Cross-linked Complexes Research Articles

Mutant huntingtin causes defective actin remodeling during stress: defining a new role for transglutaminase 2 in neurodegenerative disease

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin–actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin–actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement–Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin–cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.

Design of a stable and methanol resistant membrane with cross-linked multilayered polyelectrolyte complexes for direct methanol fuel cells

Sulfonated poly (arylene ether ketone) bearing carboxyl groups (SPAEK-C) membranes have been prepared as proton exchange membranes for applications in direct methanol fuel cells (DMFCs). Multilayered polyelectrolyte complexes (PECs) which applied as methanol barrier agents are prepared by alternate deposition of the oppositely charged amino-containing poly (ether ether ketone) (Am-PEEK) and the highly sulfonated SPAEK-C via a layer-by-layer method. The cross-linked PEC (c-PEC) is derived from a simple heat-induced cross-linking reaction between Am-PEEK and SPAEK-C. Fourier transform infrared spectroscopy confirms that Am-PEEK and SPAEK-C are assembled successfully in the multilayers. The morphology of the membranes is studied by scanning electron microscopy, which shows the presence of the thin layers coated on the SPAEK-C membrane. After PEC and c-PEC modification, the methanol permeability decreases obviously when compared to that of the pristine membrane. Notably, improved proton conductivities are obtained for the PEC modified membranes in comparison with the pristine membrane. Moreover, the selectivity of these modified membranes is one order of magnitude higher than that of Nafion 117. The thermal stability, oxidative stability, water uptake and swelling of PEC and c-PEC modified membranes are also investigated.

Crystallization of Glutaraldehyde Cross-Linked Prothrombinase Complex in its Active Form

Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, consisting of factor Xa and its cofactor factor Va bound to phosphatidylserine (PS)-containing membrane. While partial crystal structures of Xa and Va exist, attempts to crystallize XaVa complex failed. Recent studies shows that both Xa and Va2, an active isoforms of Va binding tightly to PS-membrane, binds soluble dicaproyl-phosphatidylserine (C6PS). Also, C6PS-bound form of Va2 binds with high affinity (Kd ∼ 1 nM) to Xa forming a fully active prothrombinase complex in solution. Preliminary studies show this soluble complex, when cross-linked with glutaraldehyde, remains intact after removing C6PS using size-exclusion chromatography. Depending on conditions, up to 92% of initial activity is restored after adding back C6PS. To determine optimal formation conditions of an active cross-linked XaVa complex for use in crystallization trials, we used an algorithm varying Xa/Va and glutaraldehyde concentration, incubation time, and temperature. XaVa activity was tested after four critical stages (complex formation, cross-linking with glutaraldehyde, removing C6PS, reactivation with C6PS) by a chromogenic assay, and the yield of cross-linked complex was determined by using quantitative Shodex size-exclusion chromatography. While preliminary experiments were performed using plasma-derived human Va2, greater quantities of protein are needed than can be produced by this method. Since crystallization is more successful with cloned recombinant proteins, our lab works with multiple labs to establish procedures for expressing large quantities of recombinant human Va2 (rHVa2) in Baby Hamster Kidney cells (BHK). With higher yields of Va2, we are proceeding with trials to optimize conditions for cross-linked active prothrombinase complex. If these are successful, we will continue with crystallization trials, enabling us for the first time to determine the structure of XaVa complex in its active form. Supported by grant HL072827 and a training supplement thereto.

Purification, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly(A) polymerase and a chemically modified 15-mer oligo(A) RNA

Poly(A) polymerase (PAP) synthesizes the polyadenine tail at the 3'-end of messenger RNA. A disulfide cross-linking strategy was implemented to obtain a complex between bovine PAP (bPAP) and a 15-mer oligo(A). All seven endogenous cysteines were mutated to eliminate nonspecific cross-linked complexes. A cysteine residue was introduced at several different positions and A152C was found to achieve maximum specific cross-linking efficiency. The resulting bPAP construct was active and, when mixed with a chemically modified RNA, yielded crystals of a bPAP-RNA complex. The crystals, which belonged to space group P2 and harbored two protein-RNA complexes per asymmetric unit, diffracted X-rays to 2.25 Å resolution.

Bioinformatics tools for the structural elucidation of multi-subunit protein complexes by mass spectrometric analysis of protein-protein cross-links

Multi-subunit protein complexes are involved in many essential biochemical processes including signal transduction, protein synthesis, RNA synthesis, DNA replication and protein degradation. An accurate description of the relative structural arrangement of the constituent subunits in such complexes is crucial for an understanding of the molecular mechanism of the complex as a whole. Many complexes, however, lie in the mega-Dalton range, and are not amenable to X-ray crystallographic or nuclear magnetic resonance analysis. Techniques that are suited to structural studies of such large complexes, such as cryo-electron microscopy, do not provide the resolution required for a mechanistic insight. Mass spectrometry (MS) has increasingly been applied to identify the residues that are involved in chemical cross-links in compound protein assemblies, and have provided valuable insight into the molecular arrangement, orientation and contact surfaces of subunits within such large complexes. This approach is known as MS3D, and involves the MS analysis of cross-linked di-peptides following the enzymatic cleavage of a chemically cross-linked complex. A major challenge of this approach is the identification of the cross-linked di-peptides in a composite mixture of peptides, as well as the identification of the residues involved in the cross-link. These analyses require bioinformatics tools with capabilities beyond that of general, MS-based proteomic analysis software. Many MS3D software tools have appeared, often designed for very specific experimental methods. Here, we provide a review of all major MS3D bioinformatics programmes, reviewing their applicability to different workflows, specific experimental requirements and the computational approach taken by each.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Nanoscale Structure of the Cell Wall Protecting Cellulose from Enzyme Attack

The cell wall structure protects cellulose from enzymatic attack and its successive fermentation. The nature of this protection consists in the very complex macroscopic and microscopic structure of cell wall that limits transport. Explaining this kind of protection is critical in future research to improve cell polymer availability for enzymatic attack. This research shows that the complete description of the cell wall topography at a nanoscale level allows a mechanistic understanding of cellulose protection. For this purpose, we used gas adsorption methods (CO(2) at 273 K and N(2) at 77 K) to detect mesoporosity (pore size of 1.5-30 nm diameter; MeS) and microporosity (pore size of 0.3-1.5 nm diameter; MiS) of the cell wall of five energy crops, i.e., giant cane, rivet wheat straw, miscanthus, proso millet, and sorghum. The presence of both hemicelluloses in the spaces between cellulose fibrils and the unhydrolyzable and highly cross-linked lignocarbohydrate complex (LCC) determines a microporous (80% pores having diameters below 0.8 nm) structure of the cell wall that prevents the cellulase enzymes from coming into direct contact with the cellulose, as their sizes exceed the cell wall pore size. On the other hand, the removal of the hemicelluloses and of the LCC complex determines a reduction of the MiS and an increase of the available surface for enzymatic attack, i.e., pores >5 nm diameter. This was confirmed by the good negative (r = -0.87, P < 0.001, n = 11) and positive (r = 0.78, P < 0.005, n = 11) correlations found for microporosity and mesoporosity (pores of diameters >5 nm), respectively, vs the glucose production, by cellulase enzyme attack in specific enzymatic hydrolysis tests performed on biomass samples.

Crystallographic Study of a Site-Specifically Cross-Linked Protein Complex with a Genetically Incorporated Photoreactive Amino Acid,

The benzophenone photophore is widely used to photo-cross-link macromolecules. Recent developments in genetic code expansion have allowed the biosynthesis of proteins with p-benzoyl-L-phenylalanine (pBpa) at defined sites, for covalent bonding with interacting proteins. However, the structure of a photo-cross-linked protein complex had not been revealed, and thus neither the actual structure of the "photobridge" in a complex nor the influence of this covalent bridge on the overall complex structure was known. In this study, we determine the crystal structure of the cross-linked complex of the liver oncoprotein gankyrin and the C-terminal domain of S6 proteasomal protein (S6C), at 2.05 Å resolution. First, the photoreactive amino acid was separately incorporated into gankyrin at 16 sites on the protein surface, and two variants that efficiently formed a covalent bond with S6C were found. The yield of one of the cross-linked products, with pBpa in place of Arg85 in gankyrin, was maximized for crystallization via optimization of the duration of complex exposure to 365 nm light. The structure revealed that the carbonyl group of the benzophenone of pBpa85 formed a covalent bond exclusively with the Cγ atom of Glu356 in S6C, showing the high selectivity of formation of cross-links by pBpa. In addition, the cross-linked structure exhibited little structural distortion from the native complex structure. Our results demonstrated that cross-linking with site-specifically incorporated pBpa preserves the native binding mode and is useful for probing protein-protein interactions.

Assembly of Active High Molecular Weight CD39 Complex: Role of Proteolysis and Endocytosis

AbstractAbstract 2110HUVECs, neutrophils, monocyte/macrophages and T cell subsets all express ectonucleoside triphosphate diphosphohydrolase 1 (CD39) on the cell surface. In addition, they all express both P2X and P2Y receptors and dose dependently respond to ATP. ATP enhances superoxide production in activated neutrophils and supports chemotaxis of macrophages responding to a chemoattractant. ATP also induces apoptosis of anti-inflammatory T regulatory cells and supports the differentiation of pro-inflammatory Th17 cells. Lastly, ADP drives thrombus formation by activation of platelet P2Y12 receptors. An increase in the activity of expressed CD39 would result in increased metabolism of these pro-thrombotic and pro-inflammatory nucleotides.We examined the reported relationship between CD39 cleavage and cell surface enzymatic activity. We cloned N-terminal and C-terminal V5 and VP16 tagged CD39 into eukaryotic expression vectors as well as lentiviral genomic vectors for analysis of CD39 expression in 293 cells and HUVECs. Western blots of membrane fractions prepared from HUVECs and transiently and stably transfected 293 cells identified the previously described CD39 cleavage fragments. Streptavidin precipitation of biotinylated membrane proteins demonstrated that both fragments are present on the cell surface of both transduced HUVECs and transfected 293 cells.We next separated membranes on a discontinuous sucrose gradient to yield ER, Golgi/plasma membrane, early endosome and late endosome enriched membrane fractions. Surprisingly, full length CD39 and the N- and C-terminal fragments appeared in both the Golgi/plasma membrane and early endosome fractions. In addition, the early endosome CD39 exhibited enzyme activity equal to that of the Golgi/plasma membrane CD39. We then prepared membranes from cells treated with either chloroquine or bafilomycin, reagents known to interfere with endosomal acidification and/or maturation. In each case, we observed a decrease in the fractional cleavage of full length CD39 and a proportional decrease in associated enzymatic activity. When sonicated membranes were resolved on a continuous sucrose gradient, the N- and C-terminal fragments and a fraction of full length CD39 as well as maximal enzymatic activity were found in the low density, “raft” fractions. These results suggest that the formation of an enzymatically active CD39 complex requires N- and C-terminal CD39 fragments as well as membrane cholesterol. When 293 cells that stably expressed CD39 were transiently transfected with dominant negative dynamin 2, we observed a decrease in fractional cleavage as well as a proportional decrease in enzymatic activity. This result suggested that the cleavage event occurs following endocytosis of plasma membrane expressed CD39. Finally, we treated both stably transfected 293 cells and HUVECs with the cell permeable cysteine protease inhibitor zLLY.fmk. Prepared membranes analyzed by Western blot showed a decrease in fractional cleavage of full length CD39. Apyrase assays showed a corresponding decrease in ATPase and ADPase activity.We then examined the cholesterol dependence of CD39 activity by depleting membrane cholesterol with MβCD. As expected, ATPase activity decreased in a dose dependent manner. The predominant “active” species appeared as a 1.4 megadalton complex on a 3–12% BN gel of Digitonin solubilized membranes prepared from cultured cells treated with the cleavable cross-linker DTSSP. As a result of membrane cholesterol depletion, there was a proportional decrease in the amount of full length CD39 and N- and C-terminal fragments present in the DTSSP cross-linked HMW complex. Interestingly, there was a marked increase in the abundance of lower molecular weight complexes in cholesterol depleted cells.In conclusion, we provide evidence that CD39 enzymatic activity resides in a megadalton complex formed by protein-protein interactions between full length CD39 and C- and N-terminal fragments generated by cleavage of the full length molecule. Assembly of the oligomeric protein complex requires membrane cholesterol and likely occurs on intracellular membranes. Moreover, approximately 50% of the active enzyme complex remains sequestered on intracellular membranes. These results suggest that up-regulation of CD39 metabolism of pro-thrombotic and pro-inflammatory nucleotides involves pathways independent of gene transcription. Disclosures:No relevant conflicts of interest to declare.

A region within the C‐terminal domain of Ure2p is shown to interact with the molecular chaperone Ssa1p by the use of cross‐linkers and mass spectrometry

The propagation of yeast prion phenotypes is highly dependent on molecular chaperones. We previously demonstrated that the molecular chaperone Ssa1p sequesters Ure2p in high molecular weight, assembly incompetent oligomeric species. We also determined the affinity of Ssa1p for Ure2p, and its globular domain. To map the Ure2p-Ssa1p interface, we have used chemical cross-linkers and MS. We demonstrate that Ure2p and Ssa1p form a 1 : 1 complex. An analytical strategy combining in-gel digestion of cross-linked protein complexes, and both MS and MS/MS analysis of proteolytic peptides, allowed us to identify a number of peptides that were modified because they are exposed to the solvent. A difference in the exposure to the solvent of a single lysine residue, lysine 339 of Ure2p, was detected upon Ure2p-Ssa1p complex formation. These observations strongly suggest that lysine 339 and its flanking amino acid stretches are involved in the interaction between Ure2p and Ssa1p. They also reveal that the Ure2p amino-acid stretch spanning residues 327-339 plays a central role in the assembly into fibrils.

Improved Strategies for Rapid Identification of Chemically Cross-Linked Peptides Using Protein Interaction Reporter Technology

Protein interaction reporter (PIR) technology can enable identification of in vivo protein interactions with the use of specialized chemical cross-linkers, liquid chromatography, and high-resolution mass spectrometry. PIR-cross-linkers contain labile bonds that are specifically fragmented under low energy collision or photodissociation conditions in the mass spectrometer source, thus releasing cross-linked peptides. Successful analysis of PIR-cross-linked proteins requires the use of expected mathematical relationships between cross-linked complexes and released peptides after fragmentation of the labile PIR bonds. Presented here is a next-generation software tool, BLinks, for use in the analysis and identification of PIR-cross-linked proteins. BLinks is an advancement beyond our previous efforts by incorporation of chromatographic profiles that must match between cross-linked complexes and released peptides to enable estimation of p-values to help filter true relationships from complex data sets. Additionally, BLinks was used to incorporate Mascot database searching results from subsequent MS/MS analysis of the released peptides to facilitate identification of cross-linked proteins. BLinks was used in the analysis of human serum albumin, and 46 interpeptide relationships were found spanning 30 proximal residues with a 2.2% false discovery rate. BLinks was also used to track peptides involved in multiple, coeluting relationships that make accurate identification of protein interactions difficult. An additional 10 interpeptide relationships were identified despite poor correlation using the profiling tools provided with BLinks. Additionally, BLinks can be used to globally map all interpeptide relationships from the data analysis and customize subsequent analysis to target specific peptides of interest, thus making it a useful tool for both discovery of protein interactions and mapping protein topology.

Structural model of the p14/SF3b155 · branch duplex complex.

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Electron Transfer of Site-Specifically Cross-Linked Complexes between Ferredoxin and Ferredoxin−NADP+ Reductase

Ferredoxin (Fd) and Fd-NADP(+) reductase (FNR) are redox partners responsible for the conversion between NADP(+) and NADPH in the plastids of photosynthetic organisms. Introduction of specific disulfide bonds between Fd and FNR by engineering cysteines into the two proteins resulted in 13 different Fd-FNR cross-linked complexes displaying a broad range of activity to catalyze the NADPH-dependent cytochrome c reduction. This variability in activity was thought to be mainly due to different levels of intramolecular electron transfer activity between the FNR and Fd domains. Stopped-flow analysis revealed such differences in the rate of electron transfer from the FNR to Fd domains in some of the cross-linked complexes. A group of the cross-linked complexes with high cytochrome c reduction activity comparable to dissociable wild-type Fd/FNR was shown to assume a similar Fd-FNR interaction mode as in the native Fd:FNR complex by analyses of NMR chemical shift perturbation and absorption spectroscopy. However, the intermolecular electron transfer of these cross-linked complexes with two Fd-binding proteins, nitrite reductase and photosystem I, was largely inhibited, most probably due to steric hindrance by the FNR moiety linked near the redox center of the Fd domain. In contrast, another group of the cross-linked complexes with low cytochrome c reduction activity tends to mediate higher intermolecular electron transfer activity. Therefore, reciprocal relationship of intramolecular and intermolecular electron transfer abilities was conferred by the linkage of Fd and FNR, which may explain the physiological significance of the separate forms of Fd and FNR in chloroplasts.

A Solution Model of the Complex Formed by Adrenodoxin and Adrenodoxin Reductase Determined by Paramagnetic NMR Spectroscopy

Lanthanide tags offer the opportunity to retrieve long-range distance information from NMR experiments that can be used to guide protein docking. To determine whether sufficient restraints can be retrieved for proteins with low solubility and availability, Ln tags were applied in the study of the 65 kDa membrane-associated protein complex formed by the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. The reductase is only monomeric at low concentration, and the paramagnetic iron-sulfur cluster of adrenodoxin broadens many of the resonances of nuclei in the interface. Guided by the paramagnetic restraints obtained using two Ln-tag attachment sites, protein docking yields a cluster of solutions with an rmsd of 3.2 A. The mean structure is close to the crystal structure of the cross-linked complex, with an rmsd of 4.0 A. It is concluded that with the application of Ln tags paramagnetic NMR restraints for structure determination can be retrieved even for difficult, low-concentration protein complexes.

Cleavable Cross-Linker for Protein Structure Analysis: Reliable Identification of Cross-Linking Products by Tandem MS

Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting cross-linked peptide mixture presents an alternative approach to high-resolution analysis, such as NMR spectroscopy or X-ray crystallography, to obtain low-resolution protein structures and to gain insight into protein interfaces. Here, we describe a novel urea-based cross-linker, which allows distinguishing different cross-linking products by collision-induced dissociation (CID) tandem MS experiments based on characteristic product ions and constant neutral losses. The novel cross-linker is part of our ongoing efforts in developing collision-induced dissociative reagents that allow an efficient analysis of cross-linked proteins and protein complexes. Our innovative analytical concept is exemplified for the Munc13-1 peptide and the recombinantly expressed ligand binding domain of the peroxisome proliferator-activated receptor alpha, for which cross-linking reaction mixtures were analyzed both by offline nano-HPLC/MALDI-TOF/TOF mass spectrometry and by online nano-HPLC/nano-ESI-LTQ-orbitrap mass spectrometry. The characteristic fragment ion patterns of the novel cross-linker greatly simplify the identification of different cross-linked species, namely, modified peptides as well as intrapeptide and interpeptide cross-links, from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Our novel urea-based CID cleavable cross-linker is expected to be highly advantageous for analyzing protein 3D structures and protein-protein complexes in an automated manner.

Importance of Avidity for an Endogenous Drug Carrier: An Antibody Carrier for CpG Oligonucleotides

In animal models, successful anticancer monotherapy with CpG oligodeoxynucleotide (ODN) has been limited to the intratumoral and peritumoral routes of administration. To overcome this limitation, we developed a delivery system utilizing an endogenous antibody as a carrier for CpG ODNs. When a 1:1 conjugate of 2,4-dinitrophenyl (DNP) to a CpG ODN was administered to tumor-bearing mice that were preimmunized against DNP, intravenous (iv) administration successfully inhibited tumor growth (Palma, E.; Cho, M. J. J. Controlled Release 2007, 120, 95-103). In the present studies, we reproduced the iv results and showed that a DNP derivative of a controlled ODN with scrambled nucleotide sequence failed in the same model. Perhaps more significantly, contralateral subcutaneous (sc) routes of administration also suppressed tumor growth. However, in a separate experiment, when the anti-DNP titer level was low, the antitumor effect was abolished, supporting the importance of the avidity involved in the complexation. With the low titer, a significant fraction of injected dose must have existed as unbound that is subject to rapid clearance. The present study justifies chemically cross-linked immune complexes such that the CpG ODN cannot dissociate in the body after administration.

Does Chemical Cross-Linking with NHS Esters Reflect the Chemical Equilibrium of Protein–Protein Noncovalent Interactions in Solution?

Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a K(D) range of 30 nM to >25 μM, and (2) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a buffer-dependent K(D) value between 100 and 320 μM. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1•STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about K(D) ≈ 25 μM, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak α-thrombin•BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with K(D) >> 25 μM.

Notch and Transforming Growth Factor-β (TGFβ) Signaling Pathways Cooperatively Regulate Vascular Smooth Muscle Cell Differentiation

Notch and transforming growth factor-beta (TGFbeta) play pivotal roles during vascular development and the pathogenesis of vascular disease. The interaction of these two pathways is not fully understood. The present study utilized primary human smooth muscle cells (SMC) to examine molecular cross-talk between TGFbeta1 and Notch signaling on contractile gene expression. Activation of Notch signaling using Notch intracellular domain or Jagged1 ligand induced smooth muscle alpha-actin (SM actin), smooth muscle myosin heavy chain, and calponin1, and the expression of Notch downstream effectors hairy-related transcription factors. Similarly, TGFbeta1 treatment of human aortic smooth muscle cells induced SM actin, calponin1, and smooth muscle protein 22-alpha (SM22alpha) in a dose- and time-dependent manner. Hairy-related transcription factor proteins, which antagonize Notch activity, also suppressed the TGFbeta1-induced increase in SMC markers, suggesting a general mechanism of inhibition. We found that Notch and TGFbeta1 cooperatively activate SMC marker transcripts and protein through parallel signaling axes. Although the intracellular domain of Notch4 interacted with phosphoSmad2/3 in SMC, this interaction was not observed with Notch1 or Notch2. However, we found that CBF1 co-immunoprecipitated with phosphoSmad2/3, suggesting a mechanism to link canonical Notch signaling to phosphoSmad activity. Indeed, the combination of Notch activation and TGFbeta1 treatment led to synergistic activation of a TGFbeta-responsive promoter. This increase corresponded to increased levels of phosphoSmad2/3 interaction at Smad consensus binding sites within the SM actin, calponin1, and SM22alpha promoters. Thus, Notch and TGFbeta coordinately induce a molecular and functional contractile phenotype by co-regulation of Smad activity at SMC promoters.

Visualization of exogenous delivery of nanoformulated butyrylcholinesterase to the central nervous system

Butyrylcholinesterase (BChE) is an efficient bioscavenger of highly toxic organophosphorus poisons and nerve agents. However, BChE administered into the periphery does not provide significant protection of the central nervous system (CNS) due to rejection by the blood–brain barrier. In this study, we evaluated the feasibility of delivering BChE to the CNS by packing it into a block ionomer complex of nanoscale size with a cationic poly( l-lysine)- graft-poly(ethylene oxide) (PLL- g-PEO) copolymer. The multimolecular structure of BChE/PLL- g-PEO complexes was further reinforced by formation of cross-links between the polymer chains. The resulting cross-linked complexes were stable against dilution without significant loss of BChE enzymatic activity. In some cases the BChE was labeled with fluorescent IRDye 800CW before it was incorporated into nanoparticles. BChE/PLL- g-PEO complexes were injected into mice intramuscularly and intravenously. In vivo imaging showed incorporation of the fluorescently labeled BChE in brain. Activity assays showed that BChE remained active in the brain at 72-h post-injection. It was concluded that nanocomplexes can deliver the 340 kDa BChE tetramer to the brain.

Use of a Combination of Isotopically Coded Cross-Linkers and Isotopically Coded N-Terminal Modification Reagents for Selective Identification of Inter-peptide Crosslinks

Cross-linking combined with mass spectrometry has great potential for determining three-dimensional structures of proteins and protein assemblies. One of the main analytical challenges of this method is the specific detection and identification of the inter-peptide crosslinks in the peptide mixture after enzymatic digestion of the cross-linked protein complex. These inter-peptide crosslinks are important because they provide the critical distance information needed for structural proteomics studies. In this paper, we demonstrate the use of isotopically coded N-terminal modification (ICNTM) in combination with isotopically coded cross-linkers (ICCL) for specific detection of inter-peptide crosslinks. Inter-peptide crosslinks contain two amino termini, compared to one in the case of free peptides, dead-end crosslinks, or intra-peptide crosslinks. Therefore, N-terminal modification with a 1:1 mixture of heavy and light isotopically coded reagents produces inter-peptide crosslinks with a distinct isotopic signature (a 1:2:1 ratio). Modification also occurs at the epsilon-amino groups of non-cross-linked lysine residues, resulting in two modifications per free lysine-containing peptide. However, if ICCL and ICNTM are used together, inter-peptide crosslinks can be distinguished from free lysine-containing peptides. Specialized software has also been developed for the analysis of ICCL + ICNTM experimental data. This procedure, combined with software for data analysis, provides a simple and rapid method for specific detection of inter-peptide crosslinks.