and is the third most frequent neurodegenerative disease after Alzheimer disease and dementia with Lewy bodies (1). FTLD with ubiquitin-immunoreactive inclusions is genetically, clinically, and neuropathologically heterogeneous (2, 3). FTLD-U comprises several distinct entities, including sporadic forms and familial cases caused by mutations in the genes encoding VCP (valosin-containing protein), GRN (progranulin), CHMP2B (charged multivesicular body protein 2B), TDP-43 (TAR DNA-binding protein of 43 kDa) and an unknown gene linked to chromosome 9 (2, 3). Frontotemporal dementia with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP gene located on chromosome 9p13-p12 (4-10) (Fig. 1). This multisystem disease is characterized by progressive muscle weakness and atrophy, increased osteoclastic bone resorption, and early onset frontotemporal dementia, also called FTLD (9, 11). Mutations in VCP are also associated with dilatative cardiomyopathy with ubiquitin-positive inclusions (12). Neuropathologic features of FTLD with VCP mutation include frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U). The majority of aggregates are ubiquitin- and TDP-43-positive neuronal intranuclear inclusions (NIIs); a smaller proportion is made up of TDP-43-immunoreactive dystrophic neurites (DNs) and neuronal cytoplasmic inclusions (NCIs). A small number of inclusions are VCP-immunoreactive (5, 13). Pathologic TDP-43 in inclusions links a spectrum of diseases in which TDP-43 pathology is a primary feature, including FTLD-U, motor neuron disease, including amyotrophic lateral sclerosis, FTLD with motor neuron disease, and inclusion body myopathy and Paget disease of bone, as well as an expanding spectrum of other disorders in which TDP-43 pathology is secondary (14, 15).Open in a separate windowFIGURE 1.Model of pathogenic mutations and domains in valosin-containing protein. CDC48 (magenta), located within the N terminus (residues 22-108), binds the following cofactors: p47, gp78, and Npl4-Ufd1 (23-25, 28). There are two AAA-ATPase domains (AAA; blue) at residues 240-283 and 516-569, which are joined by two linker regions (L1 and L2; red).TDP-43 proteinopathy in FTLD with VCP mutation has a biochemical signature similar to that seen in other sporadic and familial cases of FTLD-U, including sporadic amyotrophic lateral sclerosis, FTLD-motor neuron disease, FTLD with progranulin (GRN) mutation, and FTLD linked to chromosome 9p (3, 16). TDP-43 proteinopathy in these disorders is characterized by hyperphosphorylation of TDP-43, ubiquitination, and cleavage to form C-terminal fragments detected only in insoluble brain extracts from affected brain regions (16). Identification of TDP-43 as the major component of the ubiquitin-immunoreactive inclusions of FTLD with VCP mutation supports the hypothesis that VCP gene mutations cause an alteration of VCP function, leading to TDP-43 proteinopathy.VCP/p97 (valosin-containing protein) is a member of the AAA (ATPase associated with diverse cellular activities) superfamily. The N-terminal domain of VCP has been shown to be involved in cofactor binding (CDC48 (cell division cycle protein 48)) and two AAA-ATPase domains that form a hexameric complex (Fig. 1) (17). Recently, it has been shown that the N-terminal domain of VCP binds phosphoinositides (18, 19). AKT (activated serine-threonine protein kinase) phosphorylates VCP and is required for constitutive VCP function (20, 21). AKT is activated through phospholipid binding and phosphorylation via the phosphoinositide 3-kinase signaling pathway, which is involved in cell survival (22). The lipid binding domain may recruit VCP to the cell membrane where it is phosphorylated by AKT (19).The diversity of VCP functions is modulated, in part, by a variety of intracellular cofactors, including p47, gp78, and Npl4-Ufd1 (23). Cofactor p47 has been shown to play a role in the maintenance and biogenesis of both the endoplasmic reticulum (ER) and Golgi apparatus (24). The structure of p47 contains a ubiquitin regulatory X domain that binds the N-terminus of VCP, and together they act as a chaperone to deliver membrane fusion machinery to the site of adjacent membranes (25). The function of the p47-VCP complex is dependent upon cell division cycle 2 (CDC2) serine-threonine kinase phosphorylation of p47 (26, 27). Also, VCP has been found to interact with the cytosolic tail of gp78, an ER membrane-spanning E3 ubiquitin ligase that exclusively binds VCP and enhances ER-associated degradation (ERAD) (28). The Npl4-Ufd1-VCP complex is involved in nuclear envelope assembly and targeting of proteins through the ubiquitin-proteasome system (29, 30). The cell survival response of this complex has been found to be important in DNA damage repair though activation by phosphorylation and its recruitment to double-stranded breaks (20, 31). The Npl4-Ufd1-VCP cytosolic complex is also recruited to the ER membrane, interacting with Derlin 1, VCP-interacting membrane proteins (VIMP), and other complexes. At the ER membrane, these misfolded proteins are targeted to the proteasome via ERAD (32-34). VCP also targets IKKβ for ubiquitination to the ubiquitin-proteasome system, implicating VCP in the cell survival pathway and neuroprotection (21, 35-37).To investigate the mechanism of neurodegeneration caused by VCP mutations, we first tested the hypothesis that VCP mutations decrease cell viability in vitro using a neuroblastoma SHSY-5Y cell line and then investigated cellular pathways that are known to lead to neurodegeneration, including decrease in proteasome activity, caspase-mediated degeneration, and a change in cellular localization of TDP-43.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).