Abhay H. Pande

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

The phospholipase A2 (PLA2) enzymes are activated by binding to phospholipid membranes. Although the N-terminal α-helix of group I/II PLA2s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA2s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA2, we have created semisynthetic human group IB PLA2 in which the N-terminal decapeptide is joined with the 13C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA2 joined with a 13C-labeled fragment of group IB PLA2. Infrared spectral resolution of the unlabeled and 13C-labeled segments suggests that the N-terminal helix of membrane-bound IB PLA2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA2 is more rigid than that of the IB PLA2, but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA2, which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA2. Correlation is delineated between structural and membrane binding properties of PLA2s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA2s acts as a regulatory domain that mediates interfacial activation of these enzymes.

Stabilization of the Tertiary Structure of the Cholera Toxin A1 Subunit Inhibits Toxin Dislocation and Cellular Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. The catalytic subunit of CT (CTA1) then crosses the ER membrane and enters the cytosol in a process that involves the quality control mechanism of ER-associated degradation. The molecular details of this dislocation event have not been fully characterized. Here, we report that thermal instability in the CTA1 subunit-specifically, the loss of CTA1 tertiary structure at 37 degrees C-triggers toxin dislocation. Biophysical studies found that glycerol preferentially stabilized the tertiary structure of CTA1 without having any noticeable effect on the thermal stability of its secondary structure. The thermal disordering of CTA1 tertiary structure normally preceded the perturbation of its secondary structure, but in the presence of 10% glycerol the temperature-induced loss of CTA1 tertiary structure occurred at higher temperatures in tandem with the loss of CTA1 secondary structure. The glycerol-induced stabilization of CTA1 tertiary structure blocked CTA1 dislocation from the ER and instead promoted CTA1 secretion into the extracellular medium. This, in turn, inhibited CT intoxication. Glycerol treatment also inhibited the in vitro degradation of CTA1 by the core 20S proteasome. Collectively, these findings indicate that toxin thermal instability plays a key role in the intoxication process. They also suggest the stabilization of CTA1 tertiary structure is a potential goal for novel antitoxin therapeutic agents.

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera.

Contribution of Subdomain Structure to the Thermal Stability of the Cholera Toxin A1 Subunit

The catalytic A1 subunit of cholera toxin (CTA1) is an ADP-ribosyltransferase with three distinct subdomains: CTA1(1) forms the catalytic core of the toxin, CTA1(2) is an extended linker between CTA1(1) and CTA1(3), and CTA1(3) is a compact globular region. CTA1 crosses the endoplasmic reticulum (ER) membrane to enter the cytosol where it initiates a cytopathic effect. Toxin translocation involves ER-associated degradation (ERAD), a quality control system that exports misfolded proteins from the ER to the cytosol. At the physiological temperature of 37 °C, the free CTA1 subunit is in a partially unfolded conformation that triggers its ERAD-mediated translocation to the cytosol. Thus, the temperature sensitivity of CTA1 structure is an important determinant of its function. Here, we examined the contribution of CTA1 subdomain structure to the thermal unfolding of CTA1. Biophysical measurements demonstrated that the CTA1(1) subdomain is thermally unstable and that the CTA1(2) subdomain provides a degree of conformational stability to CTA1(1). The CTA1(3) subdomain does not affect the overall stability of CTA1, but the thermal unfolding of CTA1 appears to begin with a local loss of structure in the CTA1(3) subdomain: glycerol and acidic pH both inhibited the thermal disordering of full-length CTA1 but not the disordering of a CTA1 construct lacking the A1(3) subdomain. These observations provide mechanistic insight regarding the thermal unfolding of CTA1, an event which facilitates its subsequent translocation to the cytosol.

The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome.

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.

Interplay between amino acid residues at positions 192 and 115 in modulating hydrolytic activities of human paraoxonase 1

Human paraoxonase 1 (h-PON1) is a Ca(2+)-dependent serum enzyme that catalyzes the hydrolysis of different types of substrates. The crystal structure of h-PON1 is not solved yet and the molecular details of how the enzyme catalyzes different types of reactions are not clear. Literature suggests that the amino acid residues at positions 192 and 115 are important for various hydrolytic activities of h-PON1. It is proposed that catalytic residue H115 (and H134) mediates the lactonase and the arylesterase activities of the enzyme while the amino acid residue at position 192 modulates various other hydrolytic activities of the enzyme. However, the relationship between these two residues in the hydrolytic activities of h-PON1 is not studied in detail. In this study, we have expressed and purified the wild-type recombinant h-PON1 (rh-PON1(wt)) and its point mutants differing in the amino acid residues at positions 192 and/or 115 using an Escherichia coli expression system. The hydrolytic activities of the purified enzymes were compared using enzymatic assays. Our results, for the first time, show that (a) the presence of a particular amino acid residue at position 192 differentially alters the effect of the H115W substitution, and (b) H115 residue is not always needed for the lactonase and arylesterase activities of the enzyme. The results also suggest that the amino acid residues at position 192 and 115 act in conjunction in modulating the hydrolytic activities of the enzyme.

Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

Human paraoxonase 1 (h‐PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h‐PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H‐PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric‐PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h‐PON1 (rh‐PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh‐PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h‐PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h‐PON1.

Oxidized-phospholipids in reconstituted high density lipoprotein particles affect structure and function of recombinant paraoxonase 1 ☆

Paraoxonase 1 (PON1) is an HDL-associated enzyme and exhibits anti-inflammatory, anti-diabetic, and anti-atherogenic properties. Association of PON1 to HDL particles increases the stability and activity of PON1 and is important for the normal functioning of the enzyme. HDL particles are made up of lipid and protein constituents and apolipoprotein A-I (apoA-I) is a principal protein constituent of HDL that facilitates various biological activities of HDL. In many disease conditions the oxidized phospholipid (Ox-PL) content of HDL is found to be increased and an inverse correlation between the activity of PON1 and oxidation of the HDL is observed. However, the molecular details of the inhibitory action of the Ox-PL-containing HDL on the function of PON1 are not clear yet. In this study we have assembled reconstituted HDL (rHDL) particles with and without Ox-PL and compared their effect on the structure and function of (13)C-labeled recombinant PON1 ((13)C-rPON1) by employing attenuated total reflectance Fourier transformed infrared (ATR-FTIR) spectroscopy and enzymatic assay. Our results show that the presence of the Ox-PL in the rHDL particles alters the structure of rPON1 and decreases its lactonase activity.

Organophosphate-Hydrolyzing Enzymes as First-Line of Defence Against Nerve Agent-Poisoning: Perspectives and the Road Ahead

Nerve agents (NAs) are extremely neurotoxic synthetic organophosphate (OP) compounds exploited as weapons of mass destruction in terrorist attacks and chemical warfare. Considering the current world scenario, there is a persistent threat of NA-exposure to military personals and civilians. Various prophylactic and post-exposure treatments (such as atropine and oximes) available currently for NA-poisoning are inadequate and unsatisfactory and suffer from severe limitations. Hence, developing safe and effective treatment(s) against NA-poisoning is a critical necessity. With regards to counteracting NA-toxicity, the OP-hydrolyzing enzymes (OPHEs), which can hydrolyze and inactivate a variety of NAs, have emerged as promising candidates for the development of prophylactic therapy against NA-poisoning. However, there are many hurdles to be crossed before these enzymes can be brought to therapeutic use in humans. In this article, we have reviewed the various advancements in the field of development of OPHEs as prophylactic against NA-poisoning. The article majorly focuses on the toxic effects of NAs, various available therapies to counteract NA poisoning, the current status of OPHEs and attempts made to improve the various properties of these enzymes. Further, we have also briefly discussed about the prospective work that is needed to be undertaken for developing these OPHEs into those suitable for use in humans.

Improving Properties of Recombinant SsoPox by Site-Specific Pegylation.

SsoPox, a ~35 kDa enzyme from Sulfolobus solfataricus, can hydrolyze and inactivate a variety of organophosphate (OP)-compounds. The enzyme is a potential candidate for the development of prophylactic and therapeutic agent against OP-poisoning in humans. However, the therapeutic use of recombinant SsoPox suffers from certain limitations associated with the use of recombinant protein pharmaceuticals. Some of these limitations could be overcome by conjugating SsoPox enzyme with polyethylene glycol (PEG). In this study, we report generation and in vitro characterization of N-terminal mono-PEGylated rSsoPox(2p) (a variant of rSsoPox(wt) having enhanced OP-hydrolyzing activity). The enzyme was PEGylated with mPEG-propionaldehyde and the PEGylated protein was isolated using ion-exchange chromatography. Compared with the unmodified enzyme, mono-PEGylation of rSsoPox results in improvement in the thermostability and protease resistance of the enzyme. PEGylated rSsoPox(2p) can be developed as a candidate for the prevention / treatment of OP-poisoning.