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.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted α-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of thyroglobulin (Tg)² to the apical luminal cavity of thyroid follicles (1). Once secreted, Tg is iodinated via the activity of thyroid peroxidase (2). A coupling reaction involving a quinol-ether linkage especially engages di-iodinated tyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3, 4). Preferential iodination of Tg hormonogenic sites is dependent not on the specificity of the peroxidase (5) but upon the native structure of Tg (6, 7). To date, no other thyroidal proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746 amino acids) involves three regions called I-II-III comprised of disulfide-rich repeat domains held together by intradomain disulfide bonds (8, 9). The final 581 amino acids of Tg are strongly homologous to acetylcholinesterase (10–12). Rate-limiting steps in the overall process of Tg secretion involve its structural maturation within the endoplasmic reticulum (ER) (13). Interactions between regions I-II-III and the cholinesterase-like (ChEL) domain have recently been suggested to be important in this process, with ChEL functioning as an intramolecular chaperone and escort for I-II-III (14). In addition, Tg conformational maturation culminates in Tg homodimerization (15, 16) with progression to a cylindrical, and ultimately, a compact ovoid structure (17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a commonly affected site of mutation, including the recently described A2215D (20, 21), R2223H (22), G2300D, R2317Q (23), G2355V, G2356R, and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (24) (in addition to polymorphisms including P2213L, W2482R, and R2511Q that may be associated with thyroid overgrowth (25)). As best as is currently known, all of the congenital hypothyroidism-inducing Tg mutants are defective for intracellular transport (26). A homozygous G2300R mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is responsible for congenital hypothyroidism in rdw rats (27, 28), whereas we identified the Tg-L2263P point mutation as the cause of hypothyroidism in the cog mouse (29). Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33, 34) in which dimerization enhances protein stability and export (35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain) is blocked within the ER (30), whereas a secretory version of the isolated ChEL domain of Tg devoid of I-II-III undergoes rapid and efficient intracellular transport and secretion (14). A striking homology positions two predicted α-helices of the ChEL domain to the identical relative positions of the dimerization helices in acetylcholinesterase. This raises the possibility that ChEL may serve as a homodimerization domain for Tg, providing a critical function in maturation for Tg transport to the site of thyroid hormone synthesis (1).In this study, we provide unequivocal evidence for homodimerization of the ChEL domain and “hetero”-dimerization of that domain with full-length Tg, and we provide significant evidence that the predicted ChEL dimerization helices provide a nidus for Tg assembly. On the other hand, our data also suggest that upstream Tg regions known to interact with ChEL (14) actively stabilize the Tg dimer complex. Together, I-II-III and ChEL provide unique contributions to the process of intracellular transport of Tg through the secretory pathway.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

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.     

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.     

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.     

Generation of Cubic Membranes by Controlled Homotypic Interaction of Membrane Proteins in the Endoplasmic Reticulum

Cell membranes predominantly consist of lamellar lipid bilayers. When studied in vitro, however, many membrane lipids can exhibit non-lamellar morphologies, often with cubic symmetries. An open issue is how lipid polymorphisms influence organelle and cell shape. Here, we used controlled dimerization of artificial membrane proteins in mammalian tissue culture cells to induce an expansion of the endoplasmic reticulum (ER) with cubic symmetry. Although this observation emphasizes ER architectural plasticity, we found that the changed ER membrane became sequestered into large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy may be targeting irregular membrane shapes and/or aggregated protein. We suggest that membrane morphology can be controlled in cells.The observation that simple mixtures of amphiphilic (polar) lipids and water yield a rich flora of phase structures has opened a long-standing debate as to whether such membrane polymorphisms are relevant for living organisms (1–7). Lipid bilayers with planar geometry, termed lamellar symmetry, dominate the membrane structure of cells. However, this architecture comprises only a fraction of the structures seen with in vitro lipid-water systems (7–11). The propensity to form lamellar bilayers (a property exclusive to cylindrically shaped lipids) is flanked by a continuum of lipid structures that occur in a number of exotic and probably non-physiological non-bilayer configurations (3, 12). However, certain lipids, particularly those with smaller head groups and more bulky hydrocarbon chains, can adopt bilayered non-lamellar phases called cubic phases. Here the bilayer is curved everywhere in the form of saddle shapes corresponding to an energetically favorable minimal surface of zero mean curvature (1, 7). Because a substantial number of the lipids present in biological membranes, when studied as individual pure lipids, form cubic phases (13), cubic membranes have received particular interest in cell biology.Since the application of electron microscopy (EM)³ to the study of cell ultrastructure, unusual membrane morphologies have been reported for virtually every organelle (14, 15). However, interpretation of three-dimensional structures from two-dimensional electron micrographs is not easy (16). In seminal work, Landh (17) developed the method of direct template correlative matching, a technique that unequivocally assesses the presence of cubic membranes in biological specimens (16). Cubic phases adopt mathematically well defined three-dimensional configurations whose two-dimensional analogs have been derived (4, 17). In direct template correlative matching, electron micrographs are matched to these analogs. Cubic cell membrane geometries and in vitro cubic phases of purified lipid mixtures do differ in their lattice parameters; however, such deviations are thought to relate to differences in water activity and lipid to protein ratios (10, 14, 18). Direct template correlative matching has revealed thousands of examples of cellular cubic membranes in a broad survey of electron micrographs ranging from protozoa to human cells (14, 17) and, more recently, in the mitochondria of amoeba (19) and in subcellular membrane compartments associated with severe acute respiratory syndrome virus (20). Analysis of cellular cubic membranes has also been furthered by the development of EM tomography that confirmed the presence of cubic bilayers in the mitochondrial membranes of amoeba (21, 22).Although it is now clear that cubic membranes can exist in living cells, the generation of such architecture would appear tightly regulated, as evidenced by the dominance of lamellar bilayers in biology. In this light, we examined the capability and implications of generating cubic membranes in the endoplasmic reticulum (ER) of mammalian tissue culture cells. The ER is a spatially interconnected complex consisting of two domains, the nuclear envelope and the peripheral ER (23–26). The nuclear envelope surrounds the nucleus and is composed of two continuous sheets of membranes, an inner and outer nuclear membrane connected to each other at nuclear pores. The peripheral ER constitutes a network of branching trijunctional tubules that are continuous with membrane sheet regions that occur in closer proximity to the nucleus. Recently it has been suggested that the classical morphological definition of rough ER (ribosome-studded) and smooth ER (ribosome-free) may correspond to sheet-like and tubular ER domains, respectively (27). The ER has a strong potential for cubic architectures, as demonstrated by the fact that the majority of cubic cell membranes in the EM record come from ER-derived structures (14, 17). Furthermore, ER cubic symmetries are an inducible class of organized smooth ER (OSER), a definition collectively referring to ordered smooth ER membranes (=stacked cisternae on the outer nuclear membrane, also called Karmelle (28–30), packed sinusoidal ER (31), concentric membrane whorls (30, 32–34), and arrays of crystalloid ER (35–37)). Specifically, weak homotypic interactions between membrane proteins produce both a whorled and a sinusoidal OSER phenotype (38), the latter exhibiting a cubic symmetry (16, 39).We were able to produce OSER with cubic membrane morphology via induction of homo-dimerization of artificial membrane proteins. Interestingly, the resultant cubic membrane architecture was removed from the ER system by incorporation into large autophagic vacuoles. To assess whether these cubic symmetries were favored in the absence of cellular energy, we depleted ATP. To our surprise, the cells responded by forming large domains of tubulated membrane, suggesting that a cubic symmetry was not the preferred conformation of the system. Our results suggest that whereas the endoplasmic reticulum is capable of adopting cubic symmetries, both the inherent properties of the ER system and active cellular mechanisms, such as autophagy, can tightly control their appearance.     

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.     

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

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

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.     

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.     

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.