Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain.

The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.

Stabilisation of alpha-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding.

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.

Mutational analysis of the propensity for amyloid formation by a globular protein.

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme.

The thermodynamics and kinetics of folding of common-type acylphosphatase have been studied under a variety of experimental conditions and compared with those of the homologous muscle acylphosphatase. Intrinsic fluorescence and circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-state mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Nevertheless, common-type acylphosphatase folds more rapidly, suggesting that the conformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate of common-type acylphosphatase is much higher than that of the muscle enzyme, indicating that the differences in conformational stability between the two proteins are primarily determined by differences in the rate of unfolding. The equilibrium m value is markedly different for the two proteins in the pH range of maximum conformational stability (5. 0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes similar to that of the muscle enzyme. Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and mu values, respectively) are notably reduced with respect to pH 5.5. The pH-induced decrease of the m value can be attributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme. This would decrease the positive net charge of the protein, leading to a greater compactness of the denatured state. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this change in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.

Different in vitro and in vivo activity of low Mr phosphotyrosine protein phosphatase on epidermal growth factor receptor.

Low Mr phosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptor in vivo, thus reducing cellular mitogenic response to such growth factors. Following cell stimulation with platelet-derived growth factor the phosphatase undergoes a redistribution from the citosol to the Triton X-100-insoluble fraction where its activity upon the growth factor receptor is intense. Previous research uncovered evidence that low Mr phosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptor in vitro. Here we demonstrate that in vivo the enzyme is not active on the phosphorylated epidermal growth factor receptor and it does not influence the mitogenic response of cells. Since the enzyme distribution is not affected by epidermal growth factor stimulation, involvement of a recruitment mechanism in the definition of low Mr phosphotyrosine protein phosphatase substrate specificity is hypothesized.

Studies on enzymatic activity and conformational stability of muscle acylphosphatase mutated at conserved lysine residues.

An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor.

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.

Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1.

The genes of the human low Mr phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S-transferase. The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X-ray-grade, diffracted to better than 2.4 A and were suitable for high resolution X-ray structure determination.

Sequence-specific recognition of peptide substrates by the low Mr phosphotyrosine protein phosphatase isoforms.

A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) of the low Mr PTPase. The calculated k(cat), Km, and k(cat)/Km values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.

C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation.

Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.

pp60v-src phosphorylates and activates low molecular weight phosphotyrosine-protein phosphatase.

Low M(r) phosphotyrosine-protein phosphatase belongs to the non-receptor cytosolic phosphotyrosine-protein phosphatase subfamily. It has been demonstrated that this enzyme dephosphorylates receptor tyrosine kinases, namely the epidermal growth factor receptor in vitro and the platelet-derived growth factor receptor in vivo. Low M(r) phosphotyrosine-protein phosphatase is constitutively tyrosine-phosphorylated in NIH/3T3 cells transformed by pp60v-src. The same tyrosine kinase, previously immunoprecipitated, phosphorylates this enzyme in vitro as well. Phosphorylation is enhanced using phosphatase inhibitors and phenylarsine oxide-inactivated phosphatase, consistently with the existence of an auto-dephosphorylation process. Intermolecular dephosphorylation is demonstrated adding the active enzyme in a solution containing the inactivated and previously phosphorylated one. This tyrosine phosphorylation correlates with an increase in catalytic activity. Our results provide evidence of a physiological mechanism of low M(r) phosphotyrosine-protein phosphatase activity regulation.

Properties of Cys21-mutated muscle acylphosphatases.

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.

Expression, purification, and characterization of acylphosphatase muscular isoenzyme as fusion protein with glutathione S-transferase.

A genetic construct consisting of the synthetic gene coding for human muscle acylphosphatase linked to the gene for glutathione S-transferase has been prepared. This gene was transformed into and expressed by the Escherichia coli strains DB1035 and TB1, respectively. The fusion protein was purified by affinity chromatography and subsequently cleaved to the fully active acylphosphatase, which was further purified by gel filtration chromatography. Such a purification procedure is very rapid and suitable for obtaining considerable amounts of enzyme at a very high yield. The purified human muscle acylphosphatase was fully active and showed structural features, as well as kinetic and stability parameters, identical to those of the native enzyme.

Properties of N-terminus truncated and C-terminus mutated muscle acylphosphatases.

Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and 1H NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (delta 6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The delta 6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.

Antisense peptides to the 43-57 region of acylphosphatase and to the 46-60 region of two isoenzymes of a low-M(r) phosphotyrosine protein phosphatase do not interact with the corresponding proteins.

Three peptides complementary to exposed regions of two low-M(r) phosphotyrosine protein phosphatase isoenzymes and of the acylphosphatase muscle isoenzyme have been synthesized. Each peptide was synthesized on two different types of resins; the peptides were anchored to the resins by amide linkages. The peptide resins were checked by amino acid analysis and Edman degradation and directly used for enzyme purification. Despite our attempts, none of the resins was able to bind significant amounts of the corresponding protein, indicating the lack of interaction between the three proteins and the corresponding complementary peptides. This result agrees with many other reports, confirming that the molecular-recognition theory has no general validity.

Arginine-23 is involved in the catalytic site of muscle acylphosphatase.

Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.

Crystallisation of a low molecular weight phosphotyrosine protein phosphatase from bovine liver.

Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P2(1)2(1)2(1), have cell dimensions a = 46.3 A, b = 62.2 A, c = 62.7 A and diffract to better than 2.0 A resolution. The crystals are well suited for high resolution X-ray studies.