Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

Structural and Functional Elucidation of the Mechanism Promoting Error-prone Synthesis by Human DNA Polymerase �� Opposite the 7,8-Dihydro-8-oxo-2��-deoxyguanosine Adduct

Human polymerase kappa (hPol κ) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol κ bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol κ bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished “bursts” for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol κ complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol κ by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol κ to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol κ compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase η.DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery, and various mechanisms exist to either remove the resulting lesions or bypass them in a more or less mutation-prone fashion (1). Error-prone polymerases are central to trans-lesion synthesis across sites of damaged DNA (2, 3). Four so-called Y-class DNA polymerases have been identified in humans, Pol η,⁴ Pol ι, Pol κ, and Rev1, which exhibit different activities and abilities to replicate past a flurry of individual lesions (4, 5). Homologs have also been identified and characterized in other organisms, notably DinB (Pol IV) in Escherichia coli (6–8), Dbh in Sulfolobus acidocaldarius (9, 10), and Dpo4 in Sulfolobus solfataricus (11, 12). A decade of investigations directed at the structural and functional properties of bypass polymerases have significantly improved our understanding of this class of enzymes (5, 13). A unique feature of Y-class polymerases, compared with the common right-handed arrangement of palm, thumb, and finger subdomains of high fidelity (i.e. A-class) DNA polymerases (14), is a “little finger” or “PAD” (palm-associated domain) subdomain that plays a crucial role in lesion bypass (12, 15–21). In addition to the little finger subdomain at the C-terminal end of the catalytic core, both Rev1 and Pol κ exhibit an N-terminal extension that is absent in other translesion polymerases. The N-terminal extension in the structure of the ternary (human) hPol κ·DNA·dTTP complex folds into a U-shaped tether-helix-turn-helix “clasp” that is located between the thumb and little finger domains, allowing the polymerase to completely encircle the DNA (18). Although the precise role of the clasp for lesion bypass by hPol κ remains to be established, it is clear that this entity is functionally important, because mutant enzymes with partially or completely removed clasps exhibit diminished catalytic activity compared with the full-length catalytic core (hPol κ N1–526) or a core lacking the N-terminal 19 residues (hPol κ N19–526; the construct used for crystal structure determination of the ternary complex (18)).7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), found in both lower organisms and eukaryotes, is a major lesion that is a consequence of oxidative stress. The lesion is of relevance not only because of its association with cancer (22, 23), but also in connection with aging (24), hepatitis (25), and infertility (26). It is far from clear which DNA polymerases bypass 8-oxoG most often in a cellular context, but given the ubiquitous nature of the lesion it seems likely that more than one enzyme could encounter the lesion. Replicative polymerases commonly insert dATP opposite template 8-oxoG, with the lesion adopting the preferred syn conformation (e.g. 27, 28). It was recently found that the translesion polymerase Dpo4 from S. solfataricus synthesizes efficiently past 8-oxoG, inserting ≥95% dCTP > dATP opposite the lesion (29, 30). The efficient and low error bypass of the 8-oxoG lesion by Dpo4 is associated to a large extent with an arginine residue in the little finger domain (17). In the crystal structure of the ternary Dpo4·DNA·dCTP complex, the side chain of Arg-332 forms a hydrogen bond to the 8-oxygen of 8-oxoG, thus shifting the nucleoside conformational equilibrium toward the anti state and enabling a Watson-Crick binding mode with the incoming dCTP (30). The efficient and accurate replication of templates bearing 8-oxoG by yeast Pol η (31, 32) may indicate similarities between the bypass reactions catalyzed by the archaeal and eukaryotic enzymes. In contrast, bypass synthesis opposite 8-oxoG by human Pol κ is error-prone, resulting in efficient incorporation of A (33–35). The inaccurate bypass of 8-oxoG is thought to contribute to the deleterious effects associated with the lesion. These observations indicate different behaviors of the eukaryotic trans-lesion Pol κ and its archaeal “homolog” Dpo4 vis-à-vis the major oxidative stress lesion 8-oxoG. A mechanistic understanding of human DNA polymerases that bypass 8-oxoG in an error-prone fashion, such as hPol κ, is therefore of great interest.To elucidate commonalities and differences between the trans-8-oxoG syntheses of S. solfataricus Dpo4, yeast Pol η, and hPol κ, we carried out a comprehensive analysis of the bypass activity for the latter with template·DNA containing the 8-oxoG lesion, including pre-steady-state and steady-state kinetics of primer extension opposite and beyond 8-oxoG and LC-MS/MS assays of full-length extension products. We determined crystal structures of ternary hPol κ-(8-oxoG)DNA-dGTP and hPol κ-(8-oxoG)DNA-dATP complexes, apparently the first for any complex with adducted DNA for the κ enzyme reported to date. Our work demonstrates clear distinctions between genetically related translesion polymerases and provides insights into the origins of the error-prone reactions opposite 8-oxoG catalyzed by Y-family DNA polymerases.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

Crystal Structure of the LG1-3 Region of the Laminin ��2 Chain

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins.The laminins constitute a major class of cell-adhesive glycoproteins that are intimately involved in basement membrane assembly and function. Their essential roles in embryo development and tissue function have been demonstrated by numerous genetic studies and the analysis of severe human diseases resulting from mutations in laminin genes (1–4). All laminins are heterotrimers composed of three different gene products, termed α, β, and γ chains. At present, 16 mouse and human laminins are known, assembled from five α, three β, and three γ chains. The different laminins have characteristic expression patterns and functions in the embryo and adult animal (1). Laminins are cross-shaped molecules: the three short arms are composed of one chain each, while the long arm is a coiled coil of all three chains, terminating in a tandem of five laminin G-like (LG)² domains, LG1-5, contributed by the α chain (2). Basement membrane assembly requires polymerization via the short arms and cell attachment via the LG1-5 region (5, 6).Cell adhesion to laminins is mediated by multiple receptors: integrins bind to the LG1-3 region, whereas α-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids bind predominantly to sites in the LG4-5 pair (7). Integrins are heterodimers with a large extracellular domain consisting of one α and one β chain, which both span the cell membrane and engage in transmembrane signaling (8). Of the 24 mouse and human integrins, the major laminin binding integrins are α3β1, α6β1, α7β1, and α6β4, which have distinct affinities for the different laminin isoforms (9). Although some studies have reported integrin binding or integrin-mediated cell adhesion to isolated LG domains or tandems (10–12), there is strong evidence to suggest that the coiled coil region and an intact γ chain tail are required for full integrin binding to the laminin LG1-3 region (13–18). Compared with integrin binding to collagen and fibronectin, which is understood in atomic detail (19, 20), the laminin-integrin interaction remains poorly characterized in structural terms. We previously determined crystal structures of the LG4-5 region of the laminin α1 and α2 chains and defined their receptor binding sites (21–23). Here, we report the crystal structure of the remainder of the laminin α2 receptor binding region, LG1-3.     

Single Chain Variable Fragment against Nicastrin Inhibits the ��-Secretase Activity

γ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.γ-Secretase catalyzes intramembrane proteolysis of a variety of substrates including amyloid β precursor protein (APP)³ to generate amyloid β peptide (Aβ), the latter being a major component of senile plaques in the brains of Alzheimer disease patients. Thus, agents that inhibit γ-secretase activity could serve as an effective therapeutics for Alzheimer disease, whereas the γ-secretase activity plays important roles in cell signaling pathways including Notch signaling (1, 2). γ-Secretase consists of at least four integral membrane proteins, i.e. presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, all of which are essential to the proteolytic activity (3–5). Molecular cellular and chemical biological analyses have revealed that PS forms a hydrophilic pore involving the transmembrane domain 6 and 7, where conserved catalytic aspartates reside to function as catalytic residues of γ-secretase complex (6, 7). APH-1 is a multipass membrane protein that plays a role in stabilization and trafficking of the γ-secretase complex (8), and PEN-2 is a cofactor for the activation and the regulation of the γ-secretase activity (3, 9).NCT, which was identified as a PS-binding protein (10), is a single-pass membrane protein that harbors an extracellular domain (ECD) with a number of N-glycosylation sites. In mammalian cells NCT undergoes Endo H-resistant complex glycosylation and acquires trypsin resistance during the assembly process of the γ-secretase complex (11–17). Molecular and cellular analyses revealed that the trypsin resistance, presumably indicating the proper structural folding of NCT, might be directly linked to the enzymatic activity, whereas the complex glycosylation is dispensable. Moreover, multiple sequence alignment analyses revealed that NCT ECD have a similarity to an aminopeptidase (18), whereas certain catalytic residues are not conserved. Recently one study has suggested that NCT plays a critical role in substrate recognition (19). During the proteolytic process, NCT ECD captures the most N terminus of the substrate as a primary substrate receptor (i.e. exosite) for the γ-secretase via the aminopeptidase-like domain. However, this view has been recently challenged (20). Nevertheless, as structural information of NCT ECD is totally lacking, the functional role of the structural maturation of NCT in the formation and activity of the γ-secretase remains unclear.Molecular engineering of monoclonal antibodies opens a venue for the functional analyses of targeted molecule and the therapeutic intervention for several diseases (21). A single-chain antibody fragment (scFv) is comprised of heavy- and light-chain sequences of an antibody linked by a short linker and preserves binding abilities of its parental antibody. scFv can be expressed intracellularly as an intrabody (22, 23), which provides a powerful method for phenotypic knock-out of the genes. Intrabodies have been investigated as treatments for a variety of pathological conditions, including neurodegenerative diseases such as Parkinson disease and Huntington disease. Moreover, several recent publications have highlighted the therapeutic potential of intrabodies targeting intra- as well as extracellular epitopes (24–29). Here, we generated scFv against NCT from an anti-NCT monoclonal antibody. Unexpectedly, the overexpression of the anti-NCT scFv as an intrabody abolished the proteolytic activity by the destabilization of the γ-secretase complex and the inappropriate glycosylation of NCT. This is the first example showing that engineered antibody would be a useful tool for the direct modulation of the γ-secretase complex and its activity.     

Dominant-negative Inhibitors of the Clostridium perfringens ?-Toxin

The Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin.The Clostridium perfringens ϵ-toxin is one of the most potent bacterial toxins (1, 2). The ϵ-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3–6). The disease most frequently affects livestock animals, though the toxin may also affect humans (7–9). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ϵ-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10–13). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ϵ-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin.One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15–19), Bacillus anthracis anthrax toxin protective antigen (20–25), Bacillus thuringiensis Cry1Ab (26), and Escherichia coli ClyA cytotoxin (27). Like VacA, protective antigen, Cry1Ab, and ClyA, the ϵ-toxin assembles into oligomeric complexes containing multiple toxin monomers (28–30). In the case of VacA and protective antigen, the most extensively studied examples of toxins inhibited by dominant-negative mutants, the number of mutations that inactivate the toxins is substantially greater than the number of mutations that lead to a dominant-negative phenotype (16, 17, 24, 31, 32). Although many of the mutations leading to dominant-negative toxins are located within regions of the toxins that are believed to form the membrane insertion domain, some mutations that inactivate the toxins (but are not dominant-negative) also map within the predicted membrane insertion domains (24, 32). Thus, a deeper understanding of the nature of the dominant-negative phenotype is needed.In this study, we sought to generate dominant-negative mutants of the ϵ-toxin. We hypothesized that mutations within the membrane insertion domain of ϵ-toxin, particularly mutations that are expected to restrict movement of this domain, would lead to dominant-negative inhibitors. We expressed wild-type and site-specific mutants of the ϵ-toxin as recombinant proteins in E. coli. The recombinant proteins were purified, and cytotoxicity was assessed using an established cell culture assay. Using this approach, we identified mutant proteins that inhibited the activity of wild-type ϵ-toxin in vitro and determined the mechanism of inhibition.     

Amino Acid Position-specific Contributions to Amyloid ��-Protein Oligomerization

Understanding the structural and assembly dynamics of the amyloid β-protein (Aβ) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Aβ oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform “scanning PICUP.” Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Aβ40) or 42 (for Aβ42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil → α/β → β transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr¹-substituted homologues of Aβ40 and Aβ42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr¹]Aβ40 fibrils. Substitution of Aβ40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Aβ40 and Aβ42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr¹) to alter Aβ assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Aβ conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.Alzheimer disease (AD)⁴ is the most common cause of late-life dementia (1) and is estimated to afflict more than 27 million people worldwide (2). An important etiologic hypothesis is that amyloid β-protein (Aβ) oligomers are the proximate neurotoxins in AD. Substantial in vivo and in vitro evidence supports this hypothesis (3–12). Neurotoxicity studies have shown that Aβ assemblies are potent neurotoxins (5, 13–20), and the toxicity of some oligomers can be greater than that of the corresponding fibrils (21). Soluble Aβ oligomers inhibit hippocampal long term potentiation (4, 5, 13, 15, 17, 18, 22) and disrupt cognitive function (23). Compounds that bind and disrupt the formation of oligomers have been shown to block the neurotoxicity of Aβ (24, 25). Importantly, recent studies in higher vertebrates (dogs) have shown that substantial reduction in amyloid deposits in the absence of decreases in oligomer concentration has little effect on recovery of neurological function (26).Recent studies of Aβ oligomers have sought to correlate oligomer size and biological activity. Oligomers in the supernatants of fibril preparations centrifuged at 100,000 × g caused sustained calcium influx in rat hippocampal neurons, leading to calpain activation and dynamin 1 degradation (27). Aβ-derived diffusible ligand-like Aβ42 oligomers induced inflammatory responses in cultured rat astrocytes (28). A 90-kDa Aβ42 oligomer (29) has been shown to activate ERK1/2 in rat hippocampal slices (30) and bind avidly to human cortical neurons (31), in both cases causing apoptotic cell death. A comparison of the time dependence of the toxic effects of the 90-kDa assembly with that of Aβ-derived diffusible ligands revealed a 5-fold difference, Aβ-derived diffusible ligands requiring more time for equivalent effects (31). A 56-kDa oligomer, “Aβ*56,” was reported to cause memory impairment in middle-aged transgenic mice expressing human amyloid precursor protein (32). A nonamer also had adverse effects. Impaired long term potentiation in rat brain slices has been attributed to Aβ trimers identified in media from cultured cells expressing human amyloid precursor protein (33). Dimers and trimers from this medium also have been found to cause progressive loss of synapses in organotypic rat hippocampal slices (10). In mice deficient in neprilysin, an enzyme that has been shown to degrade Aβ in vivo (34), impairment in neuronal plasticity and cognitive function correlated with significant increases in Aβ dimer levels and synapse-associated Aβ oligomers (35).The potent pathologic effects of Aβ oligomers provide a compelling reason for elucidating the mechanism(s) of their formation. This has been a difficult task because of the metastability and polydispersity of Aβ assemblies (36). To obviate these problems, we introduced the use of the method of photo-induced cross-linking of unmodified proteins (PICUP) to rapidly (<1 s) and covalently stabilize oligomer mixtures (for reviews see Refs. 37, 38). Oligomers thus stabilized no longer exist in equilibrium with monomers or each other, allowing determination of oligomer frequency distributions by simple techniques such as SDS-PAGE (37). Recently, to obtain population-average information on contributions to fibril formation of amino acid residues at specific sites in Aβ, we employed a scanning intrinsic fluorescence approach (39). Tyr was used because it is a relatively small fluorophore, exists natively in Aβ, and possesses the side chain most reactive in the PICUP chemistry (40). Using this approach, we found that the central hydrophobic cluster region (Leu¹⁷–Ala²¹) was particularly important in controlling fibril formation of Aβ40, whereas the C terminus was the predominant structural element controlling Aβ42 assembly (39). Here we present results of studies in which key strategic features of the two methods have been combined to enable execution of “scanning PICUP” and the consequent revelation of site-specific effects on Aβ oligomerization.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)⁵ protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants.