Hexameric Storage Research Articles

The Design of SCI‐57: An Ultrastable, Rapid‐acting, Single‐chain Insulin Analog

There are approximately 415 million people with Diabetes Mellitus worldwide and there exists a growing need for an ultrastable, heat resistant formulation of rapid‐acting insulin to treat the millions of people who live in developing countries where temperatures routinely exceed 45ºC. The heat resistance and stability of insulin is especially crucial due to the fact that insulin degrades ten times faster for every 10°C increase in temperature above 25°C. Several rapid‐acting insulin analogs have been created, though most do not last longer than 30 days and are reliant on the expensive cold chain delivery system of insulin analogs. This is due to chemical and physical degradation by way of deamidation, transamidation and cross‐beta assembly of linear polymers, or fibrillation. Rapid‐acting analogs must also combat the formation of the inactive hexameric formulation of insulin due to the presence of zinc and an excess of hydrogen ions. A single‐chain insulin analog has been created called SCI‐57 which consists of a glycine rich peptide (6 residues in length) that connects the A and B chains of the insulin analog. Adding a peptide tether will restrict splaying of the A and B chains, thus decreasing the risk of fibrillation even under high temperatures. To ensure that the analog is rapid‐acting and will not form storage hexamers, SCI‐57 has replaced ThrA8 with His to omit an unfavorable beta‐branched amino acid and HisB10 with Asp to create a more favorable relationship with the electrostatic dipole of the B‐chain alpha helix. Replacing HisB10 with Asp also blocks zinc binding, furthermore ensuring that the analog in its active, monomeric state. Both of these actions triple the activity of two‐chain analogs which will also benefit SCI‐57 by allowing it to bind quickly to the insulin receptor. Furthermore, SCI‐57 is much more resistant to fibrillation under high temperatures than previous analogs and maintains insulin receptor affinity as well as efficiency. The Nueva School MSOE Center for Biomolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of SCI‐57.Primary Citation:Hua, Qing‐xin, et al. “Design of an Active Ultrastable Single‐Chain Insulin Analog: SYNTHESIS, STRUCTURE, AND THERAPEUTIC IMPLICATIONS.” The Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 23 May 2008This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decoding the Cryptic Active Conformation of a Protein by Synthetic Photoscanning: INSULIN INSERTS A DETACHABLE ARM BETWEEN RECEPTOR DOMAINS

Proteins evolve in a fitness landscape encompassing a complex network of biological constraints. Because of the interrelation of folding, function, and regulation, the ground-state structure of a protein may be inactive. A model is provided by insulin, a vertebrate hormone central to the control of metabolism. Whereas native assembly mediates storage within pancreatic beta-cells, the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin were probed by photocross-linking of an extensive set of azido derivatives constructed by chemical synthesis. Contacts are circumferential, suggesting that insulin is encaged within its receptor. Mapping of photoproducts to the hormone-binding domains of the insulin receptor demonstrated alternating contacts by the B-chain beta-strand (residues B24-B28). Whereas even-numbered probes (at positions B24 and B26) contact the N-terminal L1 domain of the alpha-subunit, odd-numbered probes (at positions B25 and B27) contact its C-terminal insert domain. This alternation corresponds to the canonical structure of abeta-strand (wherein successive residues project in opposite directions) and so suggests that the B-chain inserts between receptor domains. Detachment of a receptor-binding arm enables photo engagement of surfaces otherwise hidden in the free hormone. The arm and associated surfaces contain sites also required for nascent folding and self-assembly of storage hexamers. The marked compression of structural information within a short polypeptide sequence rationalizes the diversity of diabetes-associated mutations in the insulin gene. Our studies demonstrate that photoscanning mutagenesis can decode the active conformation of a protein and so illuminate cryptic constraints underlying its evolution.

A Conserved Histidine in Insulin Is Required for the Foldability of Human Proinsulin: STRUCTURE AND FUNCTION OF AN ALAB5 ANALOG

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.

Stimulation of midgut stem cell proliferation by Manduca sexta alpha-arylphorin.

Extracts of the green-colored perivisceral fat body of newly ecdysed Manduca sexta pupae stimulate mitosis in midgut stem cells of Heliothis virescens cultured in vitro. Using a combination of cation- and anion-exchange chromatography, we have isolated a protein from these fat body extracts that accounts for the observed stem cell proliferation. SDS-PAGE analysis of the protein results in a single band of 77 kDa. Sequences of tryptic peptides from this protein are identical to internal sequences of the storage hexamer alpha-arylphorin. The alpha-arylphorin isolated by our procedure represents a small fraction of the total arylphorin present in the fat body extract. However, it alone seems responsible for the stimulation of mitotic activity in H. virescens midgut stem cells.

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation.

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation.

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation

Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20-30% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.

TheDrosophila Lsp‐1βGene

In Drosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP-1), a mixed hexamer of three closely related subunits, Lsp-1 (alpha, beta and gamma) and larval serum protein 2 (LSP-2), a homohexamer of Lsp-2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of the Lsp-1 beta gene was determined for comparison with LSP-2 and a number of other arthropod hexamerins. The G + C content of the coding sequence is 55%, with 92.8% of the codons containing G or C in the third position. Conceptual translation of the Lsp-1 beta open reading frame revealed a 789-amino-acid polypeptide of 94465 Da. The amino acid sequence of Lsp-1 beta is 65.8% identical to that of calliphorin, the major hexamerin of the blowfly, Calliphora vicina, and only 35.2% identical to Drosophila Lsp-2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP-2 which is enriched to a lesser extent only in aromatic amino acids. Lsp-1 beta is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP-1 calliphorin and LSP-2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin.

Cosmic influences on the expression of a specific gene in the Colorado potato beetle: the diapause protein 1 gene.

Diapause protein 1 from the Colorado potato beetle, Leptinotarsa decemlineata, belongs to the family of insect storage hexamers. The protein accumulates in the hemolymph of last instar larvae and in adults reared under short-day (diapause-inducing) conditions. The protein disappears during metamorphosis, but remains in the hemolymph during diapause. The gene which encodes diapause protein 1 encompasses about 9 kb of chromosomal DNA and is composed of 5 exons, separated by 4 introns. The gene is expressed in last-instar larvae and in adults reared under short-day conditions. The messenger RNA for diapause protein 1 occurs only in the fat body. Gene expression is higher in the fat body from short-day adults than from last-instar larvae. Transcription is suppressed after topical application of pyriproxyfen, a JH-analog. The messenger RNA of the protein (approximately 2.3 kb) contains genetic information of a pre-protein of 702 amino acids, including a signal peptide of 17 amino acids and protein of 685 residues. The function of diapause protein 1 is unknown, but it is utilized during post-diapause development, probably for regeneration of the flight muscles.

Storage proteins in ants (Hymenoptera:Formicidae)

Storage proteins area major feature of holometabolous development in insects, accumulaüng during the larval period and disappearing during metamorphosis. In ants (Hymenoptera:Formicidae), storage proteins also play important roles in adult females. Three types of storage proteins have been characterized from ants: hexamerins, proteins high in glutamine/glutamic acid, and very high density lipoproteins (VHDLs). The hexamerins have moderately high levels of aromatic amino acids and belong to the arthropod hemocyanin family of proteins. The proteins high in glutamine/glutamic acid can form hexamers under some conditions, but the subunit size is larger than that of typical hexamerins. The VHDLs are dimeric and share features with storage chromoproteins described from Lepidoptera. In Camponotus festinatus (Formicinae), storage proteins are found in adult ants in two situations. First, lack of brood stimulates workers to accumulate the same two storage hexamers found in larvae. Second, young virgin queens store large reserves of these proteins before mating. Protein storage by queens has been confirmed in two other subfamilies of ants, indicating it is widespread. The capacity to store proteins as adults enables queens to rear brood without leaving the nest and workers to store rich reserves and regulate larval diet seasonally.

The structure of the gene encoding diapause protein 1 of the Colorado potato beetle ( Leptinotarsa decemlineata)

This paper describes the complete structure and partial sequence of the gene encoding diapause protein 1, an arylphorin-type storage hexamer of the Colorado potato beetle, Leptinotarsa decemlineata. This gene contains four introns of variable lengths, the positions of which have been determined accurately. Three of the five exons of the gene are very similar in size to those reported for storage hexamer genes from Lepidoptera. The last two exons differ significantly in size from those found for other storage protein genes. The complete gene for diapause protein 1 encompasses about 9 kb of genomic DNA. The gene encodes a pre-protein of 702 amino acids, including a signal peptide of 17 amino acids. Transcription starts 32 bp upstream from the translation initiation codon. A typical TATA-box is located 27 bp upstream from the transcription initiation site. Multiple alignment analysis of the amino acid sequence reveals extensive homology with storage hexamers from Lepidoptera. This is the first published complete nucleotide and amino acid sequence of a Coleopteran arylphorin. A simple and rapid method is described for mapping the number, size and positions of introns in genomic DNA fragments using PCR.

Storage proteins in adult ants ( Camponotus festinatus): Roles in colony founding by queens and in larval rearing by workers

Camponotus festinatus storage hexamers (Hex 1 and 2), first identified in last instar larvae, constitute two major proteins in adult queens and broodless workers. We have examined the effects of colony founding by queens and larval rearing by workers on their storage protein content. Both hexamerins accumulate in virgin queens, particularly Hex 2 in the fat body where it makes up about 70% of total soluble proteins. Colony founding causes a depletion of Hex 1 and 2 in queens. Levels of these proteins begin to decrease by the end of the claustral period when the first workers emerge, and they are depleted from fat body and hemolymph 1 month later. Larval rearing has an inhibitory effect on the accumulation of storage proteins in workers. Hex 1 occurs only in low concentrations in the hemolymph of 2- and 4-week-old workers kept with larvae, and disappears thereafter. Hex 2 does not accumulate in 2- to 8-week-old workers kept with larvae. Our results with Camponotus queens support a role of the storage hexamers in egg development and/or nourishment of the brood. These data also suggest an important role of nutrition in the synthesis and accumulation of hexamerins in adult workers.

Nucleotide and deduced amino acid sequence of a cDNA clone encoding diapause protein 1, an arylphorin-type storage hexamer of the Colorado potato beetle

Several clones encoding diapause protein 1 have been isolated by immunoscreening a cDNA library, constructed from poly(A)RNA of whole Colorado potato beetles reared under short-day conditions. The clones contained overlapping DNA sequences, but none contained the complete sequence of the protein. The nucleotide and deduced amino acid sequence of the longest clone (Dp19) is presented. This clone encodes 670 amino acids; the first five were similar to the last five of the 20 N-terminal amino acids determined by protein sequencing. Thus, the subunit of diapause protein 1 is composed of 685 amino acids with a predicted molecular weight of 81,317. The deduced amino acid composition revealed that the proportion of tyrosine/phenylalanine is 16.6%, which suggests that the protein is an arylphorin-type storage hexamer. Hybridization experiments with total RNA and poly(A)RNA from larvae and adults revealed that the gene is expressed in last instar larvae and in adults reared under short-day conditions but not in third-instar larvae and long-day adults. Topical application of pyriproxyfen suppresses the transcription of the messenger. This suggests that the gene for diapause protein 1 is transcribed only under conditions of low juvenile hormone titres. Southern blot experiments showed that the gene for diapause protein 1 occurs only once in the haploid genome of the Colorado potato beetle.

Identification of two storage hexamers in the ant, Camponotus festinatus: Accumulation in adult queenless workers

Two electrophoretically and immunologically distinct storage hexamers (Hex 1 and Hex 2) have been identified in Camponotus festinatus workers. The molecular weights of the native molecules were estimated to be 460,000 (Hex 1) and 580,000 (Hex 2) by pore limiting gradient electrophoresis. Hex 1 partially dissociates with moderate alkaline pH. Both proteins are composed of a single type of apoprotein of approx. 73 (Hex 1) and 80 kDa (Hex 2). While most of Hex 2 is sequestered by the fat body before pupation, Hex 1 remains largely in the hemolymph during the last larval and pupal stages. Both proteins were detected only in low concentrations in the hemolymph of newly emerged adults, and they gradually disappear from adult workers maintained in the colonies. In queenless workers, however, Hex 1 and Hex 2 accumulate in the hemolymph and fat body, constituting the most abundant proteins together with vitellogenin. Camponotus festinatus storage hexamers bear some homologies in their N-terminal sequence with the arylphorins of Diptera and Lepitoptera, as well as with a crab hemocyanin. However, with respect to their amino acid composition, they can not be classified as arylphorins.

The Function and Evolution of Insect Storage Hexamers

The storage hexamers are a family of insect proteins with native molecularweights around 500,000 with six homologous subunits weighing generallybetween 70,000 and 85,000 daltons. They are synthesized and secreted by thefat body of feeding larvae and nymphs and reach extraordinary concentrationsin the hemolymph just prior to metamorphosis. In holometabolous insects,they are partially recaptured by the fat body during the larval to pupal moltand stored in cytoplasmic protein granules, as well as in the hemolymph of thepupa. They disappear from these reservoirs during adult development; inmany insects, both hemi- and holometabolous, they are also utilized duringthe nymphal or larval molts. Their amino acids are primarily incorporated intonew tissues and proteins during adult development, but they may also beincorporated into cuticle as intact protein, and in one case were found to bediverted to a small degree into energy metabolism. Finally, some storagehexamers have ligand binding and transport capabilities. Functionally, de-velopmentally, and evolutionarily, they are a complex family of proteinswhose analysis promises to be exceedingly useful in the study of many basicproblems in the biology of insects.2050066-4170/91/0101-0205 $02.00Annual Reviews www.annualreviews.org/aronline

Adsorptive endocytosis of vitellogenin, lipophorin, and microvitellogenin during yolk formation in Hyalophora

AbstractYolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody‐binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.