Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Motogenic Sites in Human Fibronectin Are Masked by Long Range Interactions

Fibronectin (FN) is a large extracellular matrix glycoprotein important for development and wound healing in vertebrates. Recent work has focused on the ability of FN fragments and embryonic or tumorigenic splicing variants to stimulate fibroblast migration into collagen gels. This activity has been localized to specific sites and is not exhibited by full-length FN. Here we show that an N-terminal FN fragment, spanning the migration stimulation sites and including the first three type III FN domains, also lacks this activity. A screen for interdomain interactions by solution-state NMR spectroscopy revealed specific contacts between the Fn N terminus and two of the type III domains. A single amino acid substitution, R222A, disrupts the strongest interaction, between domains ^4–5FnI and ³FnIII, and restores motogenic activity to the FN N-terminal fragment. Anastellin, which promotes fibril formation, destabilizes ³FnIII and disrupts the observed ^4–5FnI-³FnIII interaction. We discuss these findings in the context of the control of cellular activity through exposure of masked sites.Fibronectin (FN),4 a large multidomain glycoprotein found in all vertebrates, plays a vital role in cell adhesion, tissue development, and wound healing (1). It exists in soluble form in plasma and tissue fluids but is also present in fibrillar networks as part of the extracellular matrix. The structures of many FN domains of all three types, FnI, FnII, and FnIII, are known, for example (2–4). Although interactions between domains that are close in primary sequence have been demonstrated (3, 5), studies of multidomain fragments generally assume a beads-on-string model (2). There is, however, much evidence for the presence of long range order in soluble FN as a number of functional sites, termed cryptic, are not active in the native molecule, until exposed through conformational change. These include self-association sites (5–8), sites of protein interactions (9), and sites that control cellular activity (10, 11). Low resolution studies of the FN dimer suggest a compact conformation under physiological conditions (12–14); however, attempts to define large scale structure in FN by small angle scattering or electric birefringence (15–17) have yielded contradictory results. Interpretation of domain stability changes in terms of interaction sites (18) has also not been straightforward (2), possibly because of domain stabilization through nearest-neighbor effects (19, 20).A FN splicing variant produced in fetal and cancer patient fibroblasts, termed migration stimulation factor (MSF), stimulates migration of adult skin fibroblasts into type I collagen gels (10, 21) and breast carcinoma cells using the Boyden chamber (22). MSF comprises FN domains ¹FnI to ⁹FnI, a truncated ¹FnIII, and a small C-terminal extension; a recombinant FN fragment corresponding to ¹FnI-⁹FnI (Fn70kDa) displays the same activity (10). An overview of FN domain structure and nomenclature is presented in Fig. 1a. Further experiments sub-localized full motogenic activity to the gelatin binding domain of FN (GBD, domains ⁶FnI-⁹FnI) (23) and partial activity to a shorter fragment spanning domains ^7–9FnI (24). Two IGD tripeptides of domains ⁷FnI and ⁹FnI were shown to be essential through residue substitutions and reconstitution of partial motogenic activity in synthetic peptides (10, 24, 25); however, similar IGD tripeptides outside the GBD, on domains ³FnI and ⁵FnI, appear to have little effect (10, 23). Full-length adult FN does not affect cell migration in similar assays (10, 23); thus motogenic activity sites are presumed to be masked in the conformation adopted by soluble FN, although they could be exposed by molecular rearrangement.Open in a separate windowFIGURE 1.Motogenic activity of FN fragments. a, schematic representation of the FN domain structure (top) and enlargement of the FN N terminus (bottom). Type I domains are shown as pentagons; type II domains as hexagons; and type III domains as ovals. b, comparison of motogenic activity versus protein concentration of wild-type Fn70kDa and Fn100kDa fragments. Error bars are derived from duplicate experiments, and a gray band denotes migration activity of media without additives. c, similar comparisons for mutant Fn100kDa fragments. d, analytical size exclusion chromatography of large FN fragments. The trace of UV absorbance at 280 nm versus elution volume shown here indicates a larger hydrodynamic radius for Fn100kDa R222A compared with the wild type, consistent with our model (Fig. 6a).Here we show that a recombinant fragment, closely matching a truncated form of FN identified in zebrafish (26), as well as amphibians, birds, and mammals (27), does not stimulate cell migration. This fragment is similar to MSF but includes the first three FnIII domains (^1–3FnIII), suggesting that these domains are responsible for a conformational transition that masks the activity sites in this construct and probably in full-length FN. To identify the mechanism behind this transition, we performed structural studies by solution NMR spectroscopy and identified a specific long range interaction between domains ^4–5FnI and ³FnIII as essential for this masking effect. Interestingly, this interaction does not involve direct contacts with the GBD but possibly represses motogenic activity through chain compaction, evident in analytical size exclusion assays. Intramolecular interactions thus provide a mechanism by which conformational rearrangement induced, for example, by tension or splicing variation can result in cellular activity differences.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]