Large scale refolding and purification of the catalytic domain of human BACE-2 produced in E. coli.

Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.

High yield expression of human BACE constructs in Eschericia coli for refolding, purification, and high resolution diffracting crystal forms.

BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.

The atomic-resolution structure of human caspase-8, a key activator of apoptosis.

BACKGROUND: Caspases are a family of cysteine proteases that have important intracellular roles in inflammation and apoptosis. Caspase-8 activates downstream caspases which are unable to carry out autocatalytic processing and activation. Caspase-8 is designated as an initiator caspase and is believed to sit at the apex of the Fas- or TNF-mediated apoptotic cascade. In view of this role, the enzyme is an attractive target for the design of inhibitors aimed at blocking the undesirable cell death associated with a range of degenerative disorders. RESULTS: The structure of recombinant human caspase-8, covalently modified with the inhibitor acetyl-Ile-Glu-Thr-Asp-aldehyde, has been determined by X-ray crystallography to 1.2 A resolution. The asymmetric unit contains the p18-p11 heterodimer; the biologically important molecule contains two dimers. The overall fold is very similar to that of caspase-1 and caspase-3, but significant differences exist in the substrate-binding region. The structure answers questions about the enzyme-inhibitor complex that could not be explained from earlier caspase structures solved at lower resolution. CONCLUSIONS: The catalytic triad in caspase-8 comprises Cys360, His317 and the backbone carbonyl oxygen atom of Arg258, which points towards the Nepsilon atom of His317. The oxygen atom attached to the tetrahedral carbon in the thiohemiacetal group of the inhibitor is hydrogen bonded to Ndelta of His317, and is not in a region characteristic of a classical 'oxyanion hole'. The N-acetyl group of the inhibitor is in the trans configuration. The caspase-8-inhibitor structure provides the basis for understanding structure/function relationships in this important initiator of the proteolytic cascade that leads to programmed cell death.

Cation binding to the integrin CD11b I domain and activation model assessment.

BACKGROUND: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein. RESULTS: We have determined the X-ray structure of a new crystal form of the CD11 b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11 a I-domain structures and a CD11 b I-domain complex with Mn2+. These structures define a majority conformation. CONCLUSIONS: Addition of the cations Mg2+, Mn2+ and Cd2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al, is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.

Porcine fatty acid synthase: cloning of a complementary DNA, tissue distribution of its mRNA and suppression of expression by somatotropin and dietary protein.

A cDNA for porcine fatty acid synthase was isolated and used to examine the tissue distribution of fatty acid synthase mRNA within the pig and to determine the impact of recombinant porcine somatotropin (rpSt) and the level of dietary protein on fatty acid synthase mRNA abundance in pig liver and adipose tissue. A 1.5-kb cDNA representing the thioesterase domain of porcine fatty acid synthase was isolated from a lambda gt 11 liver cDNA library. Northern analysis with total RNA extracted from adipose tissue, liver, heart, lung, kidney and intestine revealed a single major fatty acid synthase mRNA species of 8-9 kb. The amount of fatty acid synthase mRNA in hepatic tissue was 25% of the amount in adipose tissue, which suggests that the liver may be a significant site of fatty acid synthesis in the pig. Fatty acid synthase mRNA abundance was significantly reduced in the adipose tissue (P less than 0.01) and the liver (P less than 0.1) by chronic daily administration (60 micrograms/kg) of rpSt. In addition, increasing the amount of dietary protein decreased (P less than 0.1) the abundance of fatty acid synthase mRNA in adipose tissue but had no effect on liver fatty acid synthase expression. In contrast, the abundance of adipose fatty acid binding protein mRNA was unaffected by rpSt or dietary protein. These data indicate that the reduction in the level of fatty acid synthase mRNA is a factor in the pSt-mediated suppression of fatty acid synthesis in porcine adipose tissue.

Caspase 8: an efficient method for large-scale autoactivation of recombinant procaspase 8 by matrix adsorption and characterization of the active enzyme.

A gene coding for a truncated form of human procaspase 8 has been cloned and expressed in Escherichia coli. This construct contains M(206) through D(479) of human procaspase 8, preceded by an N-terminal polyhistidine tag. The recombinant protein, containing 286 amino acids, was expressed in high yield in the form of inclusion bodies (IB). The IB were solubilized in guanidinium chloride and dialyzed against 50% acetic acid. The solution was mixed with 9 volumes of H(2)O and then rapidly diluted from the acidic medium to one containing 1.0 M Tris, pH 8.0, and 5 mM DTT. SDS-PAGE analysis of the soluble, dilute protein solution (20-30 microgram of protein/ml) showed a single 33-kDa band corresponding to the nonprocessed, inactive procaspase 8. Concentration of the dilute protein to levels as high as 2 mg/ml resulted in only modest (1-10%) autocatalytic conversion to the 19- and 11-kDa polypeptide subunits which are characteristic of the activated enzyme. Further concentration of these protein solutions to a near-dry state on the ultrafiltration membrane, followed by washing of the membrane with buffer, led to extracts containing high yields of enzyme showing a specific activity of 8.43 micromol/min/mg against the chromogenic substrate Ac-IETD-pNA. SDS-PAGE, protein sequencing, and mass spectrometric analysis of these extracts showed complete conversion of the 33-kDa procaspase 8 to the 19- and 11-kDa subunits of activated caspase 8. This method allows for preparation of 100-mg quantities of highly pure and active recombinant human caspase 8. Enzyme activity was shown to be associated with a heterotetrameric complex that is converted to an inactive dimer upon storage.

Production of chemokines CTAPIII and NAP/2 by digestion of recombinant ubiquitin-CTAPIII with yeast ubiquitin C-terminal hydrolase and human immunodeficiency virus protease.

Recombinant yeast ubiquitin C-terminal hydrolase (YUH1), which has an N-terminal (His)(6) tag, and an autolysis-resistant mutant of the human immunodeficiency virus-1 protease (HIV-1 Pr) have been used as specific proteases to yield peptides from a ubiquitin conjugate. In the present example, connective tissue-activating peptide (CTAPIII) and neutrophil-activating peptide 2 (NAP/2) were generated by digestion of a ubiquitin-CTAPIII conjugate with YUH1 and HIV Pr, respectively, as indicated below: [see text] YUH1 cleaved at the peptide bond formed by the C-terminal Gly(76) of ubiquitin (Ub) and the N-terminal Asn(1) of the 85-residue peptide CTAPIII. The HIV-1 Pr cleaved between Tyr(15) and Ala(16), the N-terminal Ala of the 70-residue peptide NAP/2. Both enzymes produced authentic peptides from the Ub fusion protein, with a nearly 100% yield. The liberated CTAPIII and NAP/2 were separated from (His)(6)-Ub, the trace amounts of unreacted (His)(6)-Ub-CTAPIII, HIV-1 Pr, and the (His)(6)-YUH1 by passage over a nickel-chelate column; the final yield was about 10 mg of peptide/liter of cell culture. (His)(6)-YUH1, the HIV Pr mutant, and the (His)(6)-Ub-CTAPIII substrate were all expressed individually in Escherichia coli. (His)(6)-YUH1 and (His)(6)-Ub-CTAPIII were highly expressed in a soluble form, but about 75% of the total (His)(6)-YUH1 was also found in inclusion bodies. Both proteins from the soluble fractions were easily purified in a single step by immobilized metal ion affinity chromatography with a yield of about 27 mg of (His)(6)-Ub-CTAPIII and 13.6 mg of (His)(6)-YUH1 protein/liter of cell culture. Chemotactic factor activity, as assessed by the neutrophil shape change assay, was observed for NAP/2, but not for CTAPIII. This strategy, which employs YUH1 and the HIV-1 Pr as tools for the highly selective cleavage of the chimeric substrate, should be applicable to the large-scale production of a variety of peptides.

[W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.

Processing of the human heparanase precursor and evidence that the active enzyme is a heterodimer.

Human platelet heparanase has been purified to homogeneity and shown to consist of two, non-covalently associated polypeptide chains of molecular masses 50 and 8 kDa. Protein sequencing provided the basis for determination of the full-length cDNA for this novel protein. Based upon this information and results from protein analysis and mass spectrometry, we propose a scheme to define the structural organization of heparanase in relation to its precursor forms, proheparanase and pre-proheparanase. The 8- and 50-kDa chains which make up the active enzyme reside, respectively, at the NH(2)- and COOH-terminal regions of the inactive precursor, proheparanase. The heparanase heterodimer is produced by excision and loss of an internal linking segment. This paper is the first to suggest that human heparanase is a two-chain enzyme.

The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties.

Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.