Protein Engineering Design and Selection - current issue

The V119I polymorphism in protein L-isoaspartate O-methyltransferase alters the substrate-binding interface

Rutherford, K., Daggett, V. — Fri, 13 Nov 2009 10:03:13 PST

Protein L-isoaspartate O-methyltransferase (PIMT) repairs isoaspartate residues in damaged proteins, and it contains a Val–Ile polymorphismin in 5, ~13 Å from its active site. Val119 has lower activity and thermal stability but increased affinity for endogenous substrates. Studies suggest that heterozygosity for Val/Ile favors efficient isoaspartate repair. We have performed multiple molecular dynamics simulations of 119I and 119V PIMT. Both V119 and I119 interact with the same residues throughout all of the simulations. However, the larger Ile altered the orientations of 5 and β5, both of which have co-substrate binding residues on their distal ends. I119 increases the flexibility of several residues, loosening up the S-adenosylmethionine (SAM)-binding site. These subtle changes are propagated towards the isoaspartate-docking site via residues common to both active sites. The increased mobility in 119I PIMT reorients 3, resulting in a salt-bridge network at the substrate-binding interface that disrupts several key side-chain interactions in the isoaspartate site. In contrast, 119V PIMT remains quite rigid with little change to the co-substrate binding site, which could hinder SAM's binding and release, accounting for the decreased activity. These results shed light on the molecular basis behind the decreased activity and increased specificity for endogenous substrates of 119V PIMT relative to the 119I variant. 119I PIMT catalyzes the methylation reaction but may have difficulties recognizing and orienting specific substrates due to its distorted substrate-binding site. Heterozygosity for both the Ile and Val alleles may provide the best of both worlds, allowing the fast and specific methylation of damaged proteins.

Engineering and characterization of a baculovirus-expressed mouse/human chimeric antibody against transferrin receptor

Fri, 13 Nov 2009 10:03:13 PST

Transferrin receptor (TfR) has been explored as a target for antibody-based therapy of cancer. In the previous study, we reported a murine anti-TfR monoclonal antibody (mAb) 7579 had good anti-tumor activities in vitro. In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effector mechanism in vivo, we herein developed its chimera in the baculovirus/insect cell expression system based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The chimeric light and heavy chains, containing human IgG1 constant regions, were correctly processed and assembled in insect cells, and then secreted into the mediums as heterodimeric H₂L₂ immunoglobulins. Furthermore, analyses of antigen-binding assay and competitive binding assay indicated that the chimeric antibody possessed specificity and affinity similar to that of its parental murine antibody. Results of the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assay verified that the chimeric antibody could efficiently mediate ADCC and CDC against TfR-overexpressing tumor cells. These results suggested that this baculovirus-expressed chimeric anti-TfR IgG1 might have the potential to be used for cancer immunotherapy. Meanwhile, the MAGIC strategy, facilitating the rapid generation of chimeric mAbs, could be one of the efficient strategies for antibody engineering.

Engineering of a novel hybrid enzyme: an anti-inflammatory drug target with triple catalytic activities directly converting arachidonic acid into the inflammatory prostaglandin E2

Ruan, K.-H., Cervantes, V., So, S.-P. — Fri, 13 Nov 2009 10:03:13 PST

Cyclooxygenase isoform-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) are inducible enzymes that become up-regulated in inflammation and some cancers. It has been demonstrated that their coupling reaction of converting arachidonic acid (AA) into prostaglandin (PG) E₂ (PGE₂) is responsible for inflammation and cancers. Understanding their coupling reactions at the molecular and cellular levels is a key step toward uncovering the pathological processes in inflammation. In this paper, we describe a structure-based enzyme engineering which produced a novel hybrid enzyme that mimics the coupling reactions of the inducible COX-2 and mPGES-1 in the native ER membrane. Based on the hypothesized membrane topologies and structures, the C-terminus of COX-2 was linked to the N-terminus of mPGES-1 through a transmembrane linker to form a hybrid enzyme, COX-2-10aa-mPGES-1. The engineered hybrid enzyme expressed in HEK293 cells exhibited strong triple-catalytic functions in the continuous conversion of AA into PGG₂ (catalytic-step 1), PGH₂ (catalytic-step 2) and PGE₂ (catalytic-step 3), a pro-inflammatory mediator. In addition, the hybrid enzyme was also able to directly convert dihomo-gamma-linolenic acid (DGLA) into PGG₁, PGH₁ and then PGE₁ (an anti-inflammatory mediator). The hybrid enzyme retained similar K_d and V_max values to that of the parent enzymes, suggesting that the configuration between COX-2 and mPGES-1 (through the transmembrane domain) could mimic the native conformation and membrane topologies of COX-2 and mPGES-1 in the cells. The results indicated that the quick coupling reaction between the native COX-2 and mPGES-1 (in converting AA into PGE₂) occurred in a way so that both enzymes are localized near each other in a face-to-face orientation, where the COX-2 C-terminus faces the mPGES-1 N-terminus in the ER membrane. The COX-2-10aa-mPGES-1 hybrid enzyme engineering may be a novel approach in creating inflammation cell and animal models, which are particularly valuable targets for the next generation of NSAID screening.

Shear flow promotes amyloid-{beta} fibrilization

Dunstan, D. E., Hamilton-Brown, P., Asimakis, P., Ducker, W., Bertolini, J. — Fri, 13 Nov 2009 10:03:13 PST

The rate of formation of amyloid fibrils in an aqueous solution of amyloid-β (Aβ) is greatly increased when the solution is sheared. When Aβ solution is stirred with a magnetic stirrer bar at 37°C, a rapid increase in thioflavin T fluorescence is observed. Atomic Force Microscopy (AFM) images show the formation of aggregates, the growth of fibrils and the intertwining of the fibrils with time. Circular dichroism (CD) spectroscopy of samples taken after stirring shows a transition from random coil to -helix to β-sheet secondary structure over 20 h at 37°C. The fluorescence, AFM and CD measurements are all consistent with the formation of amyloid fibrils. Quiescent, non-stirred solutions incubated at 37°C showed no evidence of amyloid formation over a period of 3 days. Couette flow was found to accelerate the formation of amyloid fibrils demonstrating that the primary effect of stirring is not mixing but shearing. Only very small shear forces are applied to individual molecules in our experiments. Simple calculation suggests that the force is too small to support a hypothesis that shearing promotes partial unfolding of the protein as is observed.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis

Fri, 13 Nov 2009 10:03:13 PST

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca²⁺-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

Novel regulation of HIV-1 replication and pathogenicity: Rev inhibition of integration

Fri, 13 Nov 2009 10:03:13 PST

Following fusion of the human immunodeficiency virus type-1 (HIV-1) with host cells' membrane and reverse transcription of the viral RNA, the resulted cDNA is integrated into the host genome by the viral integrase enzyme (IN). Quantitative estimations have revealed that only 1–2 copies are integrated per infected cell, although many copies of the viral RNA are reverse-transcribed. The molecular mechanism that restricts the integration degree has not, so far, been elucidated. Following integration, expressed partially spliced and unspliced transcripts are exported from the nuclei by the viral Rev protein. Here, we show that in virally infected cells, the Rev interacts with the IN forming a Rev–IN complex and consequently limits the number of integration events. Disruption of the Rev–IN complex by selected IN-derived peptides or infection by a Rev-deficient virus stimulate integration resulting in large numbers of integration event/cell. Conversely, infection of Rev-expression cells blocks integration and inhibits virus production. Increased integration appears to correlate with increased cell death of infected cultures. Our results thus demonstrate a new regulatory function of Rev and probably establish a link between Rev restriction of HIV-1 integration and protection of HIV-1-infected cells from premature cell death.