Patrizia Polverino de Laureto

Probing the partly folded states of proteins by limited proteolysis

The folding of a polypeptide chain of a relatively large globular protein into its unique three-dimensional and functionally active structure occurs via folding intermediates. These partly folded states of proteins are difficult to characterize, because they are usually short lived or exist as a distribution of possible conformers. A variety of experimental techniques and approaches have been utilized in recent years in numerous laboratories for characterizing folding intermediates that occur at equilibrium, including spectroscopic techniques, solution X-ray scattering, calorimetry and gel filtration chromatography, as well as genetic methods and theoretical calculations. In this review, we focus on the use of proteolytic enzymes as probes of the structure and dynamics of folding intermediates and we show that this simple biochemical technique can provide useful information, complementing that obtained by other commonly used techniques and approaches. The key result of the proteolysis experiments is that partly folded states (molten globules) of proteins can be sufficiently rigid to prevent extensive proteolysis and appear to maintain significant native-like structure.

Oxidation of myofibrillar proteins in human heart failure

OBJECTIVES We investigated the incidence and contribution of the oxidation/nitrosylation of tropomyosin and actin to the contractile impairment and cardiomyocyte injury occurring in human end-stage heart failure (HF) as compared with nonfailing donor hearts. BACKGROUND Although there is growing evidence that augmented intracellular accumulation of reactive oxygen/nitrogen species may play a key role in causing contractile dysfunction, there is a dearth of data regarding their contractile protein targets in human HF. METHODS In left ventricular (LV) biopsies from explanted failing hearts (New York Heart Association functional class IV; HF group) and nonfailing donor hearts (NF group), carbonylation of actin and tropomyosin, disulphide cross-bridge (DCB) formation, and S-nitrosylation in tropomyosin were assessed, along with plasma troponin I and LV ejection fraction (LVEF). RESULTS The LV biopsies from the HF group had 2.14 ± 0.23-fold and 2.31 ± 0.46-fold greater levels in actin and tropomyosin carbonylation, respectively, and 1.77 ± 0.45-fold greater levels of high-molecular-weight complexes of tropomyosin due to DCB formation, compared with the NF group. Tropomyosin also underwent S-nitrosylation that was 1.3 ± 0.15-fold higher in the HF group. Notably, actin and tropomyosin carbonylation was significantly correlated with both loss of viability indicated by plasma troponin I and contractile impairment as shown by reduced LVEF. CONCLUSIONS This study demonstrated that oxidative/nitrosylative changes of actin and tropomyosin are largely increased in human failing hearts. Because these changes are inversely correlated to LVEF, actin and tropomyosin oxidation are likely to contribute to the contractile impairment evident in end-stage HF.

Differential ethylene-inducible expression of cellulase in pepper plants

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.

Global analysis of protein structural changes in complex proteomes

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ∼300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

Partly folded states of members of the lysozyme/lactalbumin superfamily: A comparative study by circular dichroism spectroscopy and limited proteolysis

The partly folded states of protein members of the lysozyme (LYS)/α‐lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123‐ to 129‐amino‐acid residues with similar three‐dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium‐binding properties and capability to adopt partly folded states or molten globules in acid solution (A‐state) or on depletion of calcium at neutral pH (apo‐state). Far‐ and near‐UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg‐white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo‐form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the β‐subdomain (approximately chain region 34–57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured α‐domain and a largely disrupted β‐subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.

The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat, trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis.

The calcium‐depleted form of α‐lactalbumin (α‐LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo‐α‐LA at neutral pH. CD spectra revealed that the molten globule of apo‐α‐LA can be obtained upon mild heating at 45°C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far‐ and near‐UV CD spectra of apo‐α‐LA are essentially identical to those of the most studied molten globule of α‐LA at pH 2.0 (A‐state). Proteolysis of the 123‐residue chain of apo‐α‐LA by proteinase K at 4°C occurs slowly as an all‐or‐none process leading to small peptides only. At 37°C, proteinase K preferentially cleaves apo‐α‐LA at peptide bonds Ser34‐Gly35, Gln39‐Ala40, Gln43‐Asn44, Phe53‐Gln54, and Asn56‐Asn57. All these peptide bonds are located at level of the β‐subdomain of the protein (chain region 34–57). Similar sites of preferential cleavage have been observed with the TFE‐ and oleic acid‐induced molten globule of apo‐α‐LA. A protein species given by the N‐terminal fragment 1–34 linked via the four disulfide bridges to the C‐terminal fragment 54–123 or 57–123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo‐α‐LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of α‐LA maintains a native‐like tertiary fold characterized by a rather well‐structured α‐domain and a disordered chain region encompassing the β‐subdomain 34–57 of the protein. Proteins 2002;49:385–397.

Dopamine quinones interact with α-synuclein to form unstructured adducts

alpha-Synuclein (alphasyn) fibril formation is considered a central event in the pathogenesis of Parkinsons disease (PD). In recent years, it has been proposed that prefibrillar annular oligomeric beta-sheet-rich species, called protofibrils, rather than fibrils themselves, may be the neurotoxic species. The oxidation products of dopamine (DAQ) can inhibit alphasyn fibril formation supporting the idea that DAQ might stabilize alphasyn protofibrils. In the present work, through different biochemical and biophysical techniques, we isolated and structurally characterized alphasyn/DAQ adducts. Contrary to protofibrils, we demonstrated that alphasyn/DAQ adducts retain an unfolded conformation. We then investigated the nature of the modifications induced on alphasyn by DAQ. Our results indicate that only a small fraction of alphasyn interacts with DAQ in a covalent way, so that non-covalent interaction appears to be the major modification induced by DAQ on alphasyn.

The oleic acid complexes of proteolytic fragments of α-lactalbumin display apoptotic activity

The complexes formed by partially folded human and bovine α‐lactalbumin with oleic acid (OA) have been reported to display selective apoptotic activity against tumor cells. These complexes were named human (HAMLET) or bovine (BAMLET) alpha‐lactalbumin made lethal to tumor cells. Here, we analyzed the OA complexes formed by fragments of bovine α‐lactalbumin obtained by limited proteolysis of the protein. Specifically, the fragments investigated were 53–103 and the two‐chain fragment species 1–40/53–123 and 1–40/104–123, these last being the N‐terminal fragment 1–40 covalently linked via disulfide bridges to the C‐terminal fragment 53–123 or 104–123. The OA complexes were obtained by mixing the fatty acid and the fragments in solution (10‐fold and 15‐fold molar excess of OA over protein fragment) or by chromatography of the fragments loaded onto an OA‐conditioned anion exchange column and salt‐induced elution of the OA complexes. Upon binding to OA, all fragments acquire an enhanced content of α‐helical secondary structure. All OA complexes of the fragment species showed apoptotic activity for Jurkat tumor cells comparable to that displayed by the OA complex of the intact protein. We conclude that the entire sequence of the protein is not required to form an apoptotic OA complex, and we suggest that the apoptotic activity of a protein–OA complex does not imply specific binding of the protein.