Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties.

Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9_29-H6 and G3-H6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H6 in comparison to 9_29-H6. Technetium-99m labeled G3-H6 demonstrated a better biodistribution profile than 9_29-H6, with several-fold lower uptake in liver. Radioiodinated G3-H6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H6 with high clinical potential for imaging of HER2.

Evolution of spectrin function in cytoskeletal and membrane networks.

Spectrin is a cytoskeletal protein thought to have descended from an alpha-actinin-like ancestor. It emerged during evolution of animals to promote integration of cells into tissues by assembling signalling and cell adhesion complexes, by enhancing the mechanical stability of membranes and by promoting assembly of specialized membrane domains. Spectrin functions as an (alphabeta([H]))(2) tetramer that cross-links transmembrane proteins, membrane lipids and the actin cytoskeleton, either directly or via adaptor proteins such as ankyrin and 4.1. In the present paper, I review recent findings on the origins and adaptations in this system. (i) The genome of the choanoflagellate Monosiga brevicollis encodes alpha-, beta- and beta(Heavy)-spectrin, indicating that spectrins evolved in the immediate unicellular precursors of animals. (ii) Ankyrin and 4.1 are not encoded in that genome, indicating that spectrin gained function during subsequent animal evolution. (iii) Protein 4.1 gained a spectrin-binding activity in the evolution of vertebrates. (iv) Interaction of chicken or mammal beta-spectrin with PtdInsP(2) can be regulated by differential mRNA splicing, which can eliminate the PH (pleckstrin homology) domain in betaI- or betaII-spectrins; in the case of mammalian betaII-spectrin, the alternative C-terminal region encodes a phosphorylation site that regulates interaction with alpha-spectrin. (v) In mammalian evolution, the single pre-existing alpha-spectrin gene was duplicated, and one of the resulting pair (alphaI) neo-functionalized for rapid make-and-break of tetramers. I hypothesize that the elasticity of mammalian non-nucleated erythrocytes depends on the dynamic rearrangement of spectrin dimers/tetramers under the shearing forces experienced in circulation.

Molecular evolution of the ankyrin gene family.

Ankyrins are membrane adaptor molecules that play important roles in coupling integral membrane proteins to the spectrin-based cytoskeleton network. Human mutations of ankyrin genes lead to severe genetic diseases such as fatal cardiac arrhythmias and hereditary spherocytosis. To elucidate the evolutionary history of ankyrins, we have identified novel ankyrin sequences in insect, fish, frog, chicken, dog, and chimpanzee genomes and explored the phylogenetic relationships of the ankyrin gene family. Our data demonstrate that duplication of ankyrin genes occurred at two different stages. The first duplication resulted from an independent evolution event specific in Arthropoda after its divergence from Chordata. Following the separation from Urochordata, expansion of ankyrins in vertebrates involved ancestral genome duplications. We did not find evidence of coordinated arrangements of gene families of ankyrin-associated membrane proteins on paralogous chromosomes. In addition, evolution of the 24 ANK-repeats strikingly correlated with the exon boundary sites of ankyrin genes, which might have occurred before its duplication in vertebrates. Such correlation is speculated to bring functional diversity and complexity. Moreover, based on the phylogenetic analysis of the ANK-repeat domain, we put forward a novel model for the putative primordial ankyrin that contains the fourth six-ANK-repeat subdomain and the spectrin-binding domain. These findings will provide guides for future studies concerning structure, function, evolutionary origins of ankyrins, and possibly other cytoskeletal proteins.

The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.