Helical Cytokines and Invertebrate Immunity: A New Field of Research

To the Editor Due to their sequence divergence, evolutionary studies on helical cytokines cannot be performed using conventional algorithms based on sequence similarity [1]. Recently, our laboratory has successfully used the Quasi Threading (QT) method, a new structure prediction algorithm developed to find helical cytokines in human databases [1, 2], on Drosophila databases. Helical cytokines represent one structural class of mammalian cytokines and include several interleukins (ILs), such as IL-2 and IL-6. These molecules play key roles in varied physiological processes, including host defense and immune regulation [3]. In mammals, helical cytokines are a divergent protein family, and their phylogenic relationship arises from the conserved protein structure, intron phases and broadly similar receptor families [4]. Due to the differences between their primary sequences, conventional gene searching methods cannot be used to look for new helical cytokines, even in the human genome. Conklin developed the QT method specifically to identify the helical cytokine fold [1, 4], and it has been used to discover new helical cytokines in humans [1, 5]. Helical proteins are extremely diffused among eukaryotes and they include also helical membrane and coiled-coil region-containing proteins. It has been calculated that in Drosophila melanogaster less than 18% of all open reading frames encode helical membrane proteins, and that approximately 8–11% of eukaryote proteins contain the coiled-coil region [6]. The QT method is very effective in its predictions as it discriminates among all the helical proteins and specifically recognizes the helical cytokine fold among the plethora of folds predicted to exist [6]. Accordingly, after searching a database of 7570 Drosophila protein sequences, only three promising candidates were observed [2]. Further analyses gave us the first evidence of the involvement of a putative helical cytokine, the Drosophila helical factor (DHF), in innate immunity in D. melanogaster (Fig. 1) [2]. The presence of cytokines concerned with both immunity and development (e.g. Spätzle, Unpaired [Upd]-1 and Upd-3) has been demonstrated in D. melanogaster [7–10], but neither in terms of the amino acidic primary sequence nor of the three-dimensional structure of the peptides is there any evidence of a close structural relationship between Drosophila and mammalian helical cytokines. The existence of cytokines apparently unrelated to those of vertebrates would argue against a common evolutionary origin of the vertebrate and protostomian invertebrate cytokine network, leaving virtually unexplained a plethora of functional evidences clearly showing the capability of mammalian cytokines to influence invertebrate immunity [11]. In this context, Beschin et al. [12] surmized that the functional similarities between invertebrate cytokine-like molecules and vertebrate cytokines is not due to a common origin, but is rather the consequence of a functional convergence. Although presenting a nucleotide ⁄ amino acidic sequence completely unrelated to that of vertebrate helical cytokines, DHF is unique in its structural similarity to mammalian cytokines, a feature established by the QT method at its highest stringency settings [2]. Moreover, initial functional evidence relates DHF with immunity in D. melanogaster third instar larvae [2], even if crystallization is required to definitely conclude DHF is a helical cytokine. Our findings suggest an exciting new focus to the field of comparative immunobiology, as they indicate that the structure of the molecule, and not its nucleotide ⁄ amino acidic sequence, would be the ‘invariant’ component that has been conserved during evolution in order to maintain the function. Confirmation of this idea through the identification of other invertebrate helical cytokines would introduce a new way to look at immune-related molecules in A