Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.     

Genetic organization of the polymorphic equine alpha globin locus and sequence of the BII alpha 1 gene.

The equine alpha globin gene complex comprises two functional alpha genes and an alpha-like pseudogene arranged in the order 5'-alpha 2-(5kb)-alpha 1-(3kb)-psi alpha-3'. A single (embryonic) zeta-like sequence lies within a 12 kb region 5' to the alpha 2 gene. We have determined the sequence of the alpha 1 gene of the BII haplotype, one of two most common haplotypes (the other being BI) which encode alpha globins with either Tyr (BI) or Phe (BII) at codon 24 in both linked alpha genes. In BI and BII the non-allelic alpha 2 and alpha 1 genes respectively code for Gln or Lys at codon 60, thus accounting for the 4 alpha globin types seen in BI/BII heterozygotes. Genomic restriction enzyme maps of the BII alpha complex (24Phe/60Lys,Gln) and the allelic BI (24Tyr/60Lys,Gln) are identical to each other, and to those of a rarer normal haplotype, A, which encodes only alpha 24Tyr/60Gln globin, and a low expression mutant of BII which encodes only 24Phe/60Lys globin. These two latter haplotypes must therefore have a linked pair of alpha genes, as in BI and BII, but with identical coding properties, and it is suggested that this has arisen by gene conversion.     

Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.     

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.     

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.     

Homology model of human interferon-alpha 8 and its receptor complex.

Human interferon-alpha 8 (HuIFN alpha 8), a type I interferon (IFN), is a cytokine belonging to the hematopoietic super-family that includes human growth hormone (HGH). Recent data identified two human type I IFN receptor components. One component (p40) was purified from human urine by its ability to bind to immobilized type I IFN. A second receptor component (IFNAR), consisting of two cytokine receptor-like domains (D200 and D200'), was identified by expression cloning. Murine cells transfected with a gene encoding this protein were able to produce an antiviral response to human IFN alpha 8. Both of these receptor proteins have been identified as members of the immunoglobulin superfamily of which HGH receptor is a member. The cytokine receptor-like structural motifs present in p40 and IFNAR were modeled based on the HGH receptor X-ray structure. Models of the complexes of HuIFN alpha 8 with the receptor subunits were built by superpositioning the conserved C alpha backbone of the HuIFN alpha 8 and receptor subunit models with HGH and its receptor complex. The HuIFN alpha 8 model was constructed from the C alpha coordinates of murine interferon-beta crystal structure. Electrostatic potentials and hydrophobic interactions appear to favor the model of HuIFN alpha 8 interacting with p40 at site 1 and the D200' domain of IFNAR at site 2 because there are regions of complementary electrostatic potential and hydrophobic interactions at both of the proposed binding interfaces. Some of the predicted receptor binding residues within HuIFN alpha 8 correspond to functionally important residues determined previously for human IFN alpha 1, IFN alpha 2, and IFN alpha 4 subtypes by site-directed mutagenesis studies. The models predict regions of interaction between HuIFN alpha 8 and each of the receptor proteins, and provide insights into interactions between other type I IFNs (IFN-alpha subtypes and IFN-beta) and their respective receptor components.     

Partially glycosylated dendrimers block MD-2 and prevent TLR4-MD-2-LPS complex mediated cytokine responses

The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2's hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4'phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamine's carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2's hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.     

Monoclonal antibody specific for chicken DNA polymerase alpha associated with DNA primase

Four monoclonal antibodies against chicken DNA polymerase alpha were obtained from mouse hybridomas (see ref. 1). Two of them, 4-2D and 4-8H, recognized different epitopes of the DNA polymerase alpha-DNA primase complex as determined by a competitive enzyme-linked immunosorbent assay. Antibody 4-8H partially (about 30%) neutralized the combined activity of primase-DNA polymerase alpha as well as the DNA polymerase alpha activity. In contrast, antibody 4-2D did not neutralize DNA polymerase alpha activity, but neutralized the primase-DNA polymerase alpha activity extensively (up to 80%). Furthermore, although an immunoaffinity column made with 4-8H antibody retained virtually all of the DNA polymerase alpha with and without associated primase, a column made with 4-2D antibody did not bind DNA polymerase alpha without the primase, but retained the enzyme associated with the primase. These results indicate that 4-8H monoclonal antibody is specific for DNA polymerase alpha and 4-2D monoclonal antibody is specific for the primase or a special structure present in the primase-DNA polymerase alpha complex.     

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.     

Preparation of integrin alpha(v)beta3-targeting Ab 38C2 constructs

This protocol describes the preparation of Ab constructs using agents that target cells expressing integrins alpha(v)beta3 and alpha(v)beta5, and the monoclonal aldolase Ab 38C2. The targeting agents are equipped with a diketone or vinylketone linker, and selectively react through the reactive Lys residues in the Ab binding sites to form 38C2 conjugates or chemically programmed 38C2 (i.e., cp38C2). The targeting agent possessing a diketone linker reacts with the Lys residues forming an enaminone derivative. By contrast, the vinylketone linker is used as the corresponding acetone adduct (i.e., a pro-vinylketone linker), and this pro-adapter undergoes a 38C2-catalyzed retro-aldol reaction to produce the vinylketone linker, which forms a Michael-type adduct with the Lys residues. The Ab construct formation is achieved in <1 h for the diketone compounds at ambient temperature, and in 2-16 h using the pro-vinylketone linker at 37 degrees C. The 38C2 constructs are retargeted to cells over-expressing integrins, and are potential candidates for immunotherapy.     

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.     

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.     

Correlation of secondary structures of bradykinin B1 receptor antagonists with their activity

The secondary structure of a bradykinin B(1)receptor antagonist B-10324 (F5C-Lys-(1)- Lys(0)-Arg(1)-Pro(2)- Hyp(3)-Gly(4)-CpG(5)- Ser(6)-DTic(7)-CpG(8)) was determined by NMR at 800MHz. The conformational data are compared with those obtained previously for two bradykinin B(1) receptor antagonists, namely B-9858 (Lys-(1)- Lys(0)-Arg(1)-Pro(2)- Hyp(3)-Gly(4)-Igl(5)- Ser(6)-DIgl(7)-Oic(8)) and B-10148 (Lys-(1)-Lys(0)-Arg(1)- Pro(2)-Hyp(3)-Gly(4)- Igl(5)-Ser(6)-DF5F(7)- Oic(8)). The abnormal amino acids are: Hyp, trans-4- hydroxyproline; Tic, 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid; Oic, (2S, 3aS, 7aS)-octahydroindole-2-carboxylic acid; Igl, alpha(2- indanyl)glycine; F5F, 2,3,4,5,6-pentafluorophenylalanine; CpG, alpha- cyclopentylglycine. F5C, pentafluorocinnamoyl, is the N-terminal protecting group and is not involved in the peptide secondary structure. B-10324 contains an N-terminal Pro(2)- CpG(5) distorted type II beta-turn whereas the rest of the peptide is random. A salt bridge is not observed between the carboxylate group at the C-terminal end and the Arg(1) side chain, in contrast to that previously observed for B-9858 and B- 10148. The conformations are correlated with the measured B(1) receptor antagonist activities (J.-F. Larrivée, L. Gera, S. Houle, J. Bouthillier, D. R. Bachvarov, J. M. Stewart and F. Marc au, Br. J. Pharmacol. 131, 885-892 (2000)). The importance of the N-terminal beta-turn is highlighted.