Direct Magnetic Resonance Evidence for Peroxymonocarbonate Involvement in the Cu,Zn-Superoxide Dismutase Peroxidase Catalytic Cycle

Cu,Zn-superoxide dismutase (SOD1) is a copper- and zinc-dependent enzyme. The main function of SOD1 is believed to be the scavenging and detoxification of superoxide radicals. Nevertheless, the last 30 years have seen a rapid accumulation of evidence indicating that SOD1 may also act as a peroxidase, an alternative function that was implicated in the onset and progression of familial amyotrophic lateral sclerosis. Although SOD1 peroxidase activity and its dependence on carbon dioxide have been well described, the molecular basis of the SOD1 peroxidase cycle remains obscure, because none of the proposed catalytic intermediates have so far been identified. In view of recent observations, we hypothesized that the SOD1 peroxidase cycle relies on two steps: 1) reduction of SOD-Cu(II) by hydrogen peroxide followed by 2) oxidation of SOD-Cu(I) by peroxymonocarbonate, the product of the spontaneous reaction of bicarbonate with hydrogen peroxide, to produce SOD-Cu(II) and carbonate radical anion. This hypothesis has been investigated through electron paramagnetic resonance and nuclear magnetic resonance to provide direct evidence for a peroxycarbonate-driven, SOD1-catalyzed carbonate radical production. The results gathered herein indicate that peroxymonocarbonate () is a key intermediate in the SOD1 peroxidase cycle and identify this species as the precursor of carbonate radical anions.Cytosolic Cu,Zn-superoxide dismutase (SOD1)² is a metal-dependent enzyme capable of accelerating the rate of spontaneous superoxide dismutation into O₂ and H₂O₂ through the redox cycling of its copper ion (1, 2). SOD1 is widely distributed in mammalian cells and tissues and has been demonstrated to be located in the cytosol and in the intermembrane space of the mitochondria (see Ref. 3 and references therein). Because of that, SOD1 is believed to be a major player in the first line defense against reactive oxygen species, in particular superoxide anion.In addition to its dismutase activity, SOD1 possesses a well described but incompletely understood peroxidase activity which is dependent on hydrogen peroxide and markedly stimulated by small oxidizable anions such as nitrite and the ubiquitous carbon dioxide (3-12). The peroxidase activity of SOD1 has been proposed to impact the onset and progression of familial amyotrophic lateral sclerosis, a severely debilitating fatal disease characterized by the selective death of motor neurons (13-18). Although several reports exist in the literature indicating the formation of SOD1 aggregates and accumulation as a potential cause in the pathology progression, conflicting hypotheses are still under debate concerning the mechanisms that lead to the formation of SOD1-protein aggregates (19-21). Although some support the suggestion that free radical-induced covalent cross-links among SOD1 amino acids play a fundamental role in aggregate formation (22, 23), others support the view that metal loss from the enzyme structure leads to an unstable apo-form of SOD1 with increased capacity to form aggregates (24, 25). A detailed understanding of the SOD1 peroxidase cycle is essential to unraveling the mechanisms through which SOD1 aggregates are produced.The SOD1 peroxidase cycle is initiated when SOD1-Cu(II) is reduced by H₂O₂ or its deprotonated form (12), the peroxide anion (HOO^-), to SOD1-Cu(I). This latter species is subsequently oxidized to a hypervalent intermediate (proposed to be either SOD1-Cu(III), SOD1-Cu(II)-·_OH, or SOD1-Cu=O) (8, 9) that remains to be characterized. The reduction of this hypervalent intermediate by small anions is supposed to close the cycle, leading to the native enzyme and diffusible highly reactive radicals derived from the anionic substrates (6, 10).During its peroxidase cycle, a considerable fraction of SOD1 is inactivated due to the oxidation of the copper-binding histidines to oxohistidine, presumably by the hypervalent intermediate, in a process that can be prevented by the presence of reducing substrates and, in their absence, unavoidably leads to copper loss (26). Although this process is well described for the heme-dependent peroxidase cycle, current literature data (9, 27-30) and the fact that the proposed SOD1-bound hypervalent copper states (Cu(III), Cu(II)=O, and Cu(II)/^._OH) have never been characterized suggested to us that an alternative mechanism may take place, leading to production from and H₂O₂ by the enzyme, a process that does not involve copper oxidation beyond the thermodynamically stable Cu(II) form. In the presence of , a significant fraction of H₂O₂ is promptly converted to through the perhydration of CO₂ (27, 28, 31, 32). The peroxo bond in peroxymonocarbonate can be cleaved by reduced metals to produce and H₂O (30 33) (Reactions 1-5),where Reactions 1 and 2 represent SOD1 reduction, Reactions 3 and 4 represent SOD1 oxidation, and Reaction 5 represents peroxycarbonate formation.Interestingly, studies employing molecular modeling of the SOD1 active site indicate that and H₂O₂ gain access to the SOD1 active site, where they react to produce in close proximity to the copper ion (29). This interaction of with Cu(I) may result in production.On the basis of these new data, we hypothesized that SOD1-Cu(I), which is the predominant form of SOD1 exposed to excess hydrogen peroxide (8, 9), is oxidized back to the native form of the enzyme by more efficiently than by H₂O₂ itself or HOO^-. The latter two oxidations would slowly produce ^.OH radicals (or the equivalent SOD1-Cu(III), SOD1-Cu(II)=O, or Cu(II)/·_OH) in the enzyme active site, leading to the observed inactivation of SOD1 (see Scheme 1). Here we present data that strongly support this hypothesis; they indicate that is a key substrate for reduced SOD1, which mediates SOD1-Cu(I) reoxidation back to the resting SOD1-Cu(II) severalfold faster than H₂O₂ itself and, in doing so, serves as the carbonate radical anion precursor.Open in a separate windowSCHEME 1.Schematic representation of the peroxidase catalytic cycle of Cu,Zn-SOD in the presence of /CO₂. Native Cu,Zn-SOD is reduced by the peroxide anion, which gains access to the copper through the enzyme''s anion channel. Reduced Cu,Zn-SOD is reoxidized by the peroxycarbonate anion (), which is in equilibrium with H₂O₂//CO₂, leading to carbonate radical anion production. Superoxide anion (), the product of HOO^--induced SOD1 reduction, can oxidize SOD1-Cu(I) back to its resting SOD-Cu(II) state at diffusion-limited rates; however, it can alternatively reduce another molecule of SOD1-Cu(II) to SOD1-Cu(I), considerably accelerating the rate of SOD1-Cu(II) reduction by H₂O₂. Whether will act as a reductant of SOD1-Cu(II) or an oxidant of SOD1-Cu(I) will depend on the ratio of SOD1-Cu(II)/SOD1-Cu(I) at a given time, because the rate constants for the reaction of with both SOD1 states are close to the diffusion limits.     

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na⁺/H⁺ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na⁺/H⁺ exchangers (NHEs)³ were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na⁺ for intracellular H⁺, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na⁺/H⁺ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.     

Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch

Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4–7 residues) have been identified in a number of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18–21 in Ure2p, plays a critical role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides containing the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.The [URE3] phenotype of Saccharomyces cerevisiae arises because of conversion of the Ure2 protein to an aggregated propagatable prion state (1, 2). Ure2p contains two regions: a poorly structured N-terminal region and a compactly folded C-terminal region (3, 4). The N-terminal region is rich in Asn and Gln residues, is highly flexible, and is without any detectable ordered secondary structure (4–6). This region is necessary and sufficient for prion behavior in vivo (2) and amyloid-forming capacity in vitro (5, 7), so it is referred to as the prion domain (PrD).² The C-terminal region has a fold similar to the glutathione S-transferase superfamily (8, 9) and possesses glutathione-dependent peroxidase activity (10). Upon fibril formation, the N-terminal region undergoes a significant conformational change from an unfolded to a thermally resistant conformation (11), whereas the glutathione S-transferase-like C-terminal domain retains its enzymatic activity, suggesting that little conformational change occurs (10, 12). Ure2p fibrils show various morphologies, including variations in thickness and the presence or absence of a periodic twist (13–16). The overall structure of the fibrils imaged by cryoelectron microscopy suggests that the intact fibrils contain a 4-nm amyloid filament backbone surrounded by C-terminal globular domains (17).It is widely accepted that disulfide bonds play a critical role in maintaining protein stability (18–21) and also affect the process of protein folding by influencing the folding pathway (22–25). A recent study shows that the presence of a disulfide bond in a protein can markedly accelerate the folding process (26). Therefore, a disulfide bond is a useful tool to study protein folding. In the study of prion and other amyloid-related proteins, cysteine scanning has been widely used to study the structure of amyloid fibrils, the driving force of amyloid formation, and the plasticity of amyloid fibrils (13, 27–31).Short segments from amyloid-related proteins, including IAPP (islet amyloid polypeptide), β₂-microglobulin, insulin, and the amyloid-β peptide, show amyloid-forming capacity (32–34). Hence, the amyloid stretch hypothesis has been proposed, which suggests that a short amino acid stretch bearing a highly amyloidogenic motif might supply most of the driving force needed to trigger the self-catalytic assembly process of a protein to form fibrils (35, 36). In support of this hypothesis, it was found that the insertion of an amyloidogenic stretch into a non-amyloid-related protein can trigger the amyloidosis of the protein (36). At the same time, the structural information obtained from microcrystals formed by amyloidogenic stretches and bearing cross-β-structure has contributed significantly to our understanding of the structure of intact fibrils at the atomic level (34, 37). However, no amyloidogenic stretches <10 amino acids have so far been identified in the yeast prion protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase of the Ure2p fibril assembly reaction upon the addition of oxidizing agents. Furthermore, we identified a 4-residue region adjacent to Arg¹⁷ as a potential amyloid stretch in Ure2p.     

Effects of Combined Phosphorylation at Ser-617 and Ser-1179 in Endothelial Nitric-oxide Synthase on EC50(Ca2+) Values for Calmodulin Binding and Enzyme Activation

We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation from the control values of 182 ± 2 and 422 ± 22 nm to 116 ± 2 and 300 ± 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC₅₀(Ca²⁺) values for calmodulin binding and enzyme activation to 77 ± 2 and 130 ± 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and l-citrulline from l-arginine and O₂, with NADPH as the electron donor (1). The role of NO generated by endothelial nitricoxide synthase (eNOS)² in the regulation of smooth muscle tone is well established and was the first of several physiological roles for this small molecule that have so far been identified (2). The nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each subunit contains a reductase and oxygenase domain (1). A significant difference between the reductase domains in eNOS and nNOS and the homologous P450 reductases is the presence of inserts in these synthase isoforms that appear to maintain them in their inactive states (3, 4). A calmodulin (CaM)-binding domain is located in the linker that connects the reductase and oxygenase domains, and the endothelial and neuronal synthases both require Ca²⁺ and exogenous CaM for activity (5, 6). When CaM is bound, it somehow counteracts the effects of the autoinhibitory insert(s) in the reductase. The high resolution structure for the complex between (Ca²⁺)₄-CaM and the isolated CaM-binding domain from eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a pair of Ca²⁺-binding sites, enfold the domain, as has been observed in several other such CaM-peptide complexes (7). Consistent with this structure, investigations of CaM-dependent activation of the neuronal synthase suggest that both CaM lobes must participate (8, 9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (10–12). There are equivalent phosphorylation sites in the human enzyme (10–12). Phosphorylation of the bovine enzyme at Thr-497, which is located in the CaM-binding domain, blocks CaM binding and enzyme activation (7, 11, 13, 14). Ser-116 can be basally phosphorylated in cells (10, 11, 13, 15), and dephosphorylation of this site has been correlated with increased NO production (13, 15). However, it has also been reported that a phosphomimetic substitution at this position has no effect on enzyme activity measured in vitro (13). Ser-1179 is phosphorylated in response to a variety of stimuli, and this has been reliably correlated with enhanced NO production in cells (10, 11). Indeed, NO production is elevated in transgenic endothelium expressing an eNOS mutant containing an S1179D substitution, but not in tissue expressing an S1179A mutant (16). Shear stress or insulin treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in endothelial cells, and this is correlated with increased NO production in the absence of extracellular Ca²⁺ (17–19). Akt-catalyzed phosphorylation or an S1179D substitution has also been correlated with increased synthase activity in cell extracts at low intracellular free [Ca²⁺] (17). Increased NO production has also been observed in cells expressing an eNOS mutant containing an S617D substitution, and physiological stimuli such as shear-stress, bradykinin, VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 (12, 13, 20). Although S617D eNOS has been reported to have the same maximum activity in vitro as the wild type enzyme (20), in our hands an S617D substitution increases the maximal CaM-dependent synthase activity of purified mutant enzyme ∼2-fold, partially disinhibits reductase activity, and reduces the EC₅₀(Ca²⁺) values for CaM binding and enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp substitution at Ser-1179 in eNOS on the Ca²⁺ dependence of CaM binding and CaM-dependent activation of reductase and synthase activities. We also describe the effects on these properties of combining this substitution with one at Ser-617. Finally, we demonstrate that Akt-catalyzed phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar functional effects. Our results suggest that phosphorylation of eNOS at Ser-617 and Ser-1179 can substantially increase synthase activity in cells at a typical basal free Ca²⁺ concentration of 50–100 nm, while single phosphorylations at these sites produce smaller activity increases, and can do so only at higher free Ca²⁺ concentrations.     

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.     

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.     

Glutathione Participates in the Regulation of Mitophagy in Yeast

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4) that target namely peroxisomes (5, 6), chromatin (7, 8), endoplasmic reticulum (9), ribosomes (10), and mitochondria (3, 11–13). Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14–16). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS⁴ production (17). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18) caused by the loss of efficiency of the maintenance process (degradation/biogenesis de novo) is often considered as a major cause of cellular aging (19–23). In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following induction/blockade of apoptosis (23), suggesting that autophagy of mitochondria was required for cell survival following mitochondria injury (14). Consistent with this idea, a direct alteration of mitochondrial permeability properties has been shown to induce mitochondrial autophagy (13, 24, 25). Furthermore, inactivation of catalase induced the autophagic elimination of altered mitochondria (26). In the yeast Saccharomyces cerevisiae, the alteration of F₀F₁-ATPase biogenesis in a conditional mutant has been shown to trigger autophagy (27). Alterations of mitochondrial ion homeostasis caused by the inactivation of the K⁺/H⁺ exchanger was shown to cause both autophagy and mitophagy (28). We have reported that treatment of cells with rapamycin induced early ROS production and mitochondrial lipid oxidation that could be inhibited by the hydrophobic antioxidant resveratrol (29). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30): these authors showed that starvation stimulated ROS production, namely H₂O₂, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H₂O₂. This provided a possible connection between the mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3, 12). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3, 12, 32, 33). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33).The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria.     

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His⁴⁶, Gln⁵⁰, and Gln⁵³) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.Lipoprotein lipase (LPL)³ is an endothelium-bound enzyme that catalyzes the hydrolysis of plasma triglyceride (TG) associated with chylomicrons and very low density lipoproteins (1, 2). This enzyme plays a major role in maintaining lipid homeostasis by promoting the clearance of TG-rich lipoproteins from the bloodstream. Abnormality in LPL functions has been associated with a number of pathological conditions, including atherosclerosis, dyslipidemia associated with diabetes, and Alzheimer disease (1).LPL is expressed in a wide variety of cell types, particularly in adipocytes and myocytes (2). As a rate-limiting enzyme for clearance of TG-rich lipoproteins, the activity of LPL is tightly modulated by multiple mechanisms in a tissue-specific manner in response to nutritional changes (3, 4). The enzymatic activity of LPL in adipose tissue is enhanced after feeding to facilitate the storage of TG, whereas it is down-regulated during fasting to increase the utilization of TG by other tissues (5). The active form of LPL is a noncovalent homodimer with the subunits associated in a head-to-tail manner, and the dissociation of its dimeric form leads to the formation of a stable inactive monomeric conformation and irreversible enzyme inactivation (6). At the post-translational level, the LPL activity is regulated by numerous apolipoprotein co-factors. For instance, apoCII, a small apolipoprotein consisting of 79 amino acid residues in human, activates LPL by directly binding to the enzyme (7, 8). By contrast, several other apolipoproteins such as apoCI, apo-CIII, and apoE have been shown to inhibit the LPL activity in vitro (3).Angiopoietin-like proteins (Angptl) are a family of secreted proteins consisting of seven members, Angptl1 to Angptl7 (9, 10). All the members of the Angptl family share a similar domain organization to those of angiopoietins, with an NH₂-terminal coiled-coil domain (CCD) and a COOH-terminal fibrinogen-like domain. Among the seven family members, only Angptl3 and Angptl4 have been shown to be involved in regulating triglyceride metabolism (10, 11). The biological functions of Angptl3 in lipid metabolism were first discovered by Koishi et al. (12) in their positional cloning of the recessive mutation gene responsible for the hypolipidemia phenotype in a strain of obese mouse KK/snk. Subsequent studies have demonstrated that Angptl3 increases plasma TG levels by inhibiting the LPL enzymatic activity (13–15). Angptl4, also known as fasting-induced adipocyte factor, hepatic fibrinogen/angiopoietin-related protein, or peroxisome proliferator-activated receptor-γ angiopoietin-related, is a secreted glycoprotein abundantly expressed in adipocyte, liver, and placenta (16–18). In addition to its role in regulating angiogenesis, a growing body of evidence demonstrated that Angptl4 is an important player of lipid metabolism (10, 11). Elevation of circulating Angptl4 by transgenic or adenoviral overexpression, or by direct supplementation of recombinant protein, leads to a marked elevation in the levels of plasma TG and low density lipoprotein cholesterol in mice (19–22). By contrast, Angptl4 knock-out mice exhibit much lower plasma TG and cholesterol levels compared with the wild type littermates (19, 20). Notably, treatment of several mouse models (such as C57BL/6J, ApoE^–/–, LDLR^–/–, and db/db obese/diabetic mice) with a neutralizing antibody against Angptl4 recapitulate the lipid phenotype found in Angptl4 knock-out mice (19). The role of Angptl4 as a physiological inhibitor of LPL is also supported by the finding that its expression levels in adipose tissue change rapidly during the fed-to-fasting transitions and correlate inversely with LPL activity (23). In humans, a genetic variant of the ANGPTL4 gene (E40K) has been found to be associated with significantly lower plasma TG levels and higher high density lipoprotein cholesterol concentrations in several ethnic groups (24–26).Angptl3 and Angptl4 share many common biochemical and functional properties (10). In both humans and rodents, Angptl3 and Angptl4 are proteolytically cleaved at the linker region and circulate in plasma as two truncated fragments, including NH₂-terminal CCD and COOH-terminal fibrinogen-like domain (14, 27–29). The effects of both Angptl3 and Angptl4 on elevating plasma TG levels are mediated exclusively by their NH₂-terminal CCDs (15, 22, 23, 27, 30). The CCDs of Angptl3 and Angptl4 have been shown to inhibit the LPL activity in vitro as well as in mice (23,30,31). Angptl4 inhibits LPL by promoting the conversion of the catalytically active LPL dimers into catalytically inactive LPL monomers, thereby leading to the inactivation of LPL (23, 31). However, the detailed structural and molecular basis underlying the LPL inhibition by Angptl3 and Angptl4 remain poorly characterized at this stage.In this study, we analyzed all known amino acid sequences of Angptl3 and Angptl4 from various species and found a short motif, LAXGLLXLGXGL (where X represents polar amino acid residues), which corresponds to amino acid residues 46–57 and 44–55 of human Angptl3 and Angptl4, respectively, is highly conserved despite the low degree of their overall homology (∼30%). Using both in vitro and in vivo approaches, we demonstrated that this 12-amino acid sequence motif, in particular the three polar amino acid residue within this motif, is essential for mediating the interactions between LPL and Angpt4, which in turn disrupts the dimerization of the enzyme.     

Energetic Requirements for Processive Elongation of Actin Filaments by FH1FH2-formins

Formin-homology (FH) 2 domains from formin proteins associate processively with the barbed ends of actin filaments through many rounds of actin subunit addition before dissociating completely. Interaction of the actin monomer-binding protein profilin with the FH1 domain speeds processive barbed end elongation by FH2 domains. In this study, we examined the energetic requirements for fast processive elongation. In contrast to previous proposals, direct microscopic observations of single molecules of the formin Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed that profilin is not required for formin-mediated processive elongation of growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin release the γ-phosphate of ATP on average >2.5 min after becoming incorporated into filaments. Therefore, the release of γ-phosphate from actin does not drive processive elongation. We compared experimentally observed rates of processive elongation by a number of different FH2 domains to kinetic computer simulations and found that actin subunit addition alone likely provides the energy for fast processive elongation of filaments mediated by FH1FH2-formin and profilin. We also studied the role of FH2 structure in processive elongation. We found that the flexible linker joining the two halves of the FH2 dimer has a strong influence on dissociation of formins from barbed ends but only a weak effect on elongation rates. Because formins are most vulnerable to dissociation during translocation along the growing barbed end, we propose that the flexible linker influences the lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament structures for diverse processes in eukaryotic cells (reviewed in Ref. 1). Formins stimulate nucleation of actin filaments and, in the presence of the actin monomer-binding protein profilin, speed elongation of the barbed ends of filaments (2-6). The ability of formins to influence elongation depends on the ability of single formin molecules to remain bound to a growing barbed end through multiple rounds of actin subunit addition (7, 8). To stay associated during subunit addition, a formin molecule must translocate processively on the barbed end as each actin subunit is added (1, 9-12). This processive elongation of a barbed end by a formin is terminated when the formin dissociates stochastically from the growing end during translocation (4, 10).The formin-homology (FH)² 1 and 2 domains are the best conserved domains of formin proteins (2, 13, 14). The FH2 domain is the signature domain of formins, and in many cases, is sufficient for both nucleation and processive elongation of barbed ends (2-4, 7, 15). Head-to-tail homodimers of FH2 domains (12, 16) encircle the barbed ends of actin filaments (9). In vitro, association of barbed ends with FH2 domains slows elongation by limiting addition of free actin monomers. This “gating” behavior is usually explained by a rapid equilibrium of the FH2-associated end between an open state competent for actin monomer association and a closed state that blocks monomer binding (4, 9, 17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for profilin to stimulate formin-mediated elongation. Individual tracks of polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer the actin directly to the FH2-associated barbed end to increase processive elongation rates (4-6, 8, 10, 17).Rates of elongation and dissociation from growing barbed ends differ widely for FH1FH2 fragments from different formin homologs (4). We understand few aspects of FH1FH2 domains that influence gating, elongation or dissociation. In this study, we examined the source of energy for formin-mediated processive elongation, and the influence of FH2 structure on elongation and dissociation from growing ends. In contrast to previous proposals (6, 18), we found that fast processive elongation mediated by FH1FH2-formins is not driven by energy from the release of the γ-phosphate from ATP-actin filaments. Instead, the data show that the binding of an actin subunit to the barbed end provides the energy for processive elongation. We found that in similar polymerizing conditions, different natural FH2 domains dissociate from growing barbed ends at substantially different rates. We further observed that the length of the flexible linker between the subunits of a FH2 dimer influences dissociation much more than elongation.     

Phosphorylation of p53 Is Regulated by TPX2-Aurora A in Xenopus Oocytes

p53 is an important tumor suppressor regulating the cell cycle at multiple stages in higher vertebrates. The p53 gene is frequently deleted or mutated in human cancers, resulting in loss of p53 activity. This leads to centrosome amplification, aneuploidy, and tumorigenesis, three phenotypes also observed after overexpression of the oncogenic kinase Aurora A. Accordingly, recent studies have focused on the relationship between these two proteins. p53 and Aurora A have been reported to interact in mammalian cells, but the function of this interaction remains unclear. We recently reported that Xenopus p53 can inhibit Aurora A activity in vitro but only in the absence of TPX2. Here we investigate the interplay between Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2 is required for nearly all Aurora A activation and for full p53 synthesis and phosphorylation in vivo during oocyte maturation. In vitro, phosphorylation mediated by Aurora A targets serines 129 and 190 within the DNA binding domain of p53. Glutathione S-transferase pull-down studies indicate that the interaction occurs via the p53 transactivation domain and the Aurora A catalytic domain around the T-loop. Our studies suggest that targeting of TPX2 might be an effective strategy for specifically inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays important roles in spindle assembly and centrosome function (1). Overexpression of either human or Xenopus Aurora A transforms mammalian cells, but only when the p53 pathway is altered (2–4). Aurora A is localized on centrosomes during mitosis, and overexpression of the protein leads to centrosome amplification and aneuploidy (2, 3, 5, 6), two likely contributors to genomic instability (7, 8). Because of its oncogenic potential and amplification in human tumors, considerable attention has been focused on the mechanism of Aurora A activation in mitosis. Evidence from several laboratories indicates that activation occurs as a result of phosphorylation of a threonine residue in the T-loop of the kinase (4, 9, 10). Purification of Aurora A-activating activity from M phase Xenopus egg extracts led to an apparent activation mechanism in which autophosphorylation at the T-loop is stimulated by binding of the targeting protein for Xklp2 (TPX2) (11–14). On the other hand, it has been shown that Aurora A activity can be inhibited by interaction with several proteins, including PP1 (protein phosphatase 1), AIP (Aurora A kinase-interacting protein), and, more recently, p53 (9, 15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest, apoptosis, or senescence when DNA is damaged or cell integrity is threatened (18, 19). In human cancers, the p53 gene is frequently deleted or mutated, leading to inactivation of p53 functions (20). p53 protein is almost undetectable in “normal cells,” mainly due to its instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in the nucleus and thereafter undergoes nuclear exclusion, allowing its ubiquitination and subsequent degradation (21). In cells under stress, p53 is stabilized through the disruption of its interaction with Mdm2 (21), leading to p53 accumulation in the nucleus and triggering different responses, as described above.Although p53 has mostly been characterized as a nuclear protein, it has also been shown to localize on centrosomes (22–24) and regulate centrosome duplication (23, 24). Centrosomes are believed to act as scaffolds that concentrate many regulatory molecules involved in signal transduction, including multiple protein kinases (25). Thus, centrosomal localization of p53 might be important for its own regulation by phosphorylation/dephosphorylation, and one of its regulators could be the mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression of these two proteins are very similar. For example, overexpression of Aurora A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and the same effects are often observed after down-regulation of p53 transactivation activity or deletion/mutation of its gene (26, 27).Several recent studies performed in mammalian models show interplay between p53 and Aurora A, with each protein having the ability to inhibit the other, depending on the stage of the cell cycle and the stress level of the cell (17, 28, 29). These studies reported that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated to be the two major Aurora A phosphorylation sites in human p53 in vitro and in vivo. Phosphorylation of Ser-215 within the DNA binding domain of human p53 inhibited both p53 DNA binding and transactivation activities (29). Recently, our group showed that Xenopus p53 is able to inhibit Aurora A kinase activity in vitro, but this inhibitory effect can be suppressed by prior binding of Aurora A to TPX2 (9). Contrary to somatic cells, where p53 is nuclear, unstable, and expressed at a very low level, p53 is highly expressed in the cytoplasm of Xenopus oocytes and stable until later stages of development (30, 31). The high concentration of both p53 and Aurora A in the oocyte provided a suitable basis for investigating p53-Aurora A interaction and also evaluating Xenopus p53 as a substrate of Aurora A.     

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.     

The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-d-aspartate Receptors

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A_ATD), removing the functional high affinity Zn²⁺ binding site. Plasmin also cleaves recombinant NR2A_ATD at lysine 317 (Lys³¹⁷), thereby producing a ∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A_ATD predicts that Lys³¹⁷ is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys³¹⁷ is functional and Zn²⁺ insensitive. Whole cell voltage-clamp recordings show that Zn²⁺ inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys³¹⁷ on NR2A to alanine blocks the effect of plasmin on Zn²⁺ inhibition. The relief of Zn²⁺ inhibition by plasmin occurs in PAR1^-/- cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn²⁺ inhibition.N-Methyl-d-aspartate (NMDA)² receptors are one of three types of ionotropic glutamate receptors that play critical roles in excitatory neurotransmission, synaptic plasticity, and neuronal death (1–3). NMDA receptors are comprised of glycine-binding NR1 subunits in combination with at least one type of glutamate-binding NR2 subunit (1, 4). Each subunit contains three transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed domain that forms the ligand binding site, and one bi-lobed amino-terminal domain (ATD), thought to share structural homology to periplasmic amino acid-binding proteins (4–6). Activation of NMDA receptors requires combined stimulation by glutamate and the co-agonist glycine in addition to membrane depolarization to overcome voltage-dependent Mg²⁺ block of the ion channel (7). The activity of NMDA receptors is negatively modulated by a variety of extracellular ions, including Mg²⁺, polyamines, protons, and Zn²⁺ ions, which can exert tonic inhibition under physiological conditions (1, 4). Several extracellular modulators such as Zn²⁺ and ifenprodil are thought to act at the ATD of the NMDA receptor (8–14).Zinc is a transition metal that plays key roles in both catalytic and structural capacities in all mammalian cells (15). Zinc is required for normal growth and survival of cells. In addition, neuronal death in hypoxia-ischemia and epilepsy has been associated with Zn²⁺ (16–18). Abnormal metabolism of zinc may contribute to induction of cytotoxicity in neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease, and amyotrophic lateral sclerosis (19). Zinc is co-released with glutamate at excitatory presynaptic terminals and inhibits native NMDA receptor activation (20, 21). Zn²⁺ inhibits NMDA receptor function through a dual mechanism, which includes voltage-dependent block and voltage-independent inhibition (22–24). Voltage-independent Zn²⁺ inhibition at low nanomolar concentrations (IC₅₀, 20 nm) is observed for NR2A-containing NMDA receptors (25–28). Evidence has accumulated that the amino-terminal domain of the NR2A subunit controls high-affinity Zn²⁺ inhibition of NMDA receptors, and several histidine residues in this region may constitute part of an NR2A-specific Zn²⁺ binding site (8, 9, 11, 12). For the NR2A subunit, several lines of evidence suggest that Zn²⁺ acts by enhancing proton inhibition (8, 11, 29, 30).Serine proteases present in the circulation, mast cells, and elsewhere signal directly to cells by cleaving protease-activated receptors (PARs), members of a subfamily of G-protein-coupled receptors. Cleavage exposes a tethered ligand domain that binds to and activates the cleaved receptors (31, 32). Protease receptor activation has been studied extensively in relation to coagulation and thrombolysis (33). In addition to their circulation in the bloodstream, some serine proteases and PARs are expressed in the central nervous system, and have been suggested to play roles in physiological conditions (e.g. long-term potentiation or memory) and pathophysiological states such as glial scarring, edema, seizure, and neuronal death (31, 34–36).Functional interactions between proteases and NMDA receptors have previously been suggested. Earlier studies reported that the blood-derived serine protease thrombin potentiates NMDA receptor response more than 2-fold through activation of PAR1 (37). Plasmin, another serine protease, similarly potentiates NMDA receptor response (38). Tissue-plasminogen activator (tPA), which catalyzes the conversion of the zymogen precursor plasminogen to plasmin and results in PAR1 activation, also interacts with and cleaves the ATD of the NR1 subunit of the NMDA receptor (39, 40). This raises the possibility that plasmin may also interact directly with the NMDA receptor subunits to modulate receptor response. We therefore investigated the ability of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar concentrations of plasmin can cleave within the ATD, a region that mediates tonic voltage-independent Zn²⁺ inhibition of NR2A-containing NMDA receptors. We hypothesized that plasmin cleavage reduces the Zn²⁺-mediated inhibition of NMDA receptors by removing the Zn²⁺ binding domain. In the present study, we have demonstrated that Zn²⁺ inhibition of agonist-evoked NMDA currents is decreased significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons. These concentrations of plasmin may be pathophysiologically relevant in situations in which the blood-brain barrier is compromised, which could allow blood-derived plasmin to enter brain parenchyma at concentrations in excess of these that can cleave NR2A. Thus, ability of plasmin to potentiate NMDA function through the relief of the Zn²⁺ inhibition could exacerbate the harmful actions of NMDA receptor overactivation in pathological situations. In addition, if newly cleaved NR2A_ATD enters the bloodstream during ischemic injury, it could serve as a biomarker of central nervous system injury.