Some Enzymatic Properties and Substrate Specificities of Peptidoglutaminase-I and II

Some enzymatic properties and substrate specificities of the two peptidoglutaminases (peptidoglutaminase-I (PGase-I) and II (PGase-II) isolated from Bacillus circulans were investigated.

The both resembled each other with respect to optimum pH and temperature for their activities, susceptibility to various reagents and effects of many metal ions. But, the pH-rate profile of PGase-II was more broad than that of PGase-I. Thermal stability of PGase-I was better than that of PGase-II at a degree of about 5°C.

However, they were definitively different each other with respect to their substrate specificities. PGase-I specifically deamidated the γ-amide of L-glutamine residing at the carboxyl terminal on peptides, and was inactive to glutamine derivative substituted at both α-amino and α-carboxyl groups. On the other hand, the best substrate for PGase-II was tri-peptides, X-Gln-Y, where X was carbobenzoxy-,t-amyloxycarbonxy- or amino acid residues, and Y was amino acids. Though L-glutamine presented in polypeptide chains composed of more than four amino acid residues was a poor substrate, two L-glutamines in oxidized insulin A chain were well attacked by the enzyme.

The both PGase were inactive to asparagine and asparaginyl peptides.     

Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.     

Puromycin-sensitive aminopeptidase is the major peptidase responsible for digesting polyglutamine sequences released by proteasomes during protein degradation

Long stretches of glutamine (Q) residues are found in many cellular proteins. Expansion of these polyglutamine (polyQ) sequences is the underlying cause of several neurodegenerative diseases (e.g. Huntington's disease). Eukaryotic proteasomes have been found to digest polyQ sequences in proteins very slowly, or not at all, and to release such potentially toxic sequences for degradation by other peptidases. To identify these key peptidases, we investigated the degradation in cell extracts of model Q-rich fluorescent substrates and peptides containing 10-30 Q's. Their degradation at neutral pH was due to a single aminopeptidase, the puromycin-sensitive aminopeptidase (PSA, cytosol alanyl aminopeptidase). No other known cytosolic aminopeptidase or endopeptidase was found to digest these polyQ peptides. Although tripeptidyl peptidase II (TPPII) exhibited limited activity, studies with specific inhibitors, pure enzymes and extracts of cells treated with siRNA for TPPII or PSA showed PSA to be the rate-limiting activity against polyQ peptides up to 30 residues long. (PSA digests such Q sequences, shorter ones and typical (non-repeating) peptides at similar rates.) Thus, PSA, which is induced in neurons expressing mutant huntingtin, appears critical in preventing the accumulation of polyQ peptides in normal cells, and its activity may influence susceptibility to polyQ diseases.     

Primary structure of peptides from bovine brain glutamine synthetase. Comparison with sequences of glutamine synthetases from other organisms

An analysis of the covalent structure of bovine brain glutamine synthetase has been initiated. Cyanogen bromide and tryptic digests have yielded peptides accounting for most of the polypeptide subunit, and sequence analysis has placed in order over half of the amino acids within these peptides. The amino terminus is acetylated and has the following partial sequence: Ac(H, S3, A2, T)-L-B-K-G-I-K-Z-V-Y-M. The carboxyl-terminal sequence is: A-L-P-Q-G-D-K-V-Q-A-M. The peptides isolated from bovine glutamine synthetase show a high degree of homology with peptides isolated from ovine and porcine brain glutamine synthetases. In contrast to the sequence homologies of the proteins from eukaryotic sources, there are no obvious amino acid sequence homologies between bovine brain glutamine synthetase and any prokaryotic glutamine synthetase. Bovine brain glutamine synthetase is inactivated by phenylglyoxal and N-ethylmaleimide. In both cases catalytic activity is protected by the presence of ATP, suggesting the presence of arginine and cysteine residues at or near the ATP binding site.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

Utilization of alanyl-L-glutamine and glycyl-L-glutamine during long-term parenteral nutrition in the growing rat

Summary Utilization of intravenously administered alanyl-L-glutamine and glycyl-L-glutamine as a source of glutamine was tested in growing rats receiving total parenteral nutrition for 15 days. In experiment A the two peptides were compared to each other, in experiment B alanyl-L-glutamine was compared to an equimolar mixture of free alanine and glutamine, and in experiment C glycyl-L-glutamine was tested against an equimolar mixture of free glycine and glutamine.There was no difference of statistical significance in weight gain or nitrogen balance between the respective groups participating in the 3 experiments. Plasma levels of free glutamine were similar during infusion with alanyl-L-glutamine or glycyl-L-glutamine. The daily urinary excretion rate recorded for alanyl-L-glutamine was 3.7% and for glycyl-L-glutamine 4.3% of the infused amount. The results show that both peptides are utilized for protein synthesis and growth to approximately the same as the corresponding free amino acids.     

The use of a novel quantitation strategy based on Reductive Isotopic Di-Ethylation (RIDE) to evaluate the effect of glufosinate on the unicellular algae Ostreococcus tauri

We report a novel stable-isotope labeling strategy for quantitative proteomics analysis. The method consists of labeling N-termini and lysine ε-amino groups through reductive amination using acetaldehyde. This allows isotope labeling using pairs of either 2H/1H or 13C/12C without mass spectrum overlap. Our labeling procedure, which is significantly different than that developed for dimethylation, can be completed with little trace of partial ethylation; non-labeled peptides represent less than 0.05% of all peptides. Co-elution of both isotopic 13C/12C peptide pairs was observed in all cases, simplifying data analysis, which can be performed using standard commercial software such as Mascot Distiller. A 13C/12C labeled mix in a 1:1 ratio from a complex extract digest of the unicellular algae Ostreococcus tauri, showed a relative standard deviation of less than 14%. This quantitative method was used to characterize O. tauri in the presence of glufosinate, an herbicide which inhibits glutamine synthetase. Blocking glutamine synthetase significantly reduced the expression of several enzymes and transporters involved in nitrogen assimilation and the expression of a number of proteins involved in various stresses including oxidative damage response were up-regulated.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide antigen

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.     

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.     

Structural features of glutamine substrates for human plasma factor XIIIa (activated blood coagulation factor XIII)

The action of human plasma factor XIIIa (thrombin-activated blood coagulation factor XIII) and guinea pig liver transglutaminase on purified caseins, fibrin, the derivatized gamma chain of fibrin, and a number of synthetic glutamine peptides, and peptide derivatives is reported. There are wide variations in the properties of the individual proteins and peptides as substrates for amine incorporation by the two transglutaminases. beta-Casein and several of its derivatives are excellent substrates for factor XIIIa. However, beta-casein is a relatively poor substrate for the liver enzyme. The primary site of amine incorporation by factor XIIIa in beta-casein was identified as glutamine 167. This was accomplished by labeling with fluorescent amine followed by proteolytic digestion and identification of labeled peptides. An 11-residue peptide and a 15-residue peptide, each containing 1 glutamine residue and each modeled after the primary site of amine incorporation in beta-casein, were prepared. A 13-residue peptide modeled after the primary crosslinking site in fibrin gamma chain was also prepared. Each of these polypeptides proved to be an efficient substrate for factor XIIIa and displayed significantly better substrate properties than a number of small glutamine peptide derivatives that are good substrates for liver transglutaminase.     

Class I and class II MHC bind self peptide sets that are strikingly different in their evolutionary characteristics

 Comparison of peptides eluted from human class I and class II major histocompatibility complex (MHC) molecules and the proteins from which they are derived (source proteins) revealed that class I MHC bind peptides derived from proteins that are highly conserved, hydrophilic, and universally expressed, while the peptides themselves are hydrophobic and even more conserved than their source proteins. In contrast, source proteins for class II-bound peptides were not significantly more conserved than a random sample of proteins. Class II-bound peptides were generally more conserved than their source proteins but were significantly less conserved than class I-bound peptides. The characteristics of class I-bound peptides can probably be explained by the selectivity of processing and transport of peptides for binding by class I, while the relative lack of selectivity of peptide binding for class II may explain the high incidence of autoimmune diseases associated with alleles of these molecules. Received: 17 May 1999 / Revised: 5 August 1999     

Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins

While tandem mass spectrometry (MS/MS) is routinely used to identify proteins from complex mixtures, certain types of proteins present unique challenges for MS/MS analyses. The major wheat gluten proteins, gliadins and glutenins, are particularly difficult to distinguish by MS/MS. Each of these groups contains many individual proteins with similar sequences that include repetitive motifs rich in proline and glutamine. These proteins have few cleavable tryptic sites, often resulting in only one or two tryptic peptides that may not provide sufficient information for identification. Additionally, there are less than 14,000 complete protein sequences from wheat in the current NCBInr release. In this paper, MS/MS methods were optimized for the identification of the wheat gluten proteins. Chymotrypsin and thermolysin as well as trypsin were used to digest the proteins and the collision energy was adjusted to improve fragmentation of chymotryptic and thermolytic peptides. Specialized databases were constructed that included protein sequences derived from contigs from several assemblies of wheat expressed sequence tags (ESTs), including contigs assembled from ESTs of the cultivar under study. Two different search algorithms were used to interrogate the database and the results were analyzed and displayed using a commercially available software package (Scaffold). We examined the effect of protein database content and size on the false discovery rate. We found that as database size increased above 30,000 sequences there was a decrease in the number of proteins identified. Also, the type of decoy database influenced the number of proteins identified. Using three enzymes, two search algorithms and a specialized database allowed us to greatly increase the number of detected peptides and distinguish proteins within each gluten protein group.     

Evaluating immobilized metal affinity chromatography for the selection of histidine-containing peptides in comparative proteomics

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed. Acylation of the amino-terminus of peptides with either succinic anhydride, N-acetoxysuccinamide, or [3-(2,5)-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium (quaternary amine) reduced the number of histidine-containing peptides bound by the Cu(II)-IMAC columns. This provides an additional possibility for sample simplification in proteomic applications. The number of acylated peptides selected decreased in the order of quaternary amine > N-acetoxysuccinamide > succinic anhydride derivatization. Although the selection of N-terminally derivatized peptides is biased toward peptides that contain more than one histidine, it is not yet possible to predict selectivity.     

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.     

Labeling of cysteine-containing peptides with 2-nitro-5-thiobenzoic acid

A method for specific labeling of cysteine-containing peptides has been developed using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Prior to cleavage with proteases or chemical reagents, proteins are reacted with DTNB, resulting in the formation of a mixed disulfide between the protein sulfhydryl group and 2-nitro-5-thiobenzoic acid (TNB). The formation of the mixed disulfide introduces a chromophore, with an absorbance peak at 328 nm. By monitoring peptide maps generated by HPLC at 210 and 328 nm, peptides containing cysteine residues are readily identified. The stability of the derivative was tested using glutathione-TNB as a model compound. Glutathione-TNB is stable to conditions used for CNBr cleavage, as well as those for tryptic cleavage. The TNB label may also increase the hydrophobicity of small peptides, which otherwise might not bind to reverse-phase matrices. This was the case for an oxidatively modified tetrapeptide isolated from Escherichia coli glutamine synthetase.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.     

The specific T-cell response to antigenic peptides is influenced by bystander peptides

T lymphocytes recognize antigens in the form of peptides presented by major histocompatibility complex (MHC) molecules on the cell surface. Only a small proportion of MHC class I and class II molecules are loaded with foreign antigenic peptides; the vast majority are loaded with thousands of different self peptides. It was suggested that MHC molecules presenting self peptides may serve either to decrease (antagonistic effect) or increase (synergistic effect) the T cell response to a specific antigen. Here, we present our finding that transfected mouse fibroblasts presenting a single antigenic peptide covalently bound to a class II MHC molecule stimulated specific mouse T cell hybridoma cells to an interleukin-2 response less efficiently than fibroblasts presenting a similar amount of antigenic peptide in the presence of class II molecules loaded with heterogenous bystander peptides.     

Effects of chain length on the aggregation of model polyglutamine peptides: molecular dynamics simulations

Aggregation in the brain of polyglutamine-containing proteins is either a cause or an associated symptom of nine hereditary neurodegenerative disorders including Huntington's disease. The molecular level mechanisms by which these proteins aggregate are still unclear. In an effort to shed light on this important phenomenon, we are investigating the aggregation of model polyglutamine peptides using molecular-level computer simulation with a simplified model of polyglutamine that we have developed. This model accounts for the most important types of intra- and inter-molecular interactions-hydrogen bonding and hydrophobic interactions-while allowing the folding process to be simulated in a reasonable time frame. The model is used to examine the folding of isolated polyglutamine peptides 16, 32, and 48 residues long and the folding and aggregation of systems of 24 model polyglutamine peptides 16, 24, 32, 36, 40, and 48 residues long. Although the isolated polyglutamine peptides did form some alpha and beta backbone-backbone hydrogen bonds they did not have as many of these bonds as they would have if they had folded into a complete alpha helix or beta sheet. In one of the simulations on the isolated polyglutamine peptide 48 residues long, we observed a structure that resembles a beta helix. In the multi-chain simulations we observed amorphous aggregates at low temperatures, ordered aggregates with significant beta sheet character at intermediate temperatures, and random coils at high temperatures. We have found that the temperature at which the model peptides undergo the transition from amorphous aggregates to ordered aggregates and the temperature at which the model peptides undergo the transition from ordered aggregates to random coils increase with increasing chain length. Our finding that the stability of the ordered aggregates increases as the peptide chain length increases may help to explain the experimentally observed relation between polyglutamine tract length and aggregation in vitro and disease progression in vivo. We have also observed in our simulations that the optimal temperature for the formation of beta sheets increases with chain length up to 36 glutamine residues but not beyond. Equivalently, at fixed temperature we find a transition from a region dominated by random coils at chain lengths less than 36 to a region dominated by relatively ordered beta sheet structures at chain lengths greater than 36. Our finding of this critical chain length of 36 glutamine residues is interesting because a critical chain length of 37 glutamine residues has been observed experimentally.     

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.