Volume 381, Issue 12

Circadian rhythms of important enzymes involved in the conversion of cholesterol to bile acids [sterol 12α-hydroxylase (12α-hydroxylase) and cholesterol 7α-hydroxylase (7α-hydroxylase)] and an albumin site D-binding protein (DBP) were examined in rats. When the animals were fed freely, they usually ate in the dark and the circadian rhythms of activities of 12α-hydroxylase and 7α-hydroxylase showed the same peaks (at 10 p.m.) and lows (at 2 p.m.). Their mRNA levels were determined at four timepoints: 3 a.m., 10 a.m., 3 p.m. and 10 p.m. A maximum of the rhythm of 12α-hydroxylase was observed at 3 p.m. and the minimum at 3 a.m. These results are distinct from those of 7α-hydroxylase, whose maximum point was at 10 p.m. and minimum at 3 p.m. When the rats were fed only in the daytime (from 9 a.m. to 5 p.m.), a marked shift of the activity and mRNA rhythms was observed with both enzymes. The circadian rhythms of the activities of both enzymes showed the same peaks (at 3 p.m.), but the mRNA levels of 12α-hydroxylase were distinct from those of 7α-hydroxylase, whose maximum point was at 3 a.m. and minimum at 10 p.m. Differences between the maximum and the minimum points of each enzyme mRNA level were statistically significant ( P < 0.01 for 12α-hydroxylase and 0.05 for 7α-hydroxylase). Moreover, circadian rhythms of DBP were also markedly shifted with the change of feeding period. The maximum mRNA level was observed at 10 p.m. instead of 10 a.m. and the minimum was at 10 a.m. instead of 10 p.m.

In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain κ NS1 , which is unfolded in unassembled molecules. We investigated the fate of κ NS1 glycosylated at position 70 (κ70) and of a double mutant (κ/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated κ NS1 chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/ calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8 + T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range ( K d < 100nm). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.

The yeast translational activator protein Cbs2p is imported into mitochondria without obvious proteolytic processing. To test the importance of amino-terminal amino acids for mitochondrial targeting we fused varying portions of the N-terminus with green fluorescent protein and examined the intracellular distribution of the reporter protein. We show that the 25 N-terminal amino acids are sufficient to direct the majority of the fusion protein into mitochondria. Cbs2p derivatives lacking 9 to 35 amino acids from the N-terminus fail to complement the respiratory deficiency of a Δ cbs2 strain, but are still imported into mitochondria. Therefore Cbs2p contains at least one independent mitochondrial targeting information in addition to the N-terminal signal. We further analyzed the effect of over-expression of Cbs2p on mitochondrial function. Elevated concentrations of Cbs2p lead to slightly impaired mitochondrial gene expression, probably as the result of the formation of inactive Cbs2p aggregates.

The tryptophan synthases from Escherichia coli and Salmonella typhimurium are tetrameric enzymes, with an elongated TrpA.TrpB.TrpB.TrpA structure. Structural studies have identified residues 277–283 of TrpB as a potentially important region for the allosteric communication between the TrpA and TrpB subunits and for the transport of indole between their active sites through a hydrophobic tunnel. To explore the functional role of this region, we analyzed the effects of 19 single and double mutations in TrpB on the tryptophan synthase (TSase) and serine deaminase (SDase) activities of the TrpB 2 dimer, either in the presence or in the absence of the TrpA subunit. The mutations of residues 273–283 could be divided into 4 classes. Mutations I278A, F280G and M282A decreased the SDase and TSase activities of TrpB 2 to similar extents. F280A decreased the SDase activity of TrpB 2 more than its TSase activity, whereas the reverse was true for Y279L. F280A decreased the activation factor of TrpB 2 by TrpA, whereas F280G increased it. The reaction steps and intramolecular contacts that could be affected by the mutations are described. The sequence 278-IYFGM-282, which is present in E. coli and S. typhimurium , is only found in 5 out of 42 organisms, whereas the sequence VLHGX is found in 21 organisms. Our results identified several mutations that could be used as structural probes to analyze precisely the roles of residues 278–282 and their evolution.

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro 456 pro 457 Ala 458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.

Granzyme B is the prototypic member of the granzymes, a family of trypsin-like serine proteinases localized in the dense cytoplasmic granules of activated natural killer cells and cytotoxic T lymphocytes. Granzyme B directly triggers apoptosis in target cells by activating the caspase pathway, and has been implicated in the etiology of rheumatoid arthritis. Human granzyme B expressed in a baculovirus system has been crystallized without inhibitor and its structure has been determined to 3.1 Å resolution, after considerably improving the diffraction power of the crystals by controlled humidity changes. The granzyme B structure reveals an overall fold similar to that found in cathepsin G and human chymase. The guanidinium group of Arg226, anchored at the back of the S1-specificity pocket, can form a salt bridge with the P1-Asp side chain of a bound peptide substrate. The architecture of the substrate binding site of granzyme B appears to be designed to accommodate and cleave hexapeptides such as the sequence Ile-Glu-Thr-Asp↓ -Ser-Gly present in the activation site of pro-caspase-3, a proven physiological substrate of granzyme B. These granzyme B crystals, with fully accessible active sites, are well suited for soaking with small synthetic inhibitors that might be used for a treatment of chronic inflammatory disorders.

The serine proteinase inhibitor (PSPI-21) isolated from potato tubers ( Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.

The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6m urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5–6 mg/l, corresponding to a concentration of at least 80–100nm in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25–0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like α 2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82 500 ± 60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co 2+ , differing in their pH optima and k cat / K m . Zn 2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III ( K i <1μM) and a preference for a hydrophobic residue at the P 1 site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.

Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S. In the present report we describe the cloning and expression of antistefin A F ab fragment A22 in E. coli. We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space. The A22 F ab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis. The recombinant A22 F ab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.

Characterization of protein damage during photosensitization of chlorin e 6 -treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen ( 1 O 2 ) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e 6 . A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e 6 . However, the 1 O 2 quenchers β-carotene and sodium azide minimized GFP photobleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e 6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e 6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e 6 vesicles. We conclude that photoactivated GFP generates endogenous 1 O 2 , inducing chromophore damage,, which can be enhanced by the cooperation of exogenous chlorine 6 .

The values of molar absorption coefficient and quantum yields of photodecomposition and peptide bond scission were determined for a number of aromatic and aliphatic dipeptides under 193 nm laser irradiation in neutral argon-saturated aqueous solution. Under these conditions we could show that no dependence of the dipeptide decomposition quantum yield on the sequence of amino acid residues exists, neither for aromatic dipeptides nor for aliphatic ones.

Chloroplast differentiation in angiosperm plants depends on the light-dependent conversion of protochlorophyllide to chlorophyllide by NADPH:protochlorophyllide oxidoreductase (PORA; EC 1.6.99.1), a nuclearly encoded protein. The protein import of the precursor form of PORA into plastids was shown previously to strictly depend on the presence of its substrate protochlorophyllide. PORA seemed to follow a novel, posttranslationally regulated import route. Here we demonstrate that the precursor of PORA from barley is imported into isolated barley plastids independently of protochlorophyllide. PORA as well as PORB import is competed for by the precursor of the small subunit of Rubisco. The data demonstrate that the PORA precursor uses the general import pathway into plastids. Furthermore, en route into chloroplasts the pea POR precursor can be crosslinked to the protein import channel in the outer envelope Toc75 from pea.

Catalase binds nitric oxide (NO) to generate ferricatalase-NO, an inhibited form of the enzyme. Superoxide (O 2 − ) is also an inactivator of the enzyme. We found, however, that O 2 − efficiently converted the inhibited ferricatalase-NO to the active ferricatalase without producing detectable intermediates. The reaction slowed down when O 2 − was disproportionated to H 2 O 2 and O 2 by superoxide dismutase, but H 2 O 2 could displace the heme-bound NO slowly to regenerate ferricatalase. Reactivation was observed even under simultaneous generation of NO and O 2 − suggesting that ferricatalase-NO reacts with O 2 − fast enough to compete with the rapid reaction of O 2 − and NO. Formation of peroxynitrite by the simultaneous generation of NO and O 2 − was only partially inhibited by ferricatalase, presumably due to slow binding of NO to catalase in comparison with the reaction of NO and O 2 − .