Intermolecular Disulfide Cross-linking Research Articles

Insight into the Coextrusion Mechanism between Whey Protein Isolate and Cysteine.

The disulfide cross-linking sites of whey protein isolate (WPI) coextruded with dissolved cysteine (Cys) at concentrations of 0, 20, 40, 60, 80, and 100 mM were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software, and the structure and gel water distribution of WPI during coextrusion (≤50 °C) were also investigated. LC/MS/MS demonstrated that α-La (6) and α-La (120) were the most active sites for intermolecular disulfide cross-linking of α-La. Meanwhile, the molecular weight of protein polymers in coextruded WPI-Cys was the largest at 100 mM Cys, and α-lactalbumin was the main reactant for polymerization from the result of SDS-PAGE and size exclusion chromatography. Additionally, the high concentration of Cys caused the secondary structure of WPI to gradually change from a highly ordered to a disordered structure during coextrusion. In addition, with an increasing concentration of Cys, the free sulfhydryl group of proteins and the binding force to immobilized water gradually increased. Therefore, this work revealed the disulfide cross-linking mechanism between WPI and Cys under low-temperature coextrusion at the molecular level, and the obtained coextruded cross-linked WPI could serve as a novel food ingredient with excellent water-holding capacity for the food industry.

BamA forms a translocation channel for polypeptide export across the bacterial outer membrane

The β-barrel assembly machine (BAM) integrates β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own β-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of β-barrel proteins, BamA functions as a polypeptide export channel. Invivo structural mapping via intermolecular disulfide crosslinking showed that the extracellular "passenger" domain of a member of the "autotransporter" superfamily of virulence factors traverses the OM through the BamA β-barrel lumen. Furthermore, we demonstrate that a highly conserved residue within autotransporter β-barrels is required to position the passenger inside BamA to initiate translocation and that during translocation, the passenger stabilizes the hybrid-barrel state. Our results not only establish a new function for BamA but also unify the divergent functions of BamA and other "Omp85" superfamily transporters.

The cataract-related S39C variant increases γS-crystallin sensitivity to environmental stress by destroying the intermolecular disulfide cross-links

γS-crystallin, a crucial structural lens protein, plays an important role in maintaining lens transparency through its solubility and stability. The S39C mutation, a proven pathogenic mutation involved in congenital cataract, resulted in progressive cataract in adolescents. In this study, using biophysical methods, we thoroughly investigated the effects of the S39C mutation on the γS-crystallin structure, stability and propensity for aggregations. The data from spectroscopy analyses did not reveal an effect of the S39C mutation on the native structure of monomeric γS-crystallin. However, when faced with oxidative conditions, the S39C mutation prevented γS-crystallin from forming stable disulfide-linked dimers and remarkably increased hydrophobicity and the propensity to aggregate and precipitate. Under UV irradiation, heat shock, and GdnHCl-induced denaturation, the S39C mutant tended to aggregate and was prone to form more deleterious aggregates than the wild type protein. Therefore, the S39C mutation significantly increased the sensitivity of γS-crystallin to environmental stress. However, the addition of αA-crystallin and lanosterol did not change the tendency of the mutant to aggregate. According to molecular dynamic (MD) simulations, the S39C mutation had little effect on the secondary or tertiary structures of monomeric γS-crystallin but disrupted the disulfide-linked structure of the γS-crystallin dimer. The cleavage of this bond might largely reduce the structural stability of γS-crystallin. The significant decrease in the structural stability along with the increasing aggregation tendency under environmental stress might be the major causes of progressive juvenile onset cataracts induced by the S39C mutation.

In Situ Photocatalyzed Oxygen Generation with Photosynthetic Bacteria to Enable Robust Immunogenic Photodynamic Therapy in Triple‐Negative Breast Cancer

AbstractHypoxia in the tumor microenvironment is a major hurdle dampening the antitumor effect of photodynamic therapy (PDT). Herein, active photosynthetic bacteria (Synechococcus 7942, Syne) are utilized for tumor‐targeted photosensitizer delivery and in situ photocatalyzed oxygen generation to achieve photosynthesis‐boosted PDT. Photosensitizer‐encapsulated nanoparticles (HSA/ICG) are assembled by intermolecular disulfide crosslinking and attached to the surface of Syne with amide bonds to form a biomimetic system (S/HSA/ICG). S/HSA/ICG combined the photosynthetic capability of Syne and the theranostic effect of HSA/ICG. Syne capable of photoautotrophy exhibit a moderate immune stimulation effect and a certain photodynamic role under 660 nm laser irradiation. Upon intravenous injection into tumor‐bearing mice, S/HSA/ICG can effectively accumulate in tumors and generate oxygen continuously under laser irradiation through photosynthesis, which remarkably relieve tumor hypoxia and enhance reactive oxygen species production, thereby completely eliminating primary tumors. This photosynthesis‐boosted PDT can also effectively reverse the tumor immunosuppressive microenvironment and robustly evoke systematic antitumor immune responses, which exhibit excellent effect on preventing tumor recurrence and metastasis inhibition in a metastatic triple‐negative breast cancer mouse model. Hence, this photosynthetic bacteria‐based photosynthesis‐boosted immunogenic PDT offers a promising approach to eliminate both local and metastatic tumors.

Effect of Methylglyoxal-Induced Glycation on the Composition and Structure of β-Lactoglobulin and α-Lactalbumin.

Glycation, and particularly reactions between aldehydes and nucleophiles (thiols, amines), can initiate changes in the structure, solubility, composition, hydrophobicity, conformation, function, and susceptibility to proteolysis of proteins. This can have adverse consequences for mammals, plants, foodstuffs, and pharmaceuticals. Low-molecular-mass dialdehydes such as methylglyoxal (MGO) are much more reactive than parent glucose and therefore potentially highly damaging. These are present at significant levels in some foods. This study investigated whether and how MGO exposure, with or without concurrent heat exposure, affected the major whey proteins β-lactoglobulin and α-lactalbumin. MGO diminished the formation of heat-induced, reducible, intermolecular disulfide cross-links for both proteins, with this being associated, at least in part, with alternative thiol consuming reactions of MGO. At long incubation times, nonreducible protein cross-links were formed in a dose-dependent manner, with LC-MS/MS and UPLC analysis showing the presence of methylglyoxal-lysine dimers (MOLD). UPLC analysis revealed MGO-dependent consumption of specific amino acids in the order Cys > Arg > Lys > Trp for both proteins, with α-lactalbumin affected to a greater extent than β-lactoglobulin. SDS-PAGE revealed altered protein mobility consistent with modification of charged residues. MGO exposure also resulted in increased binding of the hydrophobic dye, 8-anilino-1-naphthalene sulfonic acid, consistent with limited protein unfolding. Overall, these data are consistent with rapid reaction of MGO residues at Cys residues (when available) and surface accessible Arg and Lys residues, with formation of adducts and cross-linked materials. These alternative reactions of dialdehydes diminish direct heat-induced (disulfide) cross-link formation and result in limited protein unfolding.

Inactivation of human DGAT2 by oxidative stress on cysteine residues.

Diacylglycerol acyltransferases (DGATs) have a crucial role in the biosynthesis of triacylglycerol (TG), the major storage form of metabolic energy in eukaryotic organisms. Even though DGAT2, one of two distinct DGATs, has a vital role in TG biosynthesis, little is known about the regulation of DGAT2 activity. In this study, we examined the role of cysteine and its oxidation in the enzymatic activity of human DGAT2 in vitro. Human DGAT2 activity was considerably inhibited not only by thiol-modifying reagents (NEM and IA) but also by ROS-related chemicals (H2O2 and β-lapachone), while human DGAT1 and GPAT1 were little affected. Particularly, ROS-related chemicals concomitantly induced intermolecular disulfide crosslinking of human DGAT2. Both the oxidative inactivation and disulfide crosslinking were almost completely reversed by the treatment with DTT, a disulfide-reducing agent. These results clearly demonstrated the significant role of ROS-induced intermolecular crosslinking in the inactivation of human DGAT2 and also suggested DGAT2 as a redox-sensitive regulator in TG biosynthesis.

58 - DUOX1-Dependent Disulfide Bonding of Src Kinase in the Airway Epithelium

The non-receptor tyrosine kinase Src is ubiquitously expressed and is involved in regulation of cell proliferation, differentiation, and motility. Strongly linked with cancer progression, Src also contributes to non-malignant diseases such as allergic asthma. Regulation of Src activity is incompletely understood, but critically involves tyrosine (de)phosphorylation events that mediate Src “unclamping” and interactions with regulatory or substrate proteins as well as kinase activation. In addition, more recent studies indicate that Src-dependent signaling is also regulated by redox-dependent mechanisms involving reversible oxidation of one or more conserved cysteine residues. Indeed, our previous studies indicate that epithelial wound responses or innate response to airborne allergens are mediated by oxidant-mediated activation of Src by the NADPH oxidase DUOX1. However, the precise mechanisms by which cysteine oxidation regulates Src activity are still not clear, and diverse cysteine residues have been implicated. In addition to its potential role in regulating Src tyrosine kinase activity, evidence is emerging that cysteine oxidation within Src can promote intermolecular disulfide bonding, e.g. between Src monomers, with its negative regulator C-terminal Src Kinase (CSK), or its substrate cortactin. The aim of the present study was to determine whether DUOX1-dependent activation of Src oxidation in airway epithelial cells results in intermolecular disulfide cross-linking with other proteins, and to characterize Src interacting partners. Using diagonal electrophoresis to visualize intermolecular disulfides, we observed that stimulation of H292 epithelial cells with H2O2 or with ATP, which activates DUOX1, induces disulfide-based cross-linking of Src with a number of proteins. This was confirmed after immunoprecipitation of Src, and interacting proteins are being identified using mass spectrometry. We also used recombinant Src and various Cys-to-Ala variants to determine oxidant-mediated cross-linking between Src monomers, with indicated intermolecular crosslinking between Src monomers, which was diminished with Cys mutants. We seek to extend these findings in studies with epithelial cells expressing different Src Cys-to-Ala variants to gain further insight into the significance of specific cysteine oxidation events in Src-dependent activation of various signaling pathways. Supported by NIH R01 ES021476, NIH R01 HL085646, and NIH F32 HL129706.

Multitriggered Tumor-Responsive Drug Delivery Vehicles Based on Protein and Polypeptide Coassembly for Enhanced Photodynamic Tumor Ablation

Tumor-responsive nanocarriers are highly valuable and demanded for smart drug delivery particularly in the field of photodynamic therapy (PDT), where a quick release of photosensitizers in tumors is preferred. Herein, it is demonstrated that protein-based nanospheres, prepared by the electrostatic assembly of proteins and polypeptides with intermolecular disulfide cross-linking and surface polyethylene glycol coupling, can be used as versatile tumor-responsive drug delivery vehicles for effective PDT. These nanospheres are capable of encapsulation of various photosensitizers including Chlorin e6 (Ce6), protoporphyrin IX, and verteporfin. The Chlorin e6-encapsulated nanospheres (Ce6-Ns) are responsive to changes in pH, redox potential, and proteinase concentration, resulting in multitriggered rapid release of Ce6 in an environment mimicking tumor tissues. In vivo fluorescence imaging results indicate that Ce6-Ns selectively accumulate near tumors and the quick release of Ce6 from Ce6-Ns can be triggered by tumors. In tumors the fluorescence of released Ce6 from Ce6-Ns is observed at 0.5 h postinjection, while in normal tissues the fluorescence appeared at 12 h postinjection. Tumor ablation is demonstrated by in vivo PDT using Ce6-Ns and the biocompatibility of Ce6-Ns is evident from the histopathology imaging, confirming the enhanced in vivo PDT efficacy and the biocompatibility of the assembled drug delivery vehicles.

Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens*

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H(2)O(2), whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.

Conserved C272/278 in b domain regulate the function of PDI-P5 to make lysozymes trypsin-resistant forms via significant intermolecular disulfide cross-linking

Protein disulfide isomerase-P5 (P5) is thought to have important functions as an oxidoreductase, however, molecular functions of P5 have not been fully elucidated. We have reported that P5 has insulin reductase activity and inhibits lysozyme refolding by formation of lysozyme multimers with hypermolecular mass inactivated by intermolecular disulfides (hyLYS) in oxidative refolding of reduced denatured lysozyme. To explore the role of each domain of P5, we investigated the effects of domain deletion and Cys-Ala mutants of P5 on insulin reduction and the oxidative refolding of the lysozyme. The mutants of catalytic cysteines, C36/39A, C171/174A, and C36/39/171/174A inhibited the lysozyme refolding almost similarly to P5, and even b domain without catalytic cysteines showed moderate inhibitory effect, suggesting that the b domain played a key role in the inhibition. Western blotting analysis of the refolding products indicated that the catalytic cysteines in both the a and a′ domains cross-linked lysozyme comparably to form hyLYS resistant to trypsin, in which b domain was suggested to capture lysozyme for the significant sulfhydryl oxidation. The mutant of the conserved cysteines in b domain, C272/278A, did not form hyLYS, however, showed predominant reductase activity, implying that P5 functioned as a potent sulfhydryl oxidase and a predominant reductase depending on the circumstance around C272/278. These results provide new insight into the molecular basis of P5 function.

Inhibition of tau aggregation by a rosamine derivative that blocks tau intermolecular disulfide cross-linking

Abnormal tau aggregates are presumed to be neurotoxic and are an important therapeutic target for multiple neurodegenerative disorders including Alzheimer’s disease. Growing evidence has shown that tau intermolecular disulfide cross-linking is critical in generating tau oligomers that serve as a building block for higher-order aggregates. Here we report that a small molecule inhibitor prevents tau aggregation by blocking the generation of disulfide cross-linked tau oligomers. Among the compounds tested, a rosamine derivative bearing mild thiol reactivity selectively labeled tau and effectively inhibited oligomerization and fibrillization processes in vitro. Our data suggest that controlling tau oxidation status could be a new therapeutic strategy for prevention of abnormal tau aggregation.

Nanoengineering of stimuli-responsive protein-based biomimetic protocells as versatile drug delivery tools.

We present a general strategy to nanoengineer protein-based colloidal spheres (biomimetic protocells) as versatile delivery carriers with stimuli responsiveness by the electrostatic assembly of binary components (proteins and polypeptides) in association with intermolecular disulfide cross-linking. The size of the colloidal spheres, ranging from nanoscale to microscale, is readily tuned through parameters like protein and polypeptide concentration, the ratio between both, pH, and so on. Moreover, such colloidal spheres show versatile encapsulation of various guest molecules including small organic molecules and biomacromolecules. The pH and redox dual-responsiveness facilitates the rapid release of the payload in an acidic and reductant-enriched ambient such as in lysosomes. Thus, nanoengineering of protein-based biomimetic protocells opens a new alternative avenue for developing delivery vehicles with multifunctional properties towards a range of therapeutic and diagnostic applications.

Crosslinks in wheat gluten films with hexagonal close-packed protein structures

Wheat gluten/glycerol (WGG) films were extruded with aqueous ammonia/salicylic acid or urea to investigate the reactions contributing to their hexagonal close-packed protein structures and material properties. The addition of aqueous ammonia and salicylic acid increased the pH, which, in turn, increased the level of intermolecular disulfide and lanthionine cross-links in the WGG films. Increased protein cross-linking reactions resulted in higher material strength and tensile modulus. These cross-linking reactions and the resulting material properties were similar for WGG films with 7.5% and 10% aqueous ammonia. Added urea into WGG film partially degraded into cyanate and ammonium. Cyanate subsequently reacted with lysine and cysteine to ɛ-carbamyllysine and S-carbamylcysteine, respectively. Even though these reactions resulted in a more alkaline reaction environment, hereby favoring disulfide bond formation and decreasing protein extractability, they also prevented the involvement of cysteine and lysine in protein cross-linking. The alkylation of these reactive amino acids, together with the plasticizing effect of urea, led to lower material strength and elastic modulus with increasing levels of urea.

Glutathione and Ascorbate: The balance between oxidant and carbonyl stress in the aging human lens

AbstractBackground: Two major processes appear to be at play in formation of ARNC. On the one hand advanced glycation end products (AGEs) modify lens crystallins, increasing thereby their susceptibility to oxidation via unfolding, exposure of SH‐groups and formation of UV‐photosensitizers, on the other hand SH groups form disulfides and high molecular weight aggregates that scatter light. In order to find out if there are critical SH residues in the latter process, we have generated a glutathione knockout(LEGSKO) mouse and an in vitro model of crystallin aggregation upon oxidation with H2O2.Methods: Crystallins from in vitro incubation or LEGSKO mouse were analyzed using ICAT labeling methods and 2D gel electrophoresis (2D‐PAGE) for disulfide analysis. Incubation of mouse lens homogenate (LH) with 5 mM H2O2 led to opacification within a few hours. Results: Analysis of LEGSKO crystallins by 2D‐PAGE revealed an age‐related shift from mostly intramolecular to intermolecular disulfide crosslinks which mimicked the pattern observed at 20 months in wild type lenses. All oxidation‐related disulfide forming sites in 13 crystallins were identified in vitro and found quasi concordant with those forming in the LEGSKO lens.

Role of Disulfide Cross-Linking of Mutant SOD1 in the Formation of Inclusion-Body-Like Structures

BackgroundPathologic aggregates of superoxide dismutase 1 (SOD1) harboring mutations linked to familial amyotrophic lateral sclerosis (fALS) have been shown to contain aberrant intermolecular disulfide cross-links. In prior studies, we observed that intermolecular bonding was not necessary in the formation of detergent- insoluble SOD1 complexes by mutant SOD1, but we were unable to assess whether this type of bonding may be important for pathologic inclusion formation. In the present study, we visually assess the formation of large inclusions by fusing mutant SOD1 to yellow fluorescent protein (YFP).Methodology/Principal FindingsExperimental constructs possessing mutations at all cysteine residues in SOD1 (sites 6, 57, 111, and 146 to F,S,Y,R or G,S,Y,R, respectively) were shown to maintain a high propensity of inclusion formation despite the inability to form disulfide cross-links. Interestingly, although aggregates form when all cysteines were mutated, double mutants of the ALS mutation C6G with an experimental mutation C111S exhibited low aggregation propensity.Conclusions/SignificanceOverall, this study is an extension of previous work demonstrating that cysteine residues in mutant SOD1 play a role in modulating aggregation and that intermolecular disulfide bonds are not required to produce large intracellular inclusion-like structures.

Protein polymer hydrogels by in situ, rapid and reversible self-gelation

Protein-based biomaterials are an important class of materials for applications in biotechnology and medicine. The exquisite control of their composition, stereochemistry, and chain length offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here, we report the synthesis of a thermally responsive peptide polymer-based hydrogel composed of a recombinant elastin-like polypeptide (ELP) that rapidly forms a reversibly cross-linked hydrogel by the formation of intermolecular disulfide cross-links. To do so, we designed and synthesized ELPs that incorporate periodic cysteine residues (cELPs), and show that cELPs are thermally responsive protein polymers that display rapid gelation under physiologically relevant, mild oxidative conditions. Gelation of cELPs, at concentrations as low as 2.5 wt%, occurs in ∼2.5 min upon addition a low concentration of hydrogen peroxide (0.3 wt%). We show the utility of these hydrogels for the sustained release of a model protein in vitro, and demonstrate the ability of this injectable biomaterial to pervade tumors to maximize tumor coverage and retention time upon intratumoral injection. cELPs represent a new class of injectable reversibly cross-linked hydrogels with properties intermediate between ELP coacervates and chemically cross-linked ELP hydrogels that will find useful applications in drug delivery and tissue engineering.

Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

Physicochemical Stability of the Antibody−Drug Conjugate Trastuzumab-DM1: Changes due to Modification and Conjugation Processes

In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.

639 Optimizing pathological diagnosis with new biological tools: examples in breast cancer

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 °C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 °C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9–18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60–70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (>95 %) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 °C or with NaTT transferred most (>95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamidetreated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.

AMPA Receptor Ligand Binding Domain Mobility Revealed by Functional Cross Linking

Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.