Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.     

Kinetic Considerations on the Development of Binding Assays in Single-Addition Mode: Application to the Search for α2δ1 Modulators

The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.     

Diversity of Structure and Function of α1α6β3δ GABAA Receptors: COMPARISON WITH α1β3δ AND α6β3δ RECEPTORS*

δ subunit-containing γ-aminobutyric acid, type A (GABA_A)receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with α₁ and/or α₆ subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA_A receptor pentamers by concatenation. These receptors composed of α₁, α₆, β₃, and δ subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3α,21-dihydroxy-5α-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that δ can assume multiple positions in a receptor pentamer made up of α₁, α₆, β₃, and δ subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of δ subunits between two α subunits in α₁α₆β₃δ receptors. This property is shared by α₁β₃δ and α₆β₃δ receptors, but there are differences in the additionally expressed isoforms.     

Are Osteoclasts Needed for the Bone Anabolic Response to Parathyroid Hormone?: A STUDY OF INTERMITTENT PARATHYROID HORMONE WITH DENOSUMAB OR ALENDRONATE IN KNOCK-IN MICE EXPRESSING HUMANIZED RANKL*

PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb. PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and histomorphometrical indices of bone turnover, -i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices, in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK^−/− mice. PTH increased osteocalcin similarly in RANK^−/− and WT mice. It also increased BMD in RANK^−/− mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism, which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as determined by the number and activity of osteoclasts on bone surfaces.     

Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE,MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES*

The F₁F₀-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F₁F₀ activity via an interaction with the “d” subunit of F₁F₀-ATP synthase (dF₁F₀) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF₁F₀-derived peptide (NH₂-²AGRKLALKTIDWVSF¹⁶-COOH) that inhibits PKCδ binding to dF₁F₀ in overlay assays. We have also identified a second dF₁F₀-derived peptide (NH₂-¹¹¹RVREYEKQLEKIKNMI¹²⁶-COOH) that facilitates PKCδ binding to dF₁F₀. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF₁F₀ interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF₁F₀ by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F₁F₀-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF₁F₀ facilitator peptide by itself induced the PKCδ-dF₁F₀ co-immunoprecipitation and inhibited F₁F₀-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F₁F₀ inhibitor blocked PKCδ-mediated inhibition of F₁F₀-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF₁F₀ interaction in NCMs. These novel peptides will improve our understanding of cardiac F₁F₀ regulation and may have potential as therapeutics to attenuate cardiac injury.     

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.     

Polymorphisms in the α4 integrin of neotropical primates: insights for binding of natural ligands and HIV-1 gp120 to the human α4β7

The α4 integrin subunit associates with β7 and β1 and plays important roles in immune function and cell trafficking. The gut-homing receptor α4β7 has been recently described as a new receptor for HIV. Here, we describe polymorphisms of ITGA4 gene in New World primates (NWP), and tested their impact on the binding to monoclonal antibodies, natural ligands (MAdCAM and VCAM), and several gp120 HIV-1 envelope proteins. Genomic DNA of NWP specimens comprising all genera of the group had their exons 5 and 6 (encoding the region of binding to the ligands studied) analyzed. The polymorphisms found were introduced into an ITGA4 cDNA clone encoding the human α4 subunit. Mutant α4 proteins were co-expressed with β7 and were tested for binding of mAbs, MAdCAM, VCAM and gp120 of HIV-1, which was compared to the wild-type (human) α4. Mutant α4 proteins harboring the K201E/I/N substitution had reduced binding of all ligands tested, including HIV-1 gp120 envelopes. The mAbs found with reduced biding included one from which a clinically-approved drug for the treatment of neurological disorders has been derived. α4 polymorphisms in other primate species may influence outcomes in the development and treatment of infectious and autoimmune diseases in humans and in non-human primates.     

Adhesion of Lymphoma Cells to Fibronectin: Differential Use of α4β1and α5β1Integrins and Stimulation by the 9EG7 mAb against the Murine β1Integrin Subunit

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor α₄β₁, and MDAY-D2 cells in addition also α₅β₁. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn²⁺. In ESb cells, this adhesion was mediated by α₄β₁. In MDAY-D2 cells, however, only α₅β₁was involved, despite β₄β₁levels similar to ESb cells. The α₄β₁integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An α₅β₁-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by α₅β₁. These results indicate that α₄β₁function in these cells is suppressed in the presence of α₅β₁. Adhesion of ESb cells to hepatocytes was inhibited by anti-α₄antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that α₄β₁and α₅β₁function is not activated during this interaction.

The 9EG7 antibody against mouse β₁integrin was described to inhibit β₁integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that β₁stimulated Printegrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.     

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.     

Molecular Determinants of the Coupling between STIM1 and Orai Channels: DIFFERENTIAL ACTIVATION OF Orai1�C3 CHANNELS BY A STIM1 COILED-COIL MUTANT*

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca²⁺ release-activated Ca²⁺ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by ∼50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca²⁺ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel (2–6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7, 8) and later on Orai1 (9–11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca²⁺ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12, 13). Its N terminus is located in the ER lumen and contains an EF-hand Ca²⁺-binding motif, which senses the ER Ca²⁺ level, and a sterile α-motif, which is suggested to mediate homomeric STIM1 aggregation (14–16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14, 17, 18). Three recent studies have independently identified the ezrin-radixin-moesin domain as the essential Orai activating domain, named SOAR (STIM1 Orai-activating region) (20) which represents so far the shortest active fragment, OASF (Orai-activating small fragment) (21) or CAD (CRAC-activating domain) (22), which includes the second, more C terminally located coiled-coil domain and the following ∼55 amino acids. The latter amino acids are suggested to contain an additional cytosolic homomerization domain indispensable for OASF homomerization and Orai activation (21).The Orai family includes three highly Ca²⁺-selective ion channels (Orai1–3) that locate to the plasma membrane, and each protein contains four predicted transmembrane segments with cytosolic N and C termini (10). All three Orai proteins possess a conserved putative coiled-coil domain in the C terminus (23, 24), whereas only the N terminus of Orai1 consists of a proline/arginine-rich region (25). Orai1 has been assumed to act in concert with STIM1 (10, 27)-activating inward Ca²⁺ currents after store depletion. The two other members of the Orai family, Orai2 and Orai3, display similar but smaller store-operated inward Ca²⁺ currents when co-expressed with STIM1 with distinct inactivation profiles, permeability properties, and 2-aminoethoxydiphenyl borate sensitivity (28–32). Recently, we have provided evidence for a store depletion-induced, dynamic coupling of STIM1 to Orai1 that involves the putative coiled-coil domain in the C terminus of Orai1 (33). Furthermore, the C terminus of STIM1, in particular the essential cytosolic region 344–442 as narrowed down by SOAR, OASF, and CAD (20–22), has been established as the key fragment for CRAC as well as Orai1 activation, because its expression alone, without the necessity to deplete ER store, is sufficient for constitutive current activation (18, 32, 33). These fragments SOAR, OASF, and CAD when co-expressed with Orai1 (20–22) exhibit enhanced plasma membrane localization in comparison with the complete STIM1 C terminus in the presence of Orai1. Specificity of interaction of SOAR to the Orai1 C terminus has been shown by its disruption (20) employing the Orai1 L273S mutant (33). Park et al. (22) have provided additional, conclusive evidence for a direct binding by combining multiple biochemical approaches demonstrating CAD interaction with Orai1.This study focused specifically on the role of the putative coiled-coil domains of STIM1 as well as Orai proteins in their coupling. Coiled-coils generally function as protein-protein interaction sites with the ability of dynamic protein assembly and disassembly (35–37). We suggest the C-terminal, putative coiled-coil domains in all three Orai proteins and the second coiled-coil motif of STIM1 as essential for STIM1/Orai communication. Moreover, the single point coiled-coil STIM1 L373S mutant allowed for differential activation of Orai channels partially stimulating Orai2 as well as Orai3 but not Orai1.     

Photochemistry and photophysics of the endoperoxide of mesodiphenylhelianthrene: a contribution to the localization of the S1(π*σ*) state of aromatic endoperoxides

The endoperoxide of mesodiphenylhelianthrene MDHPO has been studied in detail with respect to fluorescence and photo-induced rearrangement. MDHPO proved to be non-fluorescent, although its absorption spectrum is dominated at the low energy side by a strong ππ* band with a maximum at 429.5 nm. Irradiation of that band effects rearrangement to the corresponding diepoxide MDHDO, a reaction typical for S(1)(π*σ*) excited endoperoxides (EPOs). The absorption spectrum of the product MDHDO is blue shifted by only 3.5 nm. MDHDO has the same extended planar aromatic system like its precursor MDHPO, but MDHDO fluoresces strongly. These results set the excitation energy of the S(1)(π*σ*) state of MDHPO to ≤23?000 cm(-1), which is considered to be a generally realistic value of the S(1)(π*σ*) state energy of aromatic EPOs. The main reaction of S(1)(π*σ*) excited MDHPO is, however, chemical deactivation to ground state MDHPO via an oxygen biradical. The sequence of O-O bond opening and closing is the general way of repopulation of the S(0) state of aromatic EPOs from S(1)(π*σ*) excited states.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

Enzymatic Basis for N-Glycan Sialylation: STRUCTURE OF RAT α2,6-SIALYLTRANSFERASE (ST6GAL1) REVEALS CONSERVED AND UNIQUE FEATURES FOR GLYCAN SIALYLATION*

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.