OPTIMIZATION ALIGNMENT: THE END OF MULTIPLE SEQUENCE ALIGNMENT IN PHYLOGENETICS?

Abstract— A method is described to assess directly the number of DNA sequence transformations, evolutionary events, required by a phylogenetic topology without the use of multiple sequence alignment. This is accomplished through a generalization of existing character optimization procedures to include insertion and deletion events (indels) in addition to base substitutions. The crux of the model is the treatment of indels as processes as opposed to the patterns implied by multiple sequence alignment. The results of this procedure are directly compatible with parsimony-based tree lengths. In addition to the simplicity of the method, it appears to generate more efficient (simpler) explanations of sequence variation than does multiple alignment.     

MULTIPLE MATING IN THE GLANVILLE FRITILLARY BUTTERFLY: A CASE OF WITHIN‐GENERATION BET HEDGING?

Many hypotheses have been proposed to explain multiple mating in females. One of them is bet hedging, that is avoiding having no or very few offspring in any given generation, rather than maximizing the expected number of offspring. However, within-generation bet hedging is generally believed to be an unimportant evolutionary force, except in very small populations. In this study, we derive predictions of the bet-hedging hypothesis for a case in which local insect populations are often small, offspring performance varies, for example, due to inbreeding depression, and the groups of gregarious larvae have to exceed a threshold size before they are likely to survive throughout the larval stage. These conditions exist for populations of the Glanville fritillary butterfly (Melitaea cinxia), potentially making bet-hedging benefits larger than usual. We observed matings in a field cage, which allowed detailed observations under practically natural conditions, and analyzed genetic paternity of egg clutches laid by females under direct observation. The egg-laying and survival patterns are in line with the predictions, supporting the hypothesis that multiple mating in M. cinxia presents a rare case of within-generation bet hedging.     

Paradoxical Condensation of Copper with Elevated ��-Amyloid in Lipid Rafts under Cellular Copper Deficiency Conditions: IMPLICATIONS FOR ALZHEIMER DISEASE*

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), β-amyloid peptide (Aβ) aggregation, and amyloid formation. Aβ·copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the β-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. β- and γ-secretases, and Aβ have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Aβ synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Aβ·copper complexes are more likely to form. This explains the paradoxical hypermetallation of Aβ with copper under tissue copper deficiency conditions in AD.Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).² Aberrant interactions between copper or zinc with the β-amyloid peptide (Aβ) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic Aβ oligomers and aggregation into plaques characteristic of AD brains (reviewed in Ref. 1). Copper, iron, and zinc are highly concentrated in extracellular plaques (2, 3), and yet brain tissues from AD (4–6) and human β-amyloid precursor protein (APP) transgenic mice (7–10) are paradoxically copper deficient compared with age-matched controls. Elevation of intracellular copper levels by genetic, dietary, and pharmacological manipulations in both AD transgenic animal and cell culture models is able to attenuate Aβ production (7, 9, 11–15). However, the underlying mechanism is at present unclear.Abnormal cholesterol metabolism is also a contributing factor in the pathogenesis of AD. Hypercholesterolemia increases the risk of developing AD-like pathology in a transgenic mouse model (16). Epidemiological and animal model studies show that a hypercholesterolemic diet is associated with Aβ accumulation and accelerated cognitive decline, both of which are further aggravated by high dietary copper (17, 18). In contrast, biochemical depletion of cholesterol using statins, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, and methyl-β-cyclodextrin, a cholesterol sequestering agent, inhibit Aβ production in animal and cell culture models (19–25).Cholesterol is enriched in lipid rafts, membrane microdomains implicated in Aβ generation from APP cleavage by β- and γ-secretases. Recruitment of BACE1 (β-secretase) into lipid rafts increases the production of sAPP_β and Aβ (23, 26). The β-secretase-cleaved APP C-terminal fragment (β-CTF), and γ-secretase, a multiprotein complex composed of presenilin (PS1 or PS2), nicastrin (Nct), PEN-2 and APH-1, colocalize to lipid rafts (27). The accumulation of Aβ in lipid rafts isolated from AD and APP transgenic mice brains (28) provided further evidence that cholesterol plays a role in APP processing and Aβ generation.Currently, copper and cholesterol have been reported to modulate APP processing independently. However, evidence indicates that, despite tissue copper deficiency, Aβ·Cu²⁺ complexes form in AD that catalytically oxidize cholesterol and lipid to generate H₂O₂ and lipid peroxides (e.g. hydroxynonenal and malondialdehyde), which contribute to oxidative damage observed in AD (29–35). The underlying mechanism leading to the formation of pathological Aβ·Cu²⁺ complexes is unknown. In this study, we show that copper alters the structure of lipid rafts, and attenuates Aβ synthesis in lipid rafts by inhibition of APP endocytosis. We also identify a paradoxical inverse relationship between total cellular copper levels and copper distribution to lipid rafts, which appear to possess a privileged pool of copper where Aβ is more likely to interact with Cu²⁺ under copper-deficiency conditions to form Aβ·Cu²⁺ complexes. These data provide a novel mechanism by which cellular copper deficiency in AD could foster an environment for potentially adverse interactions between Aβ, copper, and cholesterol in lipid rafts.     

CLCA Splicing Isoform Associated with Adhesion through β1-Integrin and Its Scaffolding Protein: SPECIFIC EXPRESSION IN UNDIFFERENTIATED EPITHELIAL CELLS*

We previously found that a rat CLCA homologue (rCLCA-f) modulates Ca²⁺-dependent Cl⁻ transport in the ductal cells of the rat submandibular gland. CLCA proteins have been shown to be multifunctional, with roles in, for example, cell adhesion. Here, we describe the mRNA and protein expressions of a splicing isoform of rat rCLCA (rCLCA-t). This isoform is a 514-amino acid protein containing a C-terminal 59-amino acid that is distinct from the rCLCA-f sequence. Immunohistochemistry revealed rCLCA-t to be located in the basal cells of the rat submandibular gland excretory duct and the stratum basale of rat epidermis, whereas rCLCA-f was detected in cells during the process of differentiation. In a heterologous expression system, rCLCA-t was found to be a membrane protein present predominantly in the perinuclear region, and not to be either present on the cell surface or secreted. rCLCA-t failed to enhance ionomycin-induced Cl⁻ conductance (unlike rCLCA-f). When compared with rCLCA-f, it weakened cell attachment to a greater extent and in a manner that was evidently modulated by intracellular Ca²⁺, protein kinase C, and β₁-integrin. rCLCA-t was found to associate with RACK1 (receptor for activated C kinase) and to reduce expression of mature β₁-integrin. Treatment of rat skin with rCLCA-t siRNA increased the expression of β₁-integrin in the stratum basale of the epidermis. These results are consistent with cell-specific splicing of rCLCA mRNA playing a role in the modulation of the adhesive potential of undifferentiated epithelial cells.     

Regulation of Autophagy and Its Associated Cell Death by “Sphingolipid Rheostat”: RECIPROCAL ROLE OF CERAMIDE AND SPHINGOSINE 1-PHOSPHATE IN THE MAMMALIAN TARGET OF RAPAMYCIN PATHWAY*

The role of “sphingolipid rheostat” by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(−)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(−) or C₂-ceramide. AA(−) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(−). S1P exerts biological actions via cell surface receptors, and S1P₃ among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P₃ in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(−) or C₂-ceramide. Whereas S1P treatment of S1P₃ overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(−) or C₂-ceramide. These results indicate that S1P-S1P₃ plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(−)- or C₂-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P₃ engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P₃ signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.     

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

A Novel Function of CRL4Cdt2: REGULATION OF THE SUBUNIT STRUCTURE OF DNA POLYMERASE δ IN RESPONSE TO DNA DAMAGE AND DURING THE S PHASE*

DNA polymerase δ (Pol δ4) is a heterotetrameric enzyme, whose p12 subunit is degraded in response to DNA damage, leaving behind a trimer (Pol δ3) with altered enzymatic characteristics that participate in gap filling during DNA repair. We demonstrate that CRL4^Cdt2, a key regulator of cell cycle progression that targets replication licensing factors, also targets the p12 subunit of Pol δ4 in response to DNA damage and on entry into S phase. Evidence for the involvement of CRL4^Cdt2 included demonstration that p12 possesses a proliferating cell nuclear antigen-interacting protein-degron (PIP-degron) and that knockdown of the components of the CRL4^Cdt2 complex inhibited the degradation of p12 in response to DNA damage. Analysis of p12 levels in synchronized cell populations showed that p12 is partially degraded in S phase and that this is affected by knockdowns of CUL4A or CUL4B. Laser scanning cytometry of overexpressed wild type p12 and a mutant resistant to degradation showed that the reduction in p12 levels during S phase was prevented by mutation of p12. Thus, CRL4^Cdt2 also regulates the subunit composition of Pol δ during the cell cycle. These studies reveal a novel function of CRL4^Cdt2, i.e. the direct regulation of DNA polymerase δ, adding to its known functions in the regulation of the licensing of replication origins and expanding the scope of its overall control of DNA replication. The formation of Pol δ3 in S phase as a normal aspect of cell cycle progression leads to the novel implications that it is involved in DNA replication as well as DNA repair.     

The Multifunctional Protein in Peroxisomal β-Oxidation: STRUCTURE AND SUBSTRATE SPECIFICITY OF THE ARABIDOPSIS THALIANA PROTEIN MFP2*

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal β-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

Ciliary Neurotrophic Factor Protects Mice Against Streptozotocin-induced Type 1 Diabetes through SOCS3: THE ROLE OF STAT1/STAT3 RATIO IN β-CELL DEATH*

Type 1 diabetes is characterized by a loss of islet β-cells. Ciliary neurotrophic factor (CNTF) protects pancreatic islets against cytokine-induced apoptosis. For this reason, we assessed whether CNTF protects mice against streptozotocin-induced diabetes (a model of type 1 diabetes) and the mechanism for this protection. WT and SOCS3 knockdown C57BL6 mice were treated for 5 days with citrate buffer or 0.1 mg/kg CNTF before receiving 80 mg/kg streptozotocin. Glycemia in non-fasted mice was measured weekly from days 0–28 after streptozotocin administration. Diabetes was defined as a blood glucose > 11.2 mmol/liter. Wild-type (WT) and SOCS3 knockdown MIN6 cells were cultured with CNTF, IL1β, or both. CNTF reduced diabetes incidence and islet apoptosis in WT but not in SOCS3kd mice. Likewise, CNTF inhibited apoptosis in WT but not in SOCS3kd MIN6 cells. CNTF increased STAT3 phosphorylation in WT and SOCS3kd mice and MIN6 cells but reduced STAT1 phosphorylation only in WT mice, in contrast to streptozotocin and IL1β. Moreover, CNTF reduced NFκB activation and required down-regulation of inducible NO synthase expression to exert its protective effects. In conclusion, CNTF protects mice against streptozotocin-induced diabetes by increasing pancreatic islet survival, and this protection depends on SOCS3. In addition, SOCS3 expression and β-cell fate are dependent on STAT1/STAT3 ratio.     

Functional Analysis of the Transmembrane Domains of Presenilin 1: PARTICIPATION OF TRANSMEMBRANE DOMAINS 2 AND 6 IN THE FORMATION OF INITIAL SUBSTRATE-BINDING SITE OF γ-SECRETASE*

γ-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2, which mediates intramembrane proteolysis of a range of type I transmembrane proteins. We previously analyzed the functional roles of the N-terminal transmembrane domains (TMDs) 1–6 of PS1 in the assembly and proteolytic activity of the γ-secretase using a series of TMD-swap PS1 mutants. Here we applied the TMD-swap method to all the TMDs of PS1 for the structure-function analysis of the proteolytic mechanism of γ-secretase. We found that TMD2- or -6-swapped mutant PS1 failed to bind the helical peptide-based, substrate-mimic γ-secretase inhibitor. Cross-linking experiments revealed that both TMD2 and TMD6 of PS1 locate in proximity to the TMD9, the latter being implicated in the initial substrate binding. Taken together, our data suggest that TMD2 and the luminal side of TMD6 are involved in the formation of the initial substrate-binding site of the γ-secretase complex.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.     

Crystal Structure of a Bony Fish β2-Microglobulin: INSIGHTS INTO THE EVOLUTIONARY ORIGIN OF IMMUNOGLOBULIN SUPERFAMILY CONSTANT MOLECULES*

Three-dimensional structures of β₂-microglobulin (β2m) from chicken and various mammals have been described previously, but aside from genomic sequences, very little is known about the three-dimensional structures of β2m in species other than warm-blooded vertebrates. Here, we present the first three-dimensional structure of β2m from bony fish grass carp (Ctid-β2m), resolved at 2.1 Å. The key structural differences between this new structure and previously published structures are two new hydrogen bonds at positions Ile³⁷ and Glu³⁸ in strand C and Lys⁶⁶ in strand E, and a hydrophobic pocket around the center of the protein found in Ctid-β2m. Importantly, Ctid-β2m has a short D strand and a long loop between stands C and D, rather than the flexible region found in other β2m structures that serves as a putative binding region for the major histocompatibility complex heavy chain. Comparing the Ctid-β2m structure with those of bovine and human β2ms, the Cα root mean square deviation of the latter are 1.3 Å and 1.8 Å, respectively. Compared with the constant domains of Lamprey T cell receptor-like receptor (Lamp-TCRLC) and Amphioxus V and C domain-bearing protein (Amphi-VCPC), Ctid-β2m exhibits very different topology. The three-dimensional structures of domains predicted from Amphi-VCPC/Lamp-TCRLC are distinctly lacking in strand A of β2ms. There are 18 amino acids at the N terminus of Amphi-VCPC that may have evolved into strand A of β2ms. A mutation in the BC loops of Amphi-VCPC may have led to the novel topology found in β2m. Based on these results, Ctid-β2m may well reflect evolutionary characteristics of ancestral C set molecules.     

Protein-Protein Interactions in the β-Oxidation Part of the Phenylacetate Utilization Pathway: CRYSTAL STRUCTURE OF THE PaaF-PaaG HYDRATASE-ISOMERASE COMPLEX*

Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain β-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735–10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid β-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid β-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid β-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid β-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid β-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

Role of Apolipoprotein E in β-Amyloidogenesis: ISOFORM-SPECIFIC EFFECTS ON PROTOFIBRIL TO FIBRIL CONVERSION OF Aβ IN VITRO AND BRAIN Aβ DEPOSITION IN VIVO*

Human APOE ϵ4 allele is a strong genetic risk factor of Alzheimer disease. Neuropathological and genetic studies suggested that apolipoprotein E4 (apoE4) protein facilitates deposition of amyloid β peptide (Aβ) in the brain, although the mechanism whereby apoE4 increases amyloid aggregates remains elusive. Here we show that injection of Aβ protofibrils induced Aβ deposition in the brain of APP transgenic mice, suggesting that Aβ protofibrils acted as a seed for aggregation and deposition of Aβ in vivo. Injection of Aβ protofibrils together with apoE3 significantly attenuated Aβ deposition, whereas apoE4 did not have this effect. In vitro assays revealed that the conversion of Aβ protofibrils to fibrils progressed more slowly upon coincubation with apoE2 or apoE3 compared with that with apoE4. Aβ protofibrils complexed with apoE4 were less stable than those with apoE2 or apoE3. These data suggest that the suppression effect of apoE2 or apoE3 on the structural conversion of Aβ protofibrils to fibrils is stronger than those of apoE4, thereby impeding β-amyloid deposition.     

Identification of Key Functional Residues in the Active Site of Human β1,4-Galactosyltransferase 7: A MAJOR ENZYME IN THE GLYCOSAMINOGLYCAN SYNTHESIS PATHWAY*

Glycosaminoglycans (GAGs) play a central role in many pathophysiological events, and exogenous xyloside substrates of β1,4-galactosyltransferase 7 (β4GalT7), a major enzyme of GAG biosynthesis, have interesting biomedical applications. To predict functional peptide regions important for substrate binding and activity of human β4GalT7, we conducted a phylogenetic analysis of the β1,4-galactosyltransferase family and generated a molecular model using the x-ray structure of Drosophila β4GalT7-UDP as template. Two evolutionary conserved motifs, ¹⁶³DVD¹⁶⁵ and ²²¹FWGWGREDDE²³⁰, are central in the organization of the enzyme active site. This model was challenged by systematic engineering of point mutations, combined with in vitro and ex vivo functional assays. Investigation of the kinetic properties of purified recombinant wild-type β4GalT7 and selected mutants identified Trp²²⁴ as a key residue governing both donor and acceptor substrate binding. Our results also suggested the involvement of the canonical carboxylate residue Asp²²⁸ acting as general base in the reaction catalyzed by human β4GalT7. Importantly, ex vivo functional tests demonstrated that regulation of GAG synthesis is highly responsive to modification of these key active site amino acids. Interestingly, engineering mutants at position 224 allowed us to modify the affinity and to modulate the specificity of human β4GalT7 toward UDP-sugars and xyloside acceptors. Furthermore, the W224H mutant was able to sustain decorin GAG chain substitution but not GAG synthesis from exogenously added xyloside. Altogether, this study provides novel insight into human β4GalT7 active site functional domains, allowing manipulation of this enzyme critical for the regulation of GAG synthesis. A better understanding of the mechanism underlying GAG assembly paves the way toward GAG-based therapeutics.     

Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme: INSIGHTS OF N-TERMINAL β-SANDWICH IN SUBSTRATE SPECIFICITY AND ENZYMATIC ACTIVITY*

The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an α-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q10625","term_id":"1707934","term_text":"Q10625"}}Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1→4 bond and making a new 1→6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 β-sandwich, N2 β-sandwich, a central (β/α)₈ domain that houses the catalytic site, and a C-terminal β-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbΔ108GlgB protein. The N1 β-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 β-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbΔ108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1→4 bond breakage) and isomerization (1→6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbΔ108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECΔ112GlgB).