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Results for “Protein Stability”

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PubMed2006

Alterations in expression and chromatin configuration of the alpha hemoglobin-stabilizing protein gene in erythroid Kruppel-like factor-deficient mice.

Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the beta-globin promoter, where it establishes local chromatin structure permitting beta-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the beta-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and beta-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

Acetylation↗

PubMed2003

Stage-specific translational efficiency and protein stability regulate the developmental expression of p37, an RNA binding protein from Trypanosoma brucei.

We have previously characterized two novel RNA binding proteins, p34 and p37, from Trypanosoma brucei. Their sequences do not show significant homology to other proteins but are highly homologous to one another. The p34 and p37 proteins are developmentally regulated, with p34 the predominant protein in the procyclic stage and p37 nearly exclusively expressed in the bloodstream cells. In vivo metabolic labeling of procyclic cells showed that p34 and p37 were differentially translated, with levels of p34 approximately fourfold higher than p37. The newly synthesized p34 and p37 exhibited differential stability in the procyclic stage. In vitro analysis confirmed this observation and further suggested that this differential stability may be due to a trypsin-like cysteine protease activity in procyclic extracts that selectively degraded the p37 protein. Taken together, these results indicate that the developmental regulation of the T. brucei RNA binding protein, p37, occurs at both translational and post-translational levels.

Animals↗

PubMed2000

Stabilization of proteins by enhancement of inter-residue hydrophobic contacts: lessons of T4 lysozyme and barnase.

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.

Algorithms↗

PubMed1994

Protein stability parameters measured by hydrogen exchange.

The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one-dimensional nuclear magnetic resonance and used to determine the thermodynamic parameters of global unfolding at mild solution conditions well below the melting transition. The free energy for global unfolding measured by hydrogen exchange can differ from values found by standard denaturation methods, most notably due to the slow cis-trans isomerization of the prolyl peptide bond. This difference can be quantitatively calculated from basic principles. Even with these corrections, HX experiments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denaturation data, as predicted by the denaturant binding model of Tanford.

Amines↗

PubMed2000

NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius possible determinants of protein stability.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three-dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H-NMR derived distance constraints along with 23 hydrogen-bonds, 72 phi and 27 chi1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization. BacTrx consists of a well-defined core region of five strands of beta-sheet, surrounded by four exposed alpha-helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X-ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross-linking different secondary structural elements and clamping the C-terminal alpha-helix to the core of the protein.

Amino Acid Sequence↗

PubMed2001

Relative enzymatic activity, protein stability, and tissue distribution of human steroid-metabolizing UGT2B subfamily members.

Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3alpha,17beta-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3alpha,17beta-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5alpha-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.

Cell Line↗

PubMed2006

Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.

Amino Acid Sequence↗

PubMed1999

Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens.

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711-2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.

Agrobacterium tumefaciens↗

PubMed1996

Direct measurement of salt-bridge solvation energies using a peptide model system: implications for protein stability.

The solvation energies of salt bridges formed between the terminal carboxyl of the host pentapeptide AcWL- X-LL and the side chains of Arg or Lys in the guest (X) position have been measured. The energies were derived from octanol-to-buffer transfer free energies determined between pH 1 and pH 9. 13C NMR measurements show that the salt bridges form in the octanol phase, but not in the buffer phase, when the side chains and the terminal carboxyl group are charged. The free energy of salt-bridge formation in octanol is approximately -4 kcal/mol (1 cal = 4.184 J), which is equal to or slightly larger than the sum of the solvation energies of noninteracting pairs of charged side chains. This is about one-half the free energy that would result from replacing a charge pair in octanol with a pair of hydrophobic residues of moderate size. Therefore, salt bridging in octanol can change the favorable aqueous solvation energy of a pair of oppositely charged residues to neutral or slightly unfavorable but cannot provide the same free energy decrease as hydrophobic residues. This is consistent with recent computational and experimental studies of protein stability.

Amino Acid Sequence↗

PubMed1988

Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability.

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.

Amino Acid Sequence↗

PubMed2003

Hdmx protein stability is regulated by the ubiquitin ligase activity of Mdm2.

The stability of the p53 tumor suppressor protein is critically regulated by the Hdm2 and Hdmx proteins. Hdm2 protein levels are auto-regulated by the self-ubiquitination activity of Hdm2 and on the transcriptional level by p53-activated transcription of the hdm2 gene. Little is known about the regulation of Hdmx expression levels, apart from the observation that the Mdmx protein can be cleaved by caspase-3 in a p53-inducible manner. In the functional analysis of two mutant Hdmx proteins, products of two alternatively spliced mRNAs, it was found that Hdmx proteins are targets for ubiquitination by Mdm2. The stability of the Hdmx protein is partly dependent on the presence of its internal acidic domain. Mdm2 appears only to require an intact RING domain to be able to ubiquitinate Hdmx and target it for proteasomal degradation. These findings highlight the intricate functional relationships between p53, Mdm2, and Hdmx.

Alternative Splicing↗

PubMed2005

Structural determinants of protein stabilization by solutes. The important of the hairpin loop in rubredoxins.

Despite their high sequence homology, rubredoxins from Desulfovibrio gigas and D. desulfuricans are stabilized to very different extents by compatible solutes such as diglycerol phosphate, the major osmolyte in the hyperthermophilic archaeon Archaeoglobus fulgidus[Lamosa P, Burke A, Peist R, Huber R, Liu M Y, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C and Santos H (2000) Appl Environ Microbiol66, 1974-1979]. The principal structural difference between these two proteins is the absence of the hairpin loop in the rubredoxin from D. desulfuricans. Therefore, mutants of D. gigas rubredoxin bearing deletions in the loop region were constructed to investigate the importance of this structural feature on protein intrinsic stability, as well as on its capacity to undergo stabilization by compatible solutes. The three-dimensional structure of the mutant bearing the largest deletion, Delta17/29, was determined by 1H-NMR, demonstrating that, despite the drastic deletion, the main structural features were preserved. The dependence of the NH chemical shifts on temperature and solute concentration (diglycerol phosphate or mannosylglycerate) provide evidence of subtle conformational changes induced by the solute. The kinetic stability (as assessed from the absorption decay at 494 nm) of six mutant rubredoxins was determined at 90 degrees C and the stabilizing effect exerted by both solutes was assessed. The extent of protection conferred by each solute was highly dependent on the specific mutant examined: while the half-life for iron release in the wild-type D. gigas rubredoxin increased threefold in the presence of 0.1 M diglycerol phosphate, mutant Delta23/29 was destabilized. This study provides evidence for solute-induced compaction of the protein structure and occurrence of weak, specific interactions with the protein surface. The relevance of these findings to our understanding of the molecular basis for protein stabilization is discussed.

Desulfovibrio gigas↗

PubMed1994

Application of scaled particle theory to model the hydrophobic effect: implications for molecular association and protein stability.

The energetics of alkane dissolution and partition between water and organic solvent are described in terms of the energy of cavity formation and solute-solvent interaction using scaled particle theory. Thermodynamic arguments are proposed that allow comparison of experimental measurements of the surface area with values calculated from an all-atom representation of the solute. While the surface tension relating to the accessible surface is shape dependent, it is found that for the molecular surface it is not. This model rationalizes the change in surface tension between the microscopic (20-30 cal/mol/A2) and macroscopic (70-75 cal/mol/A2) regimes without the need to invoke Flory-Huggins theory or to apply other corrections. The difference in the values arises (i) to a small extent as a result of the curvature dependence of surface tension and (ii) to a large extent due to the difference in the molecular surface derived from the experiment and that calculated from an extended all-atom model. The model suggests that the primary driving force for alkane association in water is due to the tendency of water to reduce the solute cavity surface. It is argued that to model the energetics of alkane association, the surface tension should be related to the molecular surface (rather than the accessible surface) with a surface tension near the macroscopic limit for water. This model is compared with results from theoretical simulations of the hydrophobic effect for two well-studied systems. The implications for antibody-antigen interactions and the effect of hydrophobic amino acid deletion on protein stability are discussed. The approach can be used to model the solute cavity formation energy in solution as a first step in the continuum modelling of biomolecular interactions.

Antigen-Antibody Reactions↗

PubMed2003

Inhibition of CK2 activity by TGF-beta1 promotes IkappaB-alpha protein stabilization and apoptosis of immortalized hepatocytes.

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor kappaB (IkappaB)-alpha protein following treatment of hepatocytes with transforming growth factor (TGF)-beta1 promoted NF-kappaB repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IkappaB-alpha protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-kappaB levels following treatment with TGF-beta1. We show that both messenger RNA (mRNA) and protein levels of the CK2alpha catalytic subunit are down-regulated following TGF-beta1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IkappaB protein, which is followed by the shutoff of constitutive NF-kappaB activity and induction of apoptosis. Ectopic expression of CK2alpha inhibits TGF-beta1-induced apoptosis through sustained activation of NF-kappaB. Conversely, expression of a kinase-dead mutant of CK2alpha potentiates TGF-beta1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-beta1 transgenic mice and human HCC cell lines display enhanced CK2 IkappaB kinase activity that contributes in part to an elevated NF-kappaB activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-beta1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-beta1-induced liver carcinogenesis.

Animals↗

PubMed2005

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells.

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.

Arginine↗

PubMed2004

NMDA receptor GluRepsilon/NR2 subunits are essential for postsynaptic localization and protein stability of GluRzeta1/NR1 subunit.

In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.

Animals↗

PubMed2003

Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding.

This article presents a study of the molecular charge distributions of the genetically encoded amino acids (AA), one that builds on the previous determination of their equilibrium geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quantitative expression in terms of the bond and atomic properties determined within the quantum theory of atoms-in-molecules (QTAIM) that defines atoms and bonds in terms of the observable charge density. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the atomic properties in obtaining an understanding of the biological functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with experimental physicochemical properties. A property of the AA side chains of particular importance is the charge separation index (CSI), a quantity previously defined as the sum of the magnitudes of the atomic charges and which measures the degree of separation of positive and negative charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogues, from the vapor phase to aqueous solution, yielding a linear regression equation with r2 = 0.94. The atomic volume is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calculated parameters (representing electrostatic and volume contributions) yielding linear regressions with r2 values of 0.78 and 0.89, respectively. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 approximately 0.9) were obtained between DeltaCSI upon mutation and each of the two terms DeltaDeltaH and TDeltaDeltaS taken from recent and accurate differential scanning calorimetry experiments on ubiquitin. When the two terms are summed to yield DeltaDeltaG, the quadratic terms nearly cancel, and the result is a simple linear fit between DeltaDeltaG and DeltaCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a DeltaCSI term and a term representing the change in the van der Waals volume of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI < or = 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting molecular complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concentrations and depletions defined by the topology of the Laplacian of the electron density. A display of the experimentally accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochemical code" proposed as the determinant in the folding process.

Amino Acids↗

PubMed2004

Hyperglycemia regulates hypoxia-inducible factor-1alpha protein stability and function.

Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxia-induced stabilization of HIF-1alpha protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1alpha, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1alpha function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1alpha protein levels was confirmed in vivo, where only very low levels of HIF-1alpha protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1alpha against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia.

Cell Hypoxia↗