Leonid Gaidukov

The 'evolvability' of promiscuous protein functions

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.

The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux

Serum paraoxonase (PON1) is a HDL-associated enzyme exhibiting potentially antiatherogenic properties. Here, we examined the common PON1-192R/Q human polymorphism. Despite numerous studies, the effect of this polymorphism on the antiatherogenic potential of PON1 is yet unresolved. Our structural model suggests that amino acid 192 constitutes part of the HDL-anchoring surface and active site of PON1. Based on our findings that PON1 is an interfacially activated lipolactonase that selectively binds HDL carrying apolipoprotein A-I (apoA-I) and is thereby greatly stabilized and catalytically activated, we examined the interaction of the PON1-192 isozymes with reconstituted HDL-apoA-I particles. We found that PON1 position 192 is indeed involved in HDL binding. The PON1-192Q binds HDL with a 3-fold lower affinity than the R isozyme and consequently exhibits significantly reduced stability, lipolactonase activity, and macrophage cholesterol efflux. We also observed the lower affinity and stability of the 192Q versus the 192R isozyme in sera of individuals belonging to the corresponding genotypes. The observed differences in the properties of PON1-192R/Q isozymes provide a basis for further analysis of the contribution of the 192R/Q polymorphism to the susceptibility to atherosclerosis, although other factors, such as the overall levels of PON1, may play a more significant role.

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O‐di‐isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O‐cyclohexyl methylphosphonofluoridate) and soman (O‐pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O‐isopropyl‐O‐(p‐nitrophenyl) methyl phosphonate (IMP‐pNP), DFP, and chlorpyrifos‐oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase‐inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was ∼ 10‐fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild‐type, and ninefold higher activity towards soman. L69V exhibited 100‐fold faster hydrolysis of DFP than the wild‐type. The active‐site mutant H115W exhibited 270–380‐fold enhancement toward hydrolysis of the P–S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino‐acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP‐pNP, indicating that PON1 is less active toward the more toxic optical isomers.

Effects of Acyl versus Aminoacyl Conjugation on the Properties of Antimicrobial Peptides

ABSTRACT To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptides global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.

The development of human sera tests for HDL-bound serum PON1 and its lipolactonase activity

Serum paraoxonase (PON1) is a lipolactonase that associates with HDL-apolipoprotein A-I (HDL-apoA-I) and thereby plays a role in the prevention of atherosclerosis. Current sera tests make use of promiscuous substrates and provide no indications regarding HDL-PON1 complex formation. We developed new enzymatic tests that detect total PON1 levels, irrespective of HDL status and R/Q polymorphism, as well as the degree of catalytic stimulation and increased stability that follow PON1s tight binding to HDL-apoA-I. The tests are based on measuring total PON1 levels with a fluorogenic phosphotriester, measuring the lipolactonase activity with a chromogenic lactone, and assaying the enzymes chelator-mediated inactivation rate. The latter two are affected by tight HDL binding and thereby derive the levels of the serum PON1-HDL complex. We demonstrate these new tests with a group of healthy individuals (n = 54) and show that the levels of PON1-HDL vary by a factor of 12. Whereas the traditionally applied paraoxonase and arylesterase tests weakly reflect PON1-HDL levels (R = 0.64), the lipolactonase test provides better correlation (R = 0.80). These new tests indicate the levels and activity of PON1 in a physiologically relevant context as well as the levels and quality of the HDL particles with which the enzyme is associated.

In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes

BackgroundSerum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050.MethodsFor stability studies we applied different challenges related to the in vivo disfunctionalization of HDL and PON1 and tested for inactivation of PON1s activity. We applied acute, repetitive administrations of BL-3050 in mice to assess its toxicity and adverse immune responses. The in vivo efficacy of recombinant PON1 and BL-3050 were tested with an animal model of chlorpyrifos-oxon poisoning.ResultsInactivation studies show significantly improved in vitro lifespan of the engineered rePON1 relative to human PON1. Significant sequence changes relative to human PON1 might hamper the in vivo applicability of BL-3050 due to adverse immune responses. However, we observed no toxic effects in mice subjected to repetitive administration of BL-3050, suggesting that BL-3050 could be safely used. To further evaluate the activity of BL-3050 in vivo, we applied an animal model that mimics human organophosphate poisoning. In these studies, a significant advantages of rePON1 and BL-3050 (>87.5% survival versus <37.5% in the control groups) was observed. Furthermore, BL-3050 and rePON1 were superior to the conventional treatment of atropine-2-PAM as a prophylactic treatment for OP poisoning.ConclusionIn vitro and in vivo data described here demonstrate the potential advantages of rePON1 and BL-3050 for treatment of OP toxicity and chronic cardiovascular diseases like atherosclerosis. The in vivo data also suggest that rePON1 and BL-3050 are stable and safe, and could be used for acute, and possibly repeated treatments, with no adverse effects.

Glycine Dimerization Motif in the N-terminal Transmembrane Domain of the High Density Lipoprotein Receptor SR-BI Required for Normal Receptor Oligomerization and Lipid Transport

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G15X2G18X3AX2G25, of which the submotif G18X3AX2G25 significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras). None of the SR-BI variants exhibited altered surface expression (based on antibody binding) or HDL binding. However, the G15L/G18L/G25L triple mutant exhibited reductions in cell surface homo-oligomerization (>10-fold) and the rate of selective lipid uptake (∼2-fold). Gly18 and Gly25 were necessary for normal lipid uptake activity of SR-BI and the SR-BI/croquemort chimera. The lipid uptake activity of the glycine motif-deficient SR-BI/LIMP II chimera was low but could be increased by introducing glycines at positions 18 and 25. The rate of lipid uptake mediated by SR-BI/LIMP II chimeras was proportional to the extent of receptor oligomerization. Thus, the glycine dimerization motif G18X3AX2G25 in the N-TM domain of SR-BI contributes substantially to the homo-oligomerization and lipid transport activity of SR-BI but does not influence the negative cooperativity of HDL binding. Oligomerization-independent binding cooperativity suggests that classic allostery is not involved and that the negative cooperativity is probably the consequence of a “lattice effect” (interligand steric interference accompanying binding to adjacent receptors).

ApoE Induces Serum Paraoxonase PON1 Activity and Stability Similar to ApoA-I †

Serum paraoxonase (PON1) is an anti-atherogenic interfacially activated lipo-lactonase that was shown to selectively bind high-density lipoprotein (HDL) carrying apolipoprotein A-I (apoA-I). ApoA-I binding occurs with nanomolar affinity and induces a dramatic increase in enzyme stability and lactonase activity. This study examined the association of PON1 with reconstituted HDL (rHDL) carrying apolipoprotein E, and its consequences on the stability and enzymatic activity of PON1, and on its anti-atherogenic potential. The results indicate that reconstituted HDL particles prepared with two most common isoforms of apoE (apoE3 and apoE4) associate with rePON1 in a manner and affinity similar to those of apoA-I. Binding to apoE-HDL stimulates the lactonase activity and stabilizes the enzyme, although the latter occurs to a >10-fold lesser extent compared to apoA-I-HDL particles. The anti-atherogenic potential of PON1, measured by inhibition of LDL oxidation and stimulation of macrophage cholesterol efflux, was also stimulated by apoE-HDL, at levels of 40-96% compared to apoA-I-HDL. Overall, reconstituted apoE-HDL exhibits properties similar to those of apoA-I-HDL, but with a lower capacity to stabilize PON1 and to induce its anti-atherogenic functions. ApoE, apoA-I, and to a lesser degree apoA-IV show distinct structural and functional similarities but little sequence homology. That these apolipoproteins, but not apoA-II, bind PON1 with high affinity and stimulate its activity suggests that PON1-HDL recognition is based primarily on surface properties of the apolipoproteins and that specific protein-protein interactions may play only a secondary role.