Ongoing efforts to unravel the origins of the cholesterol 5,6-secosterols (1a and 1b) in biological systems have revealed that the two known chemical routes to these oxysterols; ozonolysis of cholesterol (3) and Hock-cleavage of 5-α-hydroperoxycholesterol (4a), are distinguishable based upon the ratio of the hydrazone derivatives (2a-b) formed in each case and this ratio offers an insight into the chemical origin of the secosterols in vivo.In a recent report, Pratt and co-workers 1 have shown that Hock-cleavage of 5α-hydroperoxycholesterol (4a), that can arise from the singlet oxygen ene reaction with cholesterol (3), occurs faciley under acidic conditions in organic solvents leading to the formation of primarily cholesterol 5,6-secosterol atheronal-B (1b). Atheronal-A (1a) is either not formed at all, or is a minor component in participating solvents such as ethanol (Fig. 1).We discovered the cholesterol 5,6-secosterols 1a and 1b within human atherosclerotic plaque material in vivo 2 and surmised that, based upon the wealth of literature in the field of chemical-, biological-and auto-oxidation of cholesterol at the time, 3, 4 that only ozone was capable of generating 1a from cholesterol (3). 2 In fact, we considered the presence of 1a [measured as 2a after extraction and derivatization with 2,4-dinitrophenyl (2,4-DNP) hydrazine] within inflammatory arteries as indirect evidence that an oxidant with the chemical signature of ozone may be being generated during atherosclerosis progression. We also showed that 1a undergoes an almost instantaneous aldolization process to form 1b in whole blood and therefore the cholesterol 5,6-secosterols 1a and 1b were both potential signature molecules for cholesterol ozonolysis in vivo. 2 The oxysterols 1a and 1b are proatherogenic 5 and induce protein misfolding and amyloidogenesis 6 in a number of biologically-relevant proteins. We find that the 2,4-DNP hydrazone of atheronal-B (2b) is observed at levels above untreated hLDL (~ 0.01 % of 3), after 2,4-DNP hydrazine derivization of hLDL that has been oxidized in aqueous buffer by superoxide anion ( Tables 1-3).The photosensitized and hPMN-mediated oxidation of cholesterol in hHDL in aqueous buffer (PBS, pH 7.4) parallel the observations with hLDL in that the 2,4-DNP hydrazone of cholesterol secosterol 2b is elevated above untreated levels and no 2a is observed (ESI Tables 4-5). Similarly, the photosensitized oxidation of 3 in liposomes (DOPC 80 mol %; 3 5 mol %; PIP-2 15 mol %) in PBS (pH 7.4) implicates a 1 O 2 pathway to 2b, because the measured levels of 2b are elevated in PBS containing D 2 O, relative to H 2 O (ESI Table 6).One of the clearest observations of these aqueous hLDL oxidation studies is that the 2,4-DNP hydrazone of atheronal-A (2a) is only observed after ozonolysis (Table 1). Thus, passage of an ozone/oxygen mixture over the surface of an hLDL solution containing catalase in aqueous buffer (pH 7.4), followed by 2,4-DNP hydrazine derivatization of the lipid extract yields 2a and 2b in a ratio of ~ 4:1...
Antibody light chain (LC) aggregation in vivo leads to the systemic deposition of Ig light chain domains in the form of either amyloid fibrils (AL-amyloidosis) or amorphous deposits, light-chain deposition disease (LCDD), in mainly cardiac or renal tissue and is a pathological condition that is often fatal. Molecular factors that may contribute to the propensity of LCs to aggregate in vivo, such as the protein primary structure or local environment, are intensive areas of study. Herein, we show that the aggregation of a human antibody kappa-(kappa-MJM) and lambda-(lambda-L155) light chain (1 mg/mL) can be accelerated in vitro when they are incubated under physiologically relevant conditions, PBS, pH 7.4 and 37 degrees C, in the presence of a panel of biologically relevant lipid-derived aldehydes, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), glyoxal (GLY), atheronal-A (KA), and atheronal-B (ALD). Thioflavin-T (ThT) and Congo Red (CR) binding assays coupled with turbidity studies reveal that this aldehyde-induced aggregation can be associated with alteration of protein secondary structure to an increased beta-sheet conformation. We observed that the nature of the conformational change is primarily dependent upon the lipidic aldehyde studied, not the protein sequence. Thus, the cholesterol 5,6-secosterols, KA and ALD, cause an amorphous-type aggregation which is ThT and CR negative for both the kappa-MJM and lambda-L155 light chains, whereas 4-HNE, MDA, and GLY induce aggregates that bind both ThT and CR. TEM analysis revealed that amyloid fibrils were formed during the 4-HNE-mediated aggregation of kappa-MJM and lambda-L155 light chains, whereas ALD-induced aggregates of these LCs where amorphous in nature. Kinetic profiles of LC aggregation reveal clear differences between the aldehydes, KA and ALD, causing a classic nucleated polymerization-type aggregation, with a lag phase (of approximately 150 h) followed by a growth phase that plateaus, whereas 4-HNE, MDA, and GLY trigger a seeded-type aggregation process that has no lag phase. In-depth studies of the 4-HNE-accelerated aggregation of kappa-MJM and lambda-L155 reveal a clear aldehyde concentration dependence and a process that can be inhibited by the naturally occurring osmolyte trimethylamine N-oxide (TMAO). Given these data, we feel our recently discovered paradigm of inflammatory aldehyde-induced protein misfolding may now extend to LC aggregation.
A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, Lys H93 , that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2-5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl 2, or (ii) precoordination of 5 with CuCl 2 followed by conjugation with 38C2. The resulting 38C2-5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis-Menten kinetics [kcat(11) ؍ 2.3 min ؊1 and Km(11) 2.2 mM] with a rate enhancement [k cat(11)kuncat (11) C atalysis at metal centers plays a key role in both enzymatic and abiological reactions, providing reaction pathways, rates, and selectivity often unattainable from conventional acid, base, or nucleophilic catalysis. However, traditionally separate disciplines, the study of natural (i.e., enzymatic) and synthetic (nonbiological) catalysts has interfaced with the genesis of semisynthetic and de novo proteins (1). A number of methods have been explored for producing such hybrid species that possess metal centers, including: (i) attachment of a synthetic metal-containing cofactor to a protein (2-4); (ii) antibody elicitation using a metal-binding hapten͞protein conjugate (5-10); (iii) site-directed mutagenesis of proteins and antibodies to incorporate metal-binding sites (11-15); and (iv) panning of antibody libraries for metal binding using immobilized metal complexes (16,17).Our approach is directed toward the design and generation of novel metallo-catalytic antibodies and is inspired by the active-site structures of the many metalloenzymes that possess a coordination sphere with two or more histidine-derived imidazole ligands. The most important catalytic functions of such polyhistidyl enzymes are hydrolytic or oxidative. Illustrative of the numerous and structurally diverse polyhistidyl Zn-hydrolases (18) is carboxypeptidase A, which catalyzes the hydrolysis of C-terminal amino acid residues and features a bis-histidine͞glutamate (CO 2 Ϫ ) coordination sphere and the nucleophilic assistance of a nearby Glu (19). The Cuhydroxylases, dopamine -hydroxylase and peptide amidating hydroxylase (20), are chemically extraordinary in effecting the regio-and enantioselective hydroxylation of weakly activated COH bonds, a transformation with little precedent among nonenzymatic copper catalysts (21,22). The active sites of hydroxylase enzymes possess two Cu-polyhistidine centers, one of which appears to serve as an electron or superoxide transfer site and the other at which the substra...
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