Common Polymorphism in the Phosphatase PHLPP2 Results in Reduced Regulation of Akt and Protein Kinase C

PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt and protein kinase C (PKC) activity by specifically dephosphorylating these kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1). Here we identify a polymorphism that results in an amino acid change from a Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces phosphatase activity toward Akt both in vitro and in cells, in turn resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in breast cancer cells revealed the Ser-1016 variant is less functional toward both Akt and PKC. In pair-matched high grade breast cancer samples we observed retention of only the Ser allele from heterozygous patients (identical results were observed in a pair-matched normal and tumor cell line). Thus, we have identified a functional polymorphism that impairs the activity of PHLPP2 and correlates with elevated Akt phosphorylation and increased PKC levels.Breast cancer is diagnosed in ∼180,000 women and is the cause of 40,000 deaths each year in the U.S.² A prevalent underlying mechanism driving tumorigenesis is aberrant signal transduction pathways that result in constitutive activation of cell growth, proliferation, and survival pathways (2). A well characterized signal transduction pathway in breast cancer that promotes cellular survival, growth, and proliferation is the phosphatidylinositol 3-kinase/Akt pathway (3). This pathway is activated by a number of mechanisms, including gene amplification or gain of function mutations in upstream receptor protein-tyrosine kinases (4, 5), constitutive activation of hormone receptors (6), activating mutations in phosphatidylinositol 3-kinase and Akt (7, 8), and loss of function mutations in the regulatory phosphatase PTEN³ (phosphatase and tensin homolog on chromosome ten) (9). Thus, Akt is a major regulator of breast tumorigenesis.There are three isoforms of Akt present in humans. All three isoforms contain activating phosphorylation sites in the activation loop (Thr-308 in Akt1) and in the C-terminal hydrophobic motif (Ser-473 in Akt1) (10). Upon growth factor receptor stimulation, phosphatidylinositol 3-kinase becomes activated and phosphorylates the D3 position of, typically, phosphatidylinositol (4, 5) bisphosphate to generate phosphatidylinositol (3,4,5)-trisphosphate (11). This 3′-phosphorylated lipid recruits Akt to the plasma membrane by binding to its PH domain, resulting in conformational changes that allow access to the activation loop phosphorylation site (11). Constitutively bound phosphatidylinositol-dependent kinase-1 then phosphorylates Akt at Thr-308, accompanied by phosphorylation at Ser-473 resulting in a catalytically active kinase (12). Phosphorylation of Ser-473 depends on the mTORC2 complex (13-16). Signaling through this pathway is terminated by removal of the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate catalyzed by the phosphatase PTEN and by direct dephosphorylation of Akt by the recently-identified PHLPP family of phosphatases and protein phosphatase 2A-type phosphatases (17-20).The PHLPP family of phosphatases comprise three variants, the alternatively spliced PHLPP1α and PHLPP1β, and PHLPP2 (21). PHLPP1 and PHLPP2 specifically dephosphorylate the hydrophobic motif of specific Akt isozymes, thus decreasing Akt activity and promoting apoptosis (18, 19). PHLPP2 binds and dephosphorylates Akt1 and Akt3, whereas PHLPP1 binds and dephosphorylates Akt2 and Akt3 (18, 22). Their role in inactivating Akt suggests that both PHLPP1 and PHLPP2 could be potential tumor suppressors. Consistent with such a role, these phosphatases also dephosphorylate the hydrophobic motif of PKC, resulting in degradation of PKC. For this kinase, phosphorylation stabilizes the enzyme, so that the effect of depletion of the PHLPP phosphatases is to increase PKC protein levels (23). PKC is a well characterized oncogene, and loss of function of the PHLPP phosphatases could increase PKC protein levels and promote tumorigenesis (24). Providing further rationale that PHLPP2 could be a potential tumor suppressor, the phosphatase is located on chromosome 16q22.3, a region that encounters frequent loss of heterozygosity (LOH) in many primary and malignant breast tumors (25).Here we identify a non-synonymous polymorphism that results in an amino acid change from a Leu to a Ser at codon 1016 in the PP2C phosphatase domain of PHLPP2. Overexpression studies reveal the Ser-1016 variant has impaired phosphatase activity and is less effective at inducing apoptosis than the Leu-1016 variant. When comparing a pair-matched normal and breast cancer cell line or pair-matched normal and high grade tumor patient samples that are heterozygous, we observe preferential loss of the Leu allele in the tumor tissue or breast cancer cell line. This observation provides evidence that PHLPP2 could be one of the elusive tumor suppressor genes on chromosome 16q, and for heterozygous patients, loss of the more catalytically active Leu-1016 may promote breast tumorigenesis.     

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different G_q-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸. Transphosphorylation targeted Ser⁷⁴⁴, whereas autophosphorylation was the predominant mechanism for Ser⁷⁴⁸ in cells stimulated with G_q-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle. In this context the activation and phosphorylation of protein kinase D (PKD),⁴ the founding member of a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase (CAMK) group and separate from the previously identified PKCs (for review, see Ref. 1), are attracting intense attention. In unstimulated cells, PKD is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology (PH) domain (1–4). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (5–7). In response to cellular stimuli (1), including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8–16) that signal through G_q, G₁₂, G_i, and Rho (11, 15–19), PKD is converted into a form with high catalytic activity, as shown by in vitro kinase assays performed in the absence of lipid co-activators (5, 20).During these studies multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do not directly inhibit PKD catalytic activity (5, 20), implying that PKD activation in intact cells is mediated directly or indirectly through PKCs. Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade induced by multiple GPCR agonists and other receptor ligands in a range of cell types (for review, see Ref. 1). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (1, 4, 7, 17, 21). Collectively, these findings demonstrated the existence of a rapidly activated PKC-PKD protein kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD activation was followed by a late, PKC-independent phase of catalytic activation and phosphorylation induced by stimulation of the bombesin G_q-coupled receptor ectopically expressed in COS-7 cells (22). This study raised the possibility that PKD mediates rapid biological responses downstream of PKCs, whereas, in striking contrast, PKD could mediate long term responses through PKC-independent pathways. Despite its potential importance for defining the role of PKC and PKD in signal transduction, this hypothesis has not been tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in several cellular processes and activities, including signal transduction (14, 23–25), chromatin organization (26), Golgi function (27, 28), gene expression (29–31), immune regulation (26), and cell survival, adhesion, motility, differentiation, DNA synthesis, and proliferation (for review, see Ref. 1). In Swiss 3T3 fibroblasts, a cell line used extensively as a model system to elucidate mechanisms of mitogenic signaling (32–34), PKD expression potently enhances ERK activation, DNA synthesis, and cell proliferation induced by G_q-coupled receptor agonists (8, 14). Here, we used this model system to elucidate the role and mechanism(s) of biphasic PKD activation. First, we show that the G_q-coupled receptor agonists bombesin and vasopressin, in contrast to phorbol esters, specifically induce PKD activation through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3 cells. Subsequently, we demonstrate for the first time that the PKC-independent phase of PKD activation is responsible for promoting ERK signaling and progression to DNA synthesis through an epidermal growth factor receptor (EGFR)-dependent pathway. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.     

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the envelope of Gram-positive bacteria (1). Secreted proteins require a C-terminal sorting signal for sortase recognition such that sortase cleaves the substrate at a short peptide motif and forms a thioester-linked intermediate to its active site cysteine (2–4). Nucleophilic attack by an amino group within the bacterial envelope resolves the thioester intermediate, generating an amide bond tethering surface proteins at their C terminus onto Gram-positive bacteria (5). Four classes of sortases can be distinguished on the basis of sequence homology and substrate recognition (6, 7). Sortase A cleaves secreted protein at LPXTG sorting signals and recognizes the amino group of lipid II peptidoglycan precursors as a nucleophile (8, 9). Sortase B cleaves protein substrates at NPQTN sorting signals (10). This enzyme immobilizes proteins within fully assembled cell walls, utilizing the cell wall cross-bridge as a nucleophile (11). Sortase C cuts LPNTA sorting signals and anchors proteins to the peptidoglycan cross-bridges in sporulating bacteria (12, 13). Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14, 15). Sortase D recognizes the amino group of lysine residues within the YPKN motif of pilin subunits as nucleophiles (16). The resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow the pilus fiber (16, 17).Pili of Gram-positive bacteria comprised either two or three different pilin subunits synthesized as cytoplasmic precursors with N-terminal signal peptides and C-terminal sorting signals (P1 precursors) (14, 18). After translocation across the plasma membrane, P2 precursor species arise from removal of the signal peptide from P1 precursors by a signal peptidase (16). Bacillus cereus pili are composed of two subunits; that is, the major pilin, BcpA, and the minor pilin, BcpB (15). In contrast to BcpA, which is deposited throughout the pilus, BcpB is found at fiber tip (15). Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ε-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting signal in major pilin subunits suggest a universal pilus assembly mechanism among Gram-positive bacteria (14, 20). However, the molecular mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA pili is not known. Although the BcpB precursor harbors an N-terminal signal peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif of the major subunit (15). Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the four classes of sortases expressed by bacilli has yet to be established.     

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9–11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8–10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30–90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2–20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8–10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I)³ molecules and traffic to the cell surface for presentation to the immune system (10–12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene regulation, metabolism, cell signaling, and protein targeting (13, 14), intracellular peptides generated by proteasomes that escape degradation have been suggested to play a role in regulating protein interactions (15). Indeed, oligopeptides isolated from rat brain tissue using the catalytically inactive EP24.15 (EC 3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and were found capable of altering G protein-coupled receptor signal transduction (16). Moreover, EP24.15 overexpression itself changed both angiotensin II and isoproterenol signal transduction, suggesting a physiological function for its intracellular substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family that contains the HEXXH motif (17). This enzyme was first described as a neuropeptide-degrading enzyme present in the soluble fraction of brain homogenates (18). Whereas EP24.15 can be secreted (19, 20), its predominant location in the cytosol and nucleus suggests that the primary function of this enzyme is not the extracellular degradation of neuropeptides and hormones (21, 22). EP24.15 was shown in vivo to participate in antigen presentation through MHC-I (23–25) and in vitro to bind (26) or degrade (27) some MHC-I associated peptides. EP24.15 has also been shown in vitro to degrade peptides containing 5–17 amino acids produced after proteasome digestion of β-casein (28). EP24.15 shows substrate size restriction to peptides containing from 5 to 17 amino acids because of its catalytic center that is located in a deep channel (29). Despite the size restriction, EP24.15 has a broad substrate specificity (30), probably because a significant portion of the enzyme-binding site is lined with potentially flexible loops that allow reorganization of the active site following substrate binding (29). Recently, it has also been suggested that certain substrates may be cleaved by an open form of EP24.15 (31). This characteristic is supported by the ability of EP24.15 to accommodate different amino acid residues at subsites S4 to S3′, which even includes the uncommon post-proline cleavage (30). Such biochemical and structural features make EP24.15 a versatile enzyme to degrade structurally unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15 were isolated and identified using mass spectrometry (22). The majority of peptides captured by the inactive enzyme were intracellular protein fragments that efficiently interacted with EP24.15; the smallest peptide isolated in these assays contained 5 and the largest 17 amino acids (15, 16, 22, 32), which is within the size range previously reported for natural and synthetic substrates of EP24.15 (18, 30, 33, 34). Interestingly, the peptides released by the proteasome are in the same size range of EP24.15 competitive inhibitors/substrates (7, 35, 36). Taken altogether, these data suggest that in the intracellular environment EP24.15 could further cleave proteasome-generated peptides unrelated to MHC-I antigen presentation (15).Although the mutated inactive enzyme “capture” assay was successful in identifying several cellular protein fragments that were substrates for EP24.15, it also found some interacting peptides that were not substrates. In this study, we used several approaches to directly screen for cellular peptides that were cleaved by EP24.15. The first approach involved the extraction of cellular peptides from the HEK293 cell line, incubation in vitro with purified EP24.15, labeling with isotopic tags, and analysis by mass spectrometry to obtain quantitative data on the extent of cleavage. The second approach examined the effect of EP24.15 overexpression on the cellular levels of peptides in the HEK293 cell line. The third set of experiments tested synthetic peptides with purified EP24.15 in vitro, and examined cleavage by high pressure liquid chromatography and mass spectrometry. Collectively, these studies have identified a large number of intracellular peptides, including those that likely represent the endogenous substrates and products of EP24.15, and this original information contributes to a better understanding of the function of this enzyme in vivo.     

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.     

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.     

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.     

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.     

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.Chromosomal DNA double strand breaks (DSBs)² are potential inducers of chromosomal aberrations and tumorigenesis, and they are accurately repaired by the homologous recombinational repair (HRR) pathway, without base substitutions, deletions, and insertions (1–3). In the HRR pathway (4, 5), single-stranded DNA (ssDNA) tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue of the bacterial RecA protein, binds to the ssDNA tail and forms a helical nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact double-stranded DNA (dsDNA) to form a three-component complex, containing ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the RAD51 protein promotes recombination reactions, such as homologous pairing and strand exchange (6–9).The RAD51 protein requires auxiliary proteins to promote the homologous pairing and strand exchange reactions efficiently in cells (10–12). In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the RAD51 protein (13–17) and stimulate the RAD51-mediated homologous pairing and/or strand exchange reactions in vitro (18–21). The human RAD51AP1 protein, which directly binds to the RAD51 protein (22), was also found to stimulate RAD51-mediated homologous pairing in vitro (23, 24). The BRCA2 protein contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs (25–33), and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated RAD51-mediated strand exchange (34, 35). Most of these RAD51-interacting factors are known to be required for efficient RAD51 assembly onto DSB sites in cells treated with ionizing radiation (10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which share about 20–30% amino acid sequence similarity with the RAD51 protein (36–38). RAD51B-deficient cells are hypersensitive to DSB-inducing agents, such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the RAD51B protein is involved in the HRR pathway (39–44). Genetic experiments revealed that RAD51B-deficient cells exhibited impaired RAD51 assembly onto DSB sites (39, 44), suggesting that the RAD51B protein functions in the early stage of the HRR pathway. Biochemical experiments also suggested that the RAD51B protein participates in the early to late stages of the HRR pathway (45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein is known to be involved in cytoplasmic actin remodeling (48) and is also overexpressed in breast cancer (49). Like the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51 foci formation after DSB induction, suggesting that the EVL protein enhances RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange. The EVL protein also promoted the annealing of complementary strands. These recombination reactions that were stimulated or promoted by the EVL protein were further enhanced by the RAD51B protein. These results strongly suggested that the EVL protein is a novel factor that activates RAD51-mediated recombination reactions, probably with the RAD51B protein. We anticipate that, in addition to its involvement in cytoplasmic actin dynamics, the EVL protein may be required in homologous recombination for repairing specific DNA lesions, and it may cause tumor malignancy by inappropriate recombination enhanced by EVL overexpression in certain types of tumor cells.     

Residues Important for Nitrate/Proton Coupling in Plant and Mammalian CLC Transporters

Members of the CLC gene family either function as chloride channels or as anion/proton exchangers. The plant AtClC-a uses the pH gradient across the vacuolar membrane to accumulate the nutrient in this organelle. When AtClC-a was expressed in Xenopus oocytes, it mediated exchange and less efficiently mediated Cl^–/H⁺ exchange. Mutating the “gating glutamate” Glu-203 to alanine resulted in an uncoupled anion conductance that was larger for Cl^– than . Replacing the “proton glutamate” Glu-270 by alanine abolished currents. These could be restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4 and ClC-5 mediate stoichiometrically coupled 2Cl^–/H⁺ exchange, their transport is largely uncoupled from protons. By contrast, the AtClC-a-mediated accumulation in plant vacuoles requires tight coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this proline was mutated to serine (P160S), Cl^–/H⁺ exchange of AtClC-a proceeded as efficiently as exchange, suggesting a role of this residue in exchange. Indeed, when the corresponding serine of ClC-5 was replaced by proline, this Cl^–/H⁺ exchanger gained efficient coupling. When inserted into the model Torpedo chloride channel ClC-0, the equivalent mutation increased nitrate relative to chloride conductance. Hence, proline in the CLC pore signature sequence is important for exchange and conductance both in plants and mammals. Gating and proton glutamates play similar roles in bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either mediate electrogenic anion/proton exchange or function as chloride channels (1). In mammals, the roles of plasma membrane CLC Cl^– channels include transepithelial transport (2–5) and control of muscle excitability (6), whereas vesicular CLC exchangers may facilitate endocytosis (7) and lysosomal function (8–10) by electrically shunting vesicular proton pump currents (11). In the plant Arabidopsis thaliana, there are seven CLC isoforms (AtClC-a–AtClC-g)² (12–15), which may mostly reside in intracellular membranes. AtClC-a uses the pH gradient across the vacuolar membrane to transport the nutrient nitrate into that organelle (16). This secondary active transport requires a tightly coupled exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1 (one of the two CLC isoforms in Escherichia coli) display tightly coupled Cl^–/H⁺ exchange, but anion flux is largely uncoupled from H⁺ when is transported (17–21). The lack of appropriate expression systems for plant CLC transporters (12) has so far impeded structure-function analysis that may shed light on the ability of AtClC-a to perform efficient exchange. This dearth of data contrasts with the extensive mutagenesis work performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues (22, 23) and the investigation of mutants (17, 19–21, 24–29) have yielded important insights into their structure and function. CLC proteins form dimers with two largely independent permeation pathways (22, 25, 30, 31). Each of the monomers displays two anion binding sites (22). A third binding site is observed when a certain key glutamate residue, which is located halfway in the permeation pathway of almost all CLC proteins, is mutated to alanine (23). Mutating this gating glutamate in CLC Cl^– channels strongly affects or even completely suppresses single pore gating (23), whereas CLC exchangers are transformed by such mutations into pure anion conductances that are not coupled to proton transport (17, 19, 20). Another key glutamate, located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark of CLC anion/proton exchangers. Mutating this proton glutamate to nontitratable amino acids uncouples anion transport from protons in the bacterial EcClC-1 protein (27) but seems to abolish transport altogether in mammalian ClC-4 and -5 (21). In those latter proteins, anion transport could be restored by additionally introducing an uncoupling mutation at the gating glutamate (21).The functional complementation by AtClC-c and -d (12, 32) of growth phenotypes of a yeast strain deleted for the single yeast CLC Gef1 (33) suggested that these plant CLC proteins function in anion transport but could not reveal details of their biophysical properties. We report here the first functional expression of a plant CLC in animal cells. Expression of wild-type (WT) and mutant AtClC-a in Xenopus oocytes indicate a general role of gating and proton glutamate residues in anion/proton coupling across different isoforms and species. We identified a proline in the CLC signature sequence of AtClC-a that plays a crucial role in exchange. Mutating it to serine, the residue present in mammalian CLC proteins at this position, rendered AtClC-a Cl^–/H⁺ exchange as efficient as exchange. Conversely, changing the corresponding serine of ClC-5 to proline converted it into an efficient exchanger. When proline replaced the critical serine in Torpedo ClC-0, the relative conductance of this model Cl^– channel was drastically increased, and “fast” protopore gating was slowed.     

Familial FTDP-17 Missense Mutations Inhibit Microtubule Assembly-promoting Activity of Tau by Increasing Phosphorylation at Ser202 in Vitro

In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser²⁰², and Ser²⁰² phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser²⁰² phosphorylation and mobility shift to a ∼68-kDa band. Furthermore, Ser²⁰² phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser²⁰² phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles (NFTs)⁴ and senile plaques are the two characteristic neuropathological lesions found in the brains of patients suffering from Alzheimer disease (AD). The major fibrous component of NFTs are paired helical filaments (PHFs) (for reviews see Refs. 1–3). Initially, PHFs were found to be composed of a protein component referred to as “A68” (4). Biochemical analysis reveled that A68 is identical to the microtubule-associated protein, tau (4, 5). Some characteristic features of tau isolated from PHFs (PHF-tau) are that it is abnormally hyperphosphorylated (phosphorylated on more sites than the normal brain tau), does not bind to microtubules, and does not promote microtubule assembly in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind to and promote microtubule assembly (6, 7). Tau hyperphosphorylation is suggested to cause microtubule instability and PHF formation, leading to NFT pathology in the brain (1–3).PHF-tau is phosphorylated on at least 21 proline-directed and non-proline-directed sites (8, 9). The individual contribution of these sites in converting tau to PHFs is not entirely clear. However, some sites are only partially phosphorylated in PHFs (8), whereas phosphorylation on specific sites inhibits the microtubule assembly-promoting activity of tau (6, 10). These observations suggest that phosphorylation on a few sites may be responsible and sufficient for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on SDS-gel due to the presence of six isoforms that are phosphorylated to different extents (2). PHF-tau isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68 kDa-bands on an SDS-gel (4, 5, 11). Studies have shown that ∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau band as an ∼69-kDa band in these studies) are present only in brain areas affected by NFT degeneration (12, 13). Their amount is correlated with the NFT densities at the affected brain regions. Moreover, the increase in the amount of ∼64- and 68-kDa band tau in the brain correlated with a decline in the intellectual status of the patient. The ∼64- and 68-kDa tau bands were suggested to be the pathological marker of AD (12, 13). Biochemical analyses determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which upon dephosphorylation, migrated as normal tau on SDS-gel (4, 5, 11). Tau sites involved in the tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role in AD pathology (12, 13). It is not known whether phosphorylation at all 21 PHF-sites is required for the tau mobility shift in AD. However, in vitro the tau mobility shift on SDS-gel is sensitive to phosphorylation only on some sites (6, 14). It is therefore possible that in the AD brain, phosphorylation on some sites also causes a tau mobility shift. Identification of such sites will significantly enhance our knowledge of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients suffering from a group of neurodegenerative disorders collectively called tauopathies (2, 11). These disorders include frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, progressive supranuclear palsy, and Pick disease. Each PHF-tau isolated from autopsied brains of patients suffering from various tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands on SDS-gel, and is incapable of binding to microtubules. Upon dephosphorylation, the above referenced PHF-tau migrates as a normal tau on SDS-gel, binds to microtubules, and promotes microtubule assembly (2, 11). These observations suggest that the mechanisms of NFT pathology in various tauopathies may be similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may be an indicator of the disease. The tau gene is mutated in familial FTDP-17, and these mutations accelerate NFT pathology in the brain (15–18). Understanding how FTDP-17 mutations promote tau phosphorylation can provide a better understanding of how NFT pathology develops in AD and various tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and P301L tau mutations reduce tau phosphorylation (19, 20). In COS cells, although G272V, P301L, and V337M mutations do not show any significant affect, the R406W mutation caused a reduction in tau phosphorylation (21, 22). When expressed in SH-SY5Y cells subsequently differentiated into neurons, the R406W, P301L, and V337M mutations reduce tau phosphorylation (23). In contrast, in hippocampal neurons, R406W increases tau phosphorylation (24). When phosphorylated by recombinant GSK3β in vitro, the P301L and V337M mutations do not have any effect, and the R406W mutation inhibits phosphorylation (25). However, when incubated with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations stimulate tau phosphorylation (26). The mechanism by which FTDP-17 mutations promote tau phosphorylation leading to development of NFT pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that phosphorylates tau in the brain (27, 28). In this study, to determine how FTDP-17 missense mutations affect tau phosphorylation, we phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility shift to ∼64- and 68 kDa-bands. Although the mobility shift to a ∼64-kDa band is achieved by phosphorylation at Ser^396/404 or Ser²⁰², the mobility shift to a 68-kDa band occurs only in response to phosphorylation at Ser²⁰². We show that in vitro, FTDP-17 missense mutations, by promoting phosphorylation at Ser²⁰², enhance the mobility shift to ∼64- and 68-kDa bands and inhibit the microtubule assembly-promoting activity of tau. Our data suggest that Ser²⁰² phosphorylation is the major event leading to NFT pathology in AD and related tauopathies.     

Novel Glutaredoxin Activity of the Yeast Prion Protein Ure2 Reveals a Native-like Dimer within Fibrils

Ure2 is the protein determinant of the Saccharomyces cerevisiae prion [URE3]. Ure2 has structural similarity to glutathione transferases, protects cells against heavy metal and oxidant toxicity in vivo, and shows glutathione-dependent peroxidase activity in vitro. Here we report that Ure2 (which has no cysteine residues) also shows thiol-disulfide oxidoreductase activity similar to that of glutaredoxin enzymes. This demonstrates that disulfide reductase activity can be independent of the classical glutaredoxin CXXC/CXXS motif or indeed an intrinsic catalytic cysteine residue. The kinetics of the glutaredoxin activity of Ure2 showed positive cooperativity for the substrate glutathione in both the soluble native state and in amyloid-like fibrils, indicating native-like dimeric structure within Ure2 fibrils. Characterization of the glutaredoxin activity of Ure2 sheds light on its ability to protect yeast from heavy metal ion and oxidant toxicity and suggests a role in reversible protein glutathionylation signal transduction. Observation of allosteric enzyme behavior within amyloid-like Ure2 fibrils not only provides insight into the molecular structure of the fibrils but also has implications for the mechanism of [URE3] prion formation.The tripeptide glutathione (GSH)² is abundant in the cell. It plays an important role as a reducing agent in vivo, such as in endogenous free radical scavenging, reversible protein S-glutathionylation, and the reduction of the active sites of enzymes. One major class of enzyme that uses GSH as a reductant is glutaredoxin (GRX), which is a small protein involved in reduction of ribonucleotide reductase for the formation of deoxyribonucleotides for DNA synthesis (1), reduction of 3′-phosphoadenylylsulfate reductase (2) for generation of sulfite, signal transduction, and protection against oxidative stress (3). GRXs are ubiquitous thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily (4). GRXs also show dehydroascorbic acid (DHA) reductase (DHAR) activity (5). Yeast Saccharomyces cerevisiae has at least seven GRXs, which can be divided into two classes according to the number of cysteines in their active site motif: dithiol GRXs with the active site motif CXXC and monothiol GRXs with the motif CXXS (6–9). The dithiol GRXs catalyze protein disulfide reduction using a dithiol mechanism for which both the active site cysteines are essential. On the other hand, both the dithiol and monothiol GRXs can catalyze the reduction of GSH-protein mixed disulfides using a monothiol mechanism that only requires the N-terminal active site cysteine. This reaction and mechanism is important for reversible protein glutathionylation in redox signaling and oxidative stress (10).Glutathione S-transferases (GSTs) are a large versatile family of enzymes with multiple functions, particularly associated with cellular detoxification (11). In terms of overall structure, they belong to the thioredoxin superfamily, like GRX (4). In general, GSTs catalyze the conjugation of reduced GSH to hydrophobic substrates containing an electrophilic atom. In addition, GSTs bind a broad spectrum of ligands and show many other functions. For example, some GSTs show overlapping functions with glutathione-dependent peroxidases (GPxs), which use GSH to reduce hydrogen peroxide and/or organic hydroperoxides and thus are responsible for protection against both endogenous and exogenous oxidant toxicity (11). Interestingly Omega class and Beta class GSTs (such as Escherichia coli GST (EGST)) possess typical GRX activity toward widely used substrates, such as 2-hydroxyethyl disulfide (HEDS) (12–16). These GSTs have an active site cysteine, which is indispensable for GRX activity but not GST activity.The yeast prion protein Ure2 is composed of a disordered protease-sensitive N-terminal prion domain and a compact globular C-terminal domain, which shows high structural similarity to EGST (17). The C-terminal domain of Ure2 can be further structurally divided into two subdomains, the all-α-helix subdomain and the thioredoxin fold subdomain, which shows high structural homology to GRX. Ure2 is involved in the regulation of nitrogen metabolism and resistance to heavy metal ion toxicity (especially cadmium) and oxidative stress in S. cerevisiae (18, 19). In addition, Ure2 shows GPx activity toward both hydrogen peroxide and organic hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide (20). The discovery of the GPx activity of Ure2 (20) provides an explanation for its ability to protect yeast cells from oxidant toxicity (18). However, the reason that ure2Δ yeast cells are hypersensitive to cadmium remains unclear. In general, cadmium ions have a drastic effect on yeast cell growth, and the reasons are complicated. One possible reason for cadmium ion toxicity is that thioltransferases or GRXs can be inhibited by direct binding of cadmium to the two essential cysteine residues present in the thioltransferase active site (21). The inhibition of GRXs leads to complex effects on cell growth. Therefore, we used an in vitro assay to provide a system that allows detailed analysis of the activity of Ure2 and its relationship to that of GRX enzymes. Characterization of the allosteric behavior of the GRX activity of Ure2 revealed that Ure2 forms an active dimer within fibrils. In addition to providing information about the molecular structure of Ure2 fibrils, this also has implications for the molecular mechanism of Ure2 prion formation.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

SCAMP5 Links Endoplasmic Reticulum Stress to the Accumulation of Expanded Polyglutamine Protein Aggregates via Endocytosis Inhibition

Accumulation of expanded polyglutamine proteins is considered to be a major pathogenic biomarker of Huntington disease. We isolated SCAMP5 as a novel regulator of cellular accumulation of expanded polyglutamine track protein using cell-based aggregation assays. Ectopic expression of SCAMP5 augments the formation of ubiquitin-positive and detergent-resistant aggregates of mutant huntingtin (mtHTT). Expression of SCAMP5 is markedly increased in the striatum of Huntington disease patients and is induced in cultured striatal neurons by endoplasmic reticulum (ER) stress or by mtHTT. The increase of SCAMP5 impairs endocytosis, which in turn enhances mtHTT aggregation. On the contrary, down-regulation of SCAMP5 alleviates ER stress-induced mtHTT aggregation and endocytosis inhibition. Moreover, stereotactic injection into the striatum and intraperitoneal injection of tunicamycin significantly increase mtHTT aggregation in the striatum of R6/2 mice and in the cortex of N171-82Q mice, respectively. Taken together, these results suggest that exposure to ER stress increases SCAMP5 in the striatum, which positively regulates mtHTT aggregation via the endocytosis pathway.The expansion of CAG repeats (usually beyond a critical threshold of ∼37 glutamine repeats) encoding polyglutamine (polyQ)³ causes, to date, nine late-onset progressive neurodegenerative disorders (1, 2). Expanded polyQ-containing huntingtin is the main aggregate component in the affected neurons (3). Also, molecular chaperones, such as Hsp70, Hsp40/HDJ1 (dHDJ1), and chaperonin TRiC, perturb the aggregation of polyQ track protein and reduce polyQ track cytotoxicity in yeast and cell lines (4–6) and in Drosophila and mouse models (4, 7). Thus, it seems that HD pathology is closely correlated with the accumulation of insoluble aggregates of mutant huntingtin (mtHTT) containing expanded polyQ (2, 3, 8, 9).Endoplasmic reticulum (ER) stress is crucial in many biological responses and is generated by various signals, such as unfolded protein response, aberrant calcium regulation, oxidative stress, and inflammation (10, 11). ER stress response is generally considered an adaptive reaction of cells to environmental stress, serving as a survival signal (10). On the other hand, increasing evidence also strengthens the importance of ER stress in human diseases. A malfunction or excess of ER stress response caused by aging, genetic mutations, and environmental insults is implicated in human diseases, such as Alzheimer disease, Parkinson disease, diabetes mellitus, and inflammation (12–16). mtHTT also induces ER stress at the early stage of HD, and pathogenic ER stress from an aging or stressful environment is severe at the late stage of HD (17–19). However, the molecular event linking the aggregation of polyQ track protein to ER stress response is unknown.The ubiquitin/proteasome pathway, a major protein degradation system, is altered or impaired in the cell culture model of HD (20–22). On the contrary, autophagy employing lysosomal degradation has been recently considered as a major clearance pathway of insoluble aggregates of polyQ track protein. Thus, inhibition of autophagy has been suggested to modulate the aggregate formation of mtHTT and to affect the toxicity of polyglutamine expansions in fly and mouse models of HD (23–25). However, a key molecule controlling the aggregation and clearance of polyQ track proteins needs to be identified.To further our understanding of the regulation of polyQ track protein aggregation, we screened human full-length cDNAs and isolated SCAMP5 (secretory carrier membrane protein 5) as a modulator of polyQ track protein aggregation. SCAMP5 is up-regulated by mtHTT and ER stress and functions to inhibit endocytosis to increase mtHTT aggregation.