Decreased formation of alpha 2-macroglobulin-protease complexes in plasma of patients with cystic fibrosis.

Alpha₂-macroglobulin from patients with cystic fibrosis is shown to have reduced binding with papain, trypsin, and thrombin. The obligate heterozygotes for cystic fibrosis revealed intermediate values between the controls and the patients. Since papain and trypsin are not plasma endopeptidases, it becomes evident that the absence of α₂-macroglobulin-protease complex in cystic fibrosis is due to a molecular defect within the macroglobulin.     

Resistance of tropoelastin and elastin peptides to degradation by alpha 2-macroglobulin-protease complexes

When α-ketoglutarate is the substrate, malate is a considerably more effective inhibitor of glutamate dehydrogenase than glutamate, oxalacetate, aspartate, or glutarate. Malate is a considerably poorer inhibitor when glutamate is the substrate. Malate is competitive with α-ketoglutarate, uncompetitive with TPNH, and noncompetitive with glutamate. The above, plus the fact that malate is a considerably more potent inhibitor when TPNH rather than TPN is the coenzyme, indicates that malate is predominantly bound to the α-ketoglutarate site of the enzyme-TPNH complex and has a considerably lower affinity for the enzyme-TPN complex. Ligands which decrease binding of TPNH to the enzyme such as ADP and leucine markedly decrease inhibition by malate. Conversely, GTP, which increases binding of TPNH to the enzyme also enhances inhibition by malate. Malate also decreases interaction between mitochondrial aspartate aminotransferase and glutamate dehydrogenase. This effect of malate on enzyme-enzyme interaction is enhanced by DPNH and GTP which also increase inhibition of glutamate dehydrogenase by malate and is decreased by TPN, ADP, ATP, α-ketoglutarate, and leucine which decrease inhibition of glutamate dehydrogenase by malate. These results indicate that malate could decrease α-ketoglutarate utilization by inhibiting glutamate dehydrogenase and retarding transfer of α-ketoglutarate from the aminotransferase to glutamate dehydrogenase. These effects of malate would be most pronounced when the mitochondrial level of α-ketoglutarate is low and the level of malate and reduced pyridine nucleotide is high.     

Conformation and protease binding activity of binary and ternary human alpha 2-macroglobulin-protease complexes

Human alpha 2-macroglobulin (alpha 2M) undergoes a conformational change after reaction with proteases. In this report, it is shown that although two trypsin molecules may bind simultaneously to each alpha 2M, only one trypsin is necessary to induce alpha 2M conformational change. Ternary complexes of alpha 2M and either two radioiodinated trypsins or two nonradioiodinated trypsins were purified by gel filtration chromatography. The nonradioactive complex did not bind 125I-trypsin, even after incubation for 24 h with the free protease present at a large molar excess. Under comparable conditions, a large molar excess of nonradioactive trypsin did not cause significant dissociation of the complex prepared with radioiodinated protease. Equations are presented that distinguish between two separate models of protease binding and demonstrate that binary alpha 2M-trypsin complex retains no significant trypsin binding activity despite the presence of a vacant protease binding site. Purified alpha 2M-plasmin complex, with 1.10 mol of plasmin/mol of inhibitor, also retained no trypsin binding activity as assessed with radioiodinated protein binding experiments. These studies suggest that reactions of alpha 2M with proteases are accurately described by the "trap hypothesis" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724) independent of protease size or binding stoichiometry.     

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.     

Dissociation of alpha 2 M-macroglobulin into functional half-molecules by mild acid treatment

A slight decrease in pH below neutrality causes the dissociation of alpha 2-macroglobulin (alpha 2M) into dimers formed of two disulfide-bonded subunits. Half-dissociation occurs at pH 6.30 (50 mM NaCl), as determined by gel filtration analysis. The dissociation can be reversed either by increasing the pH or the ionic strength. The ability of alpha 2 M half-molecules at pH 5.75 to bind chymotrypsin is not too different from that of the whole molecule at pH 7.5. Furthermore, the steady-state kinetic parameters toward chromogenic substrate of chymotrypsin bound to alpha 2 M half and whole molecules are quite identical. Likewise, the accessibility of trypsin toward soybean trypsin inhibitor is also fairly similar when involved in half or whole alpha 2 M complexes. These results are consistent with the idea that alpha 2 M-half molecules on chymotrypsin binding undergo a conformational change. This change can be observed by electron microscopy.     

Evidence for active half-molecules of alpha 2-macroglobulin formed by dissociation in urea

Urea caused dissociation of alpha 2-macroglobulin (alpha 2M) into half-molecules (two disulfide-bonded subunits) as revealed by gel electrophoresis. The fraction of whole molecules remaining decreased with increasing urea concentration. Half-dissociation occurred at about 2.2 M. The ability of alpha 2M to inhibit trypsin also decreased with increasing urea concentration, but the activity-urea curve was shifted to the right as compared to the dissociation-urea curve. Thus, at 3 M urea, gel electrophoresis showed only 6.6% whole molecules, whereas the trypsin inhibitory activity was 95% of that in buffer with no urea, suggesting that half-molecules retain activity. In addition, complexes formed in urea with 125I-labeled trypsin were observed to migrate as half-molecules even though only 50% of such complexes were covalent. These results are surprising in light of the report by Gonias and Pizzo [Gonias, S., & Pizzo, S. (1983) Biochemistry 22, 536-546] that half-molecules formed by mild reduction are active; reduction is assumed to divide the molecule along an axis orthogonal to the break caused by urea. This suggests that active half-molecules can be formed by splitting either the covalent or noncovalent bonds that hold the subunits together. A model is proposed that can account for this possibility. It has the same dimensions and symmetry as a previous model of Feldman et al. [Feldman, S.R., Gonias, S.L., & Pizzo, S.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704] and accounts in a similar way for previous functional studies of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.     

Methylamine reaction and denaturation-dependent fragmentation of complement component 3. Comparison with alpha2-macroglobulin

Complement protein C-3 can covalently incoporate [14C]methylamine with a stoichiometry of 0.85 +/- 0.11 mol/mol of protein. The reactive site is located in the larger, Mr = 135,000 peptide subunit of C-3. The methylamine is incorporated as a derivative of glutamic acid, viz. as gamma-glutamylmethylamide, which was identified by high performance liquid chromatography and low resolution mass spectroscopy. C-3 was shown to undergo a specific, denaturation-dependent protein fragmentation in sodium dodecyl sulfate at 90 degrees C. The cleavage results in the partial conversion of the Mr = 135,000 subunit to fragments of Mr = 84,000 and 53,000. The cleavage is completely prevented by reaction of C-3 with methylamine prior to the 90 degrees C incubation. The site of methylamine incorporation (the glutamyl residue) and the peptide fragmentation reaction have been reported for alpha2-macroglobulin (Howard, J.B., Vermeulen, M., and Swenson, R. (1980) J. Biol. Chem. 255, 3820-3823). A comparison of the results for the two proteins suggests that they have a common reactive site.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix.

The structures of the two domains of translational initiation factor IF3 from Bacillus stearothermophilus have been solved by X-ray crystallography using single wavelength anomalous scattering and multiwavelength anomalous diffraction. Each of the two domains has an alpha/beta topology, with an exposed beta-sheet that is reminiscent of several ribosomal and other RNA binding proteins. An alpha-helix that protrudes out from the body of the N-terminal domain towards the C-terminal domain suggests that IF3 consists of two RNA binding domains connected by an alpha-helix and that it may bridge two regions of the ribosome. This represents the first high resolution structural information on a translational initiation factor.     

Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and mechanism of ATP-dependent proteases.

A general paradigm for energy-dependent proteases is emerging: ATP may be used to unfold the substrate and translocate it through a narrow channel within the enzyme into a central proteolytic chamber. Different members of the family present intriguing elaborations on this model.     

Crystallization and preliminary X-ray analysis of methylamine-treated alpha 2-macroglobulin and 3 alpha 2-macroglobulin-proteinase complexes.

Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2-macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine-treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined.     

Association of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin-plasmin complexes with isolated hepatocytes

125I-labelled alpha 2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration- and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 X 10(-8) M for alpha 2-macroglobulin-thrombin and 8.5 X 10(-8) M for alpha 2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50-60% of the bound radioactivity was internalized by the cells. alpha 2-Macroglobulin itself bound also to hepatocytes, but the affinity of the alpha 2-macroglobulin complexes was higher than that of the inhibitor alone, and alpha 2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their alpha 2-macroglobulin complexes. The alpha 2-macroglobulin-thrombin complex competed with the alpha 2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for alpha 2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.     

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.     

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the “RIP-Chip” assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.     

Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes.

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human alpha 2-macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90 degrees about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the alpha 2-macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of alpha 2-macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a "backbone" consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.     

Inhibition of proteases in coagulation, kinin-forming and complement systems by alpha2-plasmin inhibitor.

Inhibitory activities of alpha2-plasmin inhibitor against various proteases were investigated. The inhibitor promptly inhibited the esterolytic activity of alpha-chymotrypsin and progressively inhibited the esterolytic or amidolytic activities of bovine plasma kallikrein, bovine thrombin and bovine activated factor X. Heparin had no effect on the reaction of the inhibitor with thrombin or activated factor X. However, the inhibitor had no effect on the activities of human C-1-esterase, papain and snake venom kininogenase. On the basis of its rapid inhibition of kallikrein, alpha2-plasmin inhibitor is considered to exert some regulating effect on kallikrein activity in plasma.