Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.     

Modeling protein loops using a phi i + 1, psi i dimer database.

We present an automated method for modeling backbones of protein loops. The method samples a database of phi i + 1 and psi i angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles phi i + 1 and psi i completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed phi i + 1, psi i pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N-terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of phi i + 1, psi i for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three-dimensional structures. Typically, 10,000-50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50-80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near-native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near-native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop-closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near-native loop conformations.     

Russian Article pp. 151–158

Carrier facilitated transport of oxygen by hemoglobin in blood helps biotechnologists to create aeration systems using the phenomenon of labile bonds of oxygen for the intensification of the fermentation processes. This paper discusses the mechanism and the kinetics of oxygen mass transfer in the presence of carriers. For the modelling of the oxygen transport the corresponding system of differential equations of oxygen balance is used. The results presented here show the utility of carriers for the intensification of the production of SCP, enzymes and antibiotics.     

Cloning of cDNAs coding for the Gαil and Gαi2 G-proteins from chick brain

We have cloned and characterized several cDNAs coding for G-protein inhibitory α subunits (Gai) from a chick brain cDNA library. Based on homology to Ga subunits from other eukaryotes, these clones were designated chick Gαil and Gαi2. On the deduced amino-acid level, Gαi1 and Gαi2 were found to be 98 and 95% identical to rat Goal and Gαi2, respectively. Using RNase protection analysis, the Gαi1 and Gαi2 mRNAs were found to be expressed in chick atria, ventricle, lung, liver, brain and kidney.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

A study of 2-component i,i + 3 peptide stapling using thioethers

Peptides are promising scaffolds for use as therapeutics, targeting interactions previously considered to be “undruggable” by small molecules. While short peptides are generally unstructured in solution and rapidly degraded by proteases in the cell cytosol, peptide stapling offers an effective method to both stabilize peptides in a helical structure and increase resistance to proteolytic degradation. Most studies of peptide stapling have focused on residues with i, i?+?4 and i, i?+?7 spacing, while stapling of residues with i, i?+?3 spacing has been understudied. Herein, we evaluated a suite of bifunctional linkers for stapling between residues with i, i?+?3 spacing, comparing the ability of each compound to react with the peptide and the degree of helicity conferred. Finally, we evaluated the ability of the stapling to increase proteolytic resistance in cell lysates, comparing stapling of i, i?+?3 and i, i?+?4 spacing, with i, i?+?3 spacing resulting in a greater increase in peptide half-life in the model system. This presents an effective stapling strategy, adding to the peptide stapling toolbox.     

The 1,3‐diyne linker as a rigid “i,i+7” staple for α‐helix stabilization: Stereochemistry at work

Short alphahelical peptide sequences were stabilized through Glaser‐Hay couplings of propargylated l ‐ and/or d ‐serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d ‐Ser (i), l ‐Ser (i+7) combination was applied. In case two l ‐Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.     

Origin of the different strengths of the (i,i+4) and (i,i+3) leucine pair interactions in helices

Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different chi(1) leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.     

Synthesis, and carbon-13 n.m.r. study, of O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose and O-α- -rhamnopyranosyl-(1→3)-O-α- -rhamnopyranosyl (1→3)- -rhamnopyranose, constituents of bacterial, cell-wall polysaccharides

O-α- -Rhamnopyranosyl-(1→3)- -rhamnopyranose (19) and O-α- -rhamnopyranosyl-(1→2)- -rhamnopyranose were obtained by reaction of benzyl 2,4- (7) and 3,4-di-O-benzyl-α- -rhamnopyranoside (8) with 2,3,4-tri-O-acetyl-α- -rhamnopyranosyl bromide, followed by deprotection. The per-O-acetyl α-bromide (18) of 19 yielded, by reaction with 8 and 7, the protected derivatives of the title trisaccharides (25 and 23, respectively), from which 25 and 23 were obtained by Zemplén deacetylation and catalytic hydrogenolysis, With benzyl 2,3,4-tri-O-benzyl-β- -galactopyranoside, compound 18 gave an ≈3:2 mixture of benzyl 2,3,4-tri-O-benzyl-6-O-[2,4-di-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-α- -rhamnopyranosyl]-β- -galactopyranoside and 4-O-acetyl-3-O-(2,3,4-tri-O-acetyl-α- -rhamnopyranosyl)-β- -rhamnopyranose 1,2-(1,2,3,4-tetra-O-benzyl-β- -galactopyranose-6-yl (orthoacetate). The downfield shift at the α-carbon atom induced by α- -rhamnopyranosylation at HO-2 or -3 of a free α- -rhamnopyranose is 7.4-8.2 p.p.m., ≈1 p.p.m. higher than when the (reducing-end) rhamnose residue is benzyl-protected (6.6-6.9 p.p.m.). α- -Rhamnopyranosylation of HO-6 of gb- -galactopyranose deshields the C-6 atom by 5.7 p.p.m. The 1 2-orthoester ring structure [O₂,C(me)OR] gives characteristic resonances at 24.5 ±0.2 p.p.m. for the methyl, and at 124.0 ±0.5 p.p.m. for the quaternary, carbon atom.