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S Q Hu

Publications and source records attributed to S Q Hu.

17 recordsLinked to original sources
PubMed2001

Hierarchical protein folding: asymmetric unfolding of an insulin analogue lacking the A7-B7 interchain disulfide bridge.

The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (Cys(A7)-Cys(B7)) are characterized as a model of a late intermediate. Untethering cystine A7-B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6-A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20-B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains native-like supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.

Circular Dichroism↗
PubMed2001

Activities of monomeric insulin analogs at position A8 are uncorrelated with their thermodynamic stabilities.

Previous studies have demonstrated that the potency and thermodynamic stability of human insulin are enhanced in concert by substitution of Thr(A8) by arginine or histidine. These surface substitutions stabilize the N-terminal alpha-helix of the A chain, a key element of hormone-receptor recognition. Does enhanced stability necessarily imply enhanced activity? Here, we test by structure-based mutagenesis the relationship between the stability and activity of the hormone. To circumvent confounding effects of insulin self-association, A chain analogs were combined with a variant B chain (Asp(B10), Lys(B28), and Pro(B29) (DKP)) to create a monomeric template. Five analogs were obtained by chain combination; disulfide pairing proceeded in each case with native yield. CD and (1)H NMR spectra of the DKP analogs are essentially identical to those of DKP-insulin, indicating a correspondence of structures. Receptor binding affinities were determined by competitive displacement of (125)I-insulin from human placental membranes. Thermodynamic stabilities were measured by CD titration; unfolding was monitored as a function of guanidine concentration. In this broader collection of analogs receptor binding affinities are uncorrelated with stability. We suggest that receptor binding affinities of A8 analogs reflect local features of the hormone-receptor interface rather than the stability of the free hormone or the intrinsic C-capping propensity of the A8 side chain.

Amino Acid Sequence↗
PubMed2001

[Expression and purification of murine interleukin 18 in Escherichia coli and its antitumor effects].

Total RNA was extracted from murine hepatocytes, and the cDNA of interleukin 18(IL-18) was amplified by RT-PCR. The cDNA was introduced into the expression vector pJW2 and sequenced. Under heat induction, the recombinant murine IL-18(rmIL-18) was expressed in inclusion bodies in E. coli with the yield accounting for 18% of total bacteria proteins. The inclusion bodies were dissolved with 5 mol/L urea, and rmIL-18 was purified using Sephadex G-100 column chromatography. In the presence of 0.5 mg/L Con A, the purified rmIL-18 showed dose-dependent IFN-gamma-inducing activity in murine splenocytes. The purified rmIL-18 exhibited significant antitumor effects in Kunming mice challenged intraperitoneally (i.p.) with H22 hepatocarcinoma when administered 10 micrograms rmIL-18 i.p. on days 1, 4 after challenge, and the mice survived resisted the rechallenged with H22 cells.

Animals↗
PubMed1998

Mini-proinsulin and mini-IGF-I: homologous protein sequences encoding non-homologous structures.

Protein minimization highlights essential determinants of structure and function. Minimal models of proinsulin and insulin-like growth factor I contain homologous A and B domains as single-chain analogues. Such models (designated mini-proinsulin and mini-IGF-I) have attracted wide interest due to their native foldability but complete absence of biological activity. The crystal structure of mini-proinsulin, determined as a T3R3 hexamer, is similar to that of the native insulin hexamer. Here, we describe the solution structure of a monomeric mini-proinsulin under physiologic conditions and compare this structure to that of the corresponding two-chain analogue. The two proteins each contain substitutions in the B-chain (HisB10-->Asp and ProB28-->Asp) designed to destabilize self-association by electrostatic repulsion; the proteins differ by the presence or absence of a peptide bond between LysB29 and GlyA1. The structures are essentially identical, resembling in each case the T-state crystallographic protomer. Differences are observed near the site of cross-linking: the adjoining A1-A8 alpha-helix (variable among crystal structures) is less well-ordered in mini-proinsulin than in the two-chain variant. The single-chain analogue is not completely inactive: its affinity for the insulin receptor is 1500-fold lower than that of the two-chain analogue. Moreover, at saturating concentrations mini-proinsulin retains the ability to stimulate lipogenesis in adipocytes (native biological potency). These results suggest that a change in the conformation of insulin, as tethered by the B29-A1 peptide bond, optimizes affinity but is not integral to the mechanism of transmembrane signaling. Surprisingly, the tertiary structure of mini-proinsulin differs from that of mini-IGF-I (main-chain rms deviation 4.5 A) despite strict conservation of non-polar residues in their respective hydrophobic cores (side-chain rms deviation 4.9 A). Three-dimensional profile scores suggest that the two structures each provide acceptable templates for threading of insulin-like sequences. Mini-proinsulin and mini-IGF-I thus provide examples of homologous protein sequences encoding non-homologous structures.

Amino Acid Sequence↗
PubMed1996

Mapping the functional surface of insulin by design: structure and function of a novel A-chain analogue.

Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.

Amino Acid Sequence↗
PubMed1994

Insulin-like compounds related to the amphioxus insulin-like peptide.

Three insulin-like compounds consisting of two disulfide-linked polypeptide chains have been synthesized. The A-chains of these compounds correspond either to the A- or to the A + D-domain of the putative amphioxus insulin-like peptide (amphioxus ILP), and their B-chains correspond either to the B-chain of insulin or to a slightly modified (i.e., [1-Thr]) B-domain of amphioxus ILP. The biological potency of these compounds was evaluated in mammalian cells or cell fractions containing either human or rat insulin receptors or human or mouse insulin-like growth factor I (IGF-I) receptors, with respect to binding affinity, insulin-like metabolic activity (lipogenesis), and growth factor activity (mitogenesis). Amphioxus ILP A/bovine insulin B and amphioxus ILP A + D/bovine insulin B exhibited potencies ranging from 2.0 to 9.8% relative to natural insulin, and both compounds were full agonists in lipogenesis assays, stimulating lipogenesis to the same maximal extent as seen with natural insulin. Amphioxus ILP A/amphioxus ILP [1-Thr]B stimulated lipogenesis with a potency of 0.01% relative to natural insulin. We consider this compound also likely to be a full agonist. In assays measuring binding to IGF-I receptors and stimulation of mitogenesis, these compounds displayed some activity although the activity was too low for exact quantification. These results suggest that amphioxus ILP has retained an overall structural similarity to mammalian insulin and IGF-I but has also accumulated substantial mutations which markedly reduce its ability to bind and activate their cognate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

[Changes in water-soluble, urea-soluble and membrane intrinsic proteins in human senile cataract].

Gel filtration of water-soluble protein shows a substantial increase in HM+ alpha-crystallin and a marked decrease in beta- and gamma-crystallins in cortical cataract. A decrease in beta 1-crystallin in cortical punctate opaque lenses is also striking. In nuclear cataractous lenses HM+ alpha- and beta-crystallin increase, while gamma-crystallin decreases. The urea-soluble protein from clear lenses contains mainly of alpha beta chain, whereas in cataractous lenses the relative amounts of the 28 and 23ku polypeptides (the components of beta-crystallin) increased markedly. In cataractous lenses the relative amount of membrane intrinsic proteins decreases slightly and it has little statistical meaning.

Cataract↗
PubMed1993

Steric requirements at position B12 for high biological activity in insulin.

The alpha-helix formed by the amino acid residues 9-19 of the B-chain of insulin is involved in the stabilization of its three-dimensional structure. We have shown that modification at positions B9, B10, B12, and B16 results in analogues possessing biological activities ranging from ca. 0.2% to ca. 500% relative to that of natural insulin. The lowest potency was displayed by [B12 Asn]insulin, in which the hydrophobic B12 Val residue was replaced by the hydrophilic Asn residue. We now report the synthesis of four insulin analogues in which hydrophobicity is retained, and only the spatial arrangement of atoms in the B12 region is altered. Substitution of B12 Val with alpha-aminoisobutyric acid (Aib), D-Ala, and Phe led to analogues possessing biological activities, in lipogenesis assays, of 8.5%, 2%, and 0.2%, respectively, relative to that of natural insulin. Inversion of the B11-B12 sequence, -Leu-Val-, led to an analogue displaying 3.3% activity. A synthetic B-chain in which the B11 Leu-B12 Val sequence was replaced by B11 Ala-B12 Ile was incapable of combining with the natural A-chain. We conclude that the Val residue in the B12 position in insulin fulfills special side-chain packing requirements involved in the stability of the structure of insulin. Even slight steric alteration at position B12 results in a distortion of the overall conformation of the B-chain which affects its ability to combine with the natural A-chain. This distortion is retained in the corresponding analogue, which is reflected in diminished biological potency.

Amino Acid Sequence↗
PubMed1993

Contribution of the B16 and B26 tyrosine residues to the biological activity of insulin.

We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30-50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.

Amino Acid Sequence↗
PubMed1991

Insulin analogues with modifications in the beta-turn of the B-chain.

The beta-turn formed by the amino acid residues 20-23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of beta-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the beta-turn. In two analogues, we have substituted alpha-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potency ca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the beta-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.

Adipose Tissue↗
PubMed1991

Effects of pre-exposures to a rotating optokinetic drum on adaptation to motion sickness.

The purpose of this study was to investigate the effects of two different pre-exposure procedures on adaptation to motion sickness in a rotating circular vection drum. The 45 subjects were randomly divided into three groups. The control group only had a standard 16-min exposure to the drum rotating at 60 degrees/s with no pre-exposure. The incremental exposure group had two separated 4-min pre-exposure periods at 15 degrees/s and 30 degrees/s in the rotating drum immediately prior to the standard 16-min exposure period in the drum rotating at 60 degrees/s. The abrupt-exposure group had the same pre-exposure procedure except the pre-exposure drum rotation speed was 60 degrees/s and was followed by the same standard exposure periods. Subjective motion sickness reports and a measure of gastric myoelectric activity (electrogastrogram, EGG) were obtained during the standard 16-min drum rotation period in all three groups. The results showed that subjects in the incremental exposure group reported significantly fewer motion sickness symptoms during the standard 16-min rotation period than did the subjects in the abrupt exposure group and the control group. Subjects in the incremental exposure group also had less tachyarrhythmia, abnormal gastric myoelectric activity associated with nausea, during the 16-min rotation period than did the subjects in the control and abrupt exposure group. Incremental exposure to motion stimuli may be a useful method for training resistance to visually-induced motion sickness.

Adaptation, Physiological↗
PubMed1990

Superactive insulins.

The substitution of aspartic acid for the naturally-occurring histidine residue in position B10 in human insulin results in an insulin analogue which displays an in vitro potency 4- to 5-fold greater than the parent compound. This substitution has been introduced into six insulin analogues which, before modification, display potencies ranging from less than 0.01-fold to 3-fold relative to natural insulin. In each case, the resulting aspartic acid-substituted analogue is substantially more potent than the parent compound. Thus, it is now possible to prepare "tailor-made" insulins with enhanced potency.

Aspartic Acid↗
PubMed1989

Adaptation to vection-induced symptoms of motion sickness.

The visual-vestibular-proprioceptive sensory mismatch of vection provokes motion sickness in approximately 60% of healthy subjects. Approximately 60% of astronauts experience motion sickness in microgravity where vestibular/otolith function is altered. The purpose of this study was to determine the extent to which symptoms of motion sickness and tachygastria, an abnormal 4-9 cpm rhythm of the stomach, decrease or adapt during three repeated exposures to a rotating circular vection drum. Subjects sat in the drum for 45 min: 15 min baseline, 15 min drum rotation at 60 degrees.s-1, and 15 min recovery. Gastric myoelectric activity was continuously recorded with the electrogastrogram (EGG). Symptom reports were obtained during rotation. In Experiment I, 10 subjects were exposed to the drum 3 times with intersession intervals of 4-24 d. They failed to show adaptation based on subjective reports and all showed tachygastria. In Experiment II, 14 new subjects were exposed to the drum 3 times with intersession intervals of 48 h. The group experienced a reduction in symptoms and tachygastria with repeated exposure to the drum. Thus, symptomatic and physiological improvement occurred after training in subjects susceptible to vection-induced motion sickness. Preflight adaptation to visual-vestibular sensory mismatch may reduce motion sickness experienced in the environment of microgravity.

Adaptation, Physiological↗