Conversion of a c type cytochrome to a b type that spontaneously forms in vitro from apo protein and heme: Implications for c type cytochrome biogenesis and folding

Cytochrome c552 from Hydrogenobacter thermophilus, a thermophilic bacterium, has been converted into a b type cytochrome, after mutagenesis of both heme-binding cysteines to alanine and expression in the cytoplasm of Escherichia coli. The b type variant is less stable, with the guanidine hydrochloride unfolding midpoint occurring at a concentration 2 M lower than for the wild-type protein. The reduction potential is 75 mV lower than that of the recombinant wild-type protein. The heme can be removed from the b type variant, thus generating an apo protein that has, according to circular dichroism spectroscopy, an α-helical content different from that of the holo b type protein. The latter is readily reformed in vitro by addition of heme to the apo protein. This reforming suggests that previously observed assembly of cytochrome c552, which has the typical class I cytochrome c fold, in the E. coli cytoplasm is a consequence of spontaneous thioether bond formation after binding of heme to a prefolded polypeptide. These observations have implications for the general problem of c type cytochrome biogenesis.

Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the precursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.

Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: Application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K

The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code 1CLB), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.

Tumor dormancy and cell signaling: anti-mu-induced apoptosis in human B-lymphoma cells is not caused by an APO-1-APO-1 ligand interaction.

Signal transduction initiated by crosslinking of antigen-specific receptors on T- and B-lymphoma cells induces apoptosis. In T-lymphoma cells, such crosslinking results in upregulation of the APO-1 ligand, which then interacts with induced or constitutively expressed APO-1, thereby triggering apoptosis. Here we show that crosslinking the membrane immunoglobulin on human lymphoma cells (Daudi) (that constitutively express APO-1) does not induce synthesis of APO-1 ligand. Further, a noncytotoxic fragment of anti-APO-1 antibody that blocks T-cell-receptor-mediated apoptosis in T-lymphoma cells does not block anti-mu-induced apoptosis. Hence, in B-lymphoma cells, apoptosis induced by signaling via membrane IgM is not mediated by the APO-1 ligand.

Th1 CD4+ lymphocytes delete activated macrophages through the Fas/APO-1 antigen pathway.

The Fas/APO-1 cytotoxic pathway plays an important role in the regulation of peripheral immunity. Recent evidence indicates that this regulatory function operates through deletion of activated T and B lymphocytes by CD4+ T cells expressing the Fas ligand. Because macrophages play a key role in peripheral immunity, we asked whether Fas was involved in T-cell-macrophage interactions. Two-color flow cytometry revealed that Fas receptor (FasR) was expressed on resting murine peritoneal macrophages. FasR expression was upregulated after activation of macrophages with cytokines or lipopolysaccharide, although only tumor necrosis factor-alpha rendered macrophages sensitive to anti-FasR antibody-mediated death. To determine the consequence of antigen presentation by macrophages to CD4+ T cells, macrophages were pulsed with antigen and then incubated with either Th1 or Th2 cell lines or clones. Th1, but not Th2, T cells induced lysis of 60-80% of normal macrophages, whereas macrophages obtained from mice with mutations in the FasR were totally resistant to Th1-mediated cytotoxicity. Macrophage cytotoxicity depended upon specific antigen recognition by T cells and was major histocompatibility complex restricted. These findings indicate that, in addition to deletion of activated lymphocytes, Fas plays an important role in deletion of activated macrophages after antigen presentation to Th1 CD4+ T cells. Failure to delete macrophages that constitutively present self-antigens may contribute to the expression of autoimmunity in mice deficient in FasR (lpr) or Fas ligand (gld).

Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae.

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER α and ER β

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs α and β and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER α and ER β ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR−/−] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/−)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR−/−, (ii) LDLR−/−;Tg(apoa+/−), (iii) LDLR−/−;Tg(apoB+/+), and (iv)LDLR−/−;Tg(apoB+/+);Tg(apo+/−). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR−/− and LDLR−/−;Tg(apoa+/−) mice (1.0 ± 0.2% vs. 1.4 ± 0.3%). However, the LDLR−/−;Tg(apoB+/+) mice had ≈15-fold greater mean lesion area than the LDLR−/− mice. No significant difference was found in percent lesion area in the LDLR−/−;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 ± 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 ± 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR−/−;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR−/−; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.

Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol

Scavenger receptor BI (SR-BI) is a cell surface receptor that binds high density lipoproteins (HDL) and mediates selective uptake of HDL cholesteryl esters (CE) in transfected cells. To address the physiological role of SR-BI in HDL cholesterol homeostasis, mice were generated bearing an SR-BI promoter mutation that resulted in decreased expression of the receptor in homozygous mutant (designated SR-BI att) mice. Hepatic expression of the receptor was reduced by 53% with a corresponding increase in total plasma cholesterol levels of 50–70% in SR-BI att mice, attributable almost exclusively to elevated plasma HDL. In addition to increased HDL-CE, HDL phospholipids and apo A-1 levels were elevated, and there was an increase in HDL particle size in mutant mice. Metabolic studies using HDL bearing nondegradable radiolabels in both the protein and lipid components demonstrated that reducing hepatic SR-BI expression by half was associated with a decrease of 47% in selective uptake of CE by the liver, and a corresponding reduction of 53% in selective removal of HDL-CE from plasma. Taken together, these findings strongly support a pivotal role for hepatic SR-BI expression in regulating plasma HDL levels and indicate that SR-BI is the major molecule mediating selective CE uptake by the liver. The inverse correlation between plasma HDL levels and atherosclerosis further suggests that SR-BI may influence the development of coronary artery disease.

Structural basis for chemical inhibition of human blood coagulation factor Xa

Factor Xa, the converting enzyme of prothrombin to thrombin, has emerged as an alternative (to thrombin) target for drug discovery for thromboembolic diseases. An inhibitor has been synthesized and the crystal structure of the complex between Des[1–44] factor Xa and the inhibitor has been determined by crystallographic methods in two different crystal forms to 2.3- and 2.4-Å resolution. The racemic mixture of inhibitor FX-2212, (2RS)-(3′-amidino-3-biphenylyl)-5-(4-pyridylamino)pentanoic acid, inhibits factor Xa activity by 50% at 272 nM in vitro. The S-isomer of FX-2212 (FX-2212a) was found to bind to the active site of factor Xa in both crystal forms. The biphenylamidine of FX-2212a occupies the S1-pocket, and the pyridine ring makes hydrophobic interactions with the factor Xa aryl-binding site. Several water molecules meditate inhibitor binding to residues in the active site. In contrast to the earlier crystal structures of factor Xa, such as those of apo-Des[1–45] factor Xa and Des[1–44] factor Xa in complex with a naphthyl inhibitor DX-9065a, two epidermal growth factor-like domains of factor Xa are well ordered in both our crystal forms as well as the region between the two domains, which recently was found to be the binding site of the effector cell protease receptor-1. This structure provides a basis for designing next generation inhibitors of factor Xa.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Fibrinogen deficiency reduces vascular accumulation of apolipoprotein(a) and development of atherosclerosis in apolipoprotein(a) transgenic mice

To test directly whether fibrin(ogen) is a key binding site for apolipoprotein(a) [apo(a)] in vessel walls, apo(a) transgenic mice and fibrinogen knockout mice were crossed to generate fibrin(ogen)-deficient apo(a) transgenic mice and control mice. In the vessel wall of apo(a) transgenic mice, fibrin(ogen) deposition was found to be essentially colocalized with focal apo(a) deposition and fatty-streak type atherosclerotic lesions. Fibrinogen deficiency in apo(a) transgenic mice decreased the average accumulation of apo(a) in vessel walls by 78% and the average lesion (fatty streak type) development by 81%. Fibrinogen deficiency in wild-type mice did not significantly reduce lesion development. Our results suggest that fibrin(ogen) provides one of the major sites to which apo(a) binds to the vessel wall and participates in the generation of atherosclerosis.

Delayed loss of cholesterol from a localized lipoprotein depot in apolipoprotein A-I-deficient mice

The anti-atherogenic role of high density lipoprotein is well known even though the mechanism has not been established. In this study, we have used a novel model system to test whether removal of lipoprotein cholesterol from a localized depot will be affected by apolipoprotein A-I (apo A-I) deficiency. We compared the egress of cholesterol injected in the form of cationized low density lipoprotein into the rectus femoris muscle of apo A-I K-O and control mice. When the injected lipoprotein had been labeled with [3H]cholesterol, the t½ of labeled cholesterol loss from the muscle was about 4 days in controls and more than 7 days in apo A-I K-O mice. The loss of cholesterol mass had an initial slow (about 4 days) and a later more rapid component; after day 4, the disappearance curves for apo A-I K-O and controls began to diverge, and by day 7, the loss of injected cholesterol was significantly slower in apo A-I K-O than in controls. The injected lipoprotein cholesterol is about 70% in esterified form and undergoes hydrolysis, which by day 4 was similar in control and apo A-I K-O mice. The efflux potential of serum from control and apo A-I K-O mice was studied using media containing 2% native or delipidated serum. A significantly lower efflux of [3H]cholesterol from macrophages was found with native and delipidated serum from apo A-I K-O mice. In conclusion, these findings show that lack of apo A-I results in a delay in cholesterol loss from a localized depot in vivo and from macrophages in culture. These results provide support for the thesis that anti-atherogenicity of high density lipoprotein is related in part to its role in cholesterol removal.

Convergent evolution of apolipoprotein(a) in primates and hedgehog

Apolipoprotein(a) [apo(a)] is the distinguishing protein component of lipoprotein(a), a major inherited risk factor for atherosclerosis. Human apo(a) is homologous to plasminogen. It contains from 15 to 50 repeated domains closely related to plasminogen kringle four, plus single kringle five-like and inactive protease-like domains. This expressed gene is confined to a subset of primates. Although most mammals lack apo(a), hedgehogs produce an apo(a)-like protein composed of highly repeated copies of a plasminogen kringle three-like domain, with complete absence of protease domain sequences. Both human and hedgehog apo(a)-like proteins form covalently linked lipoprotein particles that can bind to fibrin and other substrates shared with plasminogen. DNA sequence comparisons and phylogenetic analysis indicate that the human type of apo(a) evolved from a duplicated plasminogen gene during recent primate evolution. In contrast, the kringle three-based type of apo(a) evolved from an independent duplication of the plasminogen gene approximately 80 million years ago. In a type of convergent evolution, the plasminogen gene has been independently remodeled twice during mammalian evolution to produce similar forms of apo(a) in two widely divergent groups of species.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.