José M. Delfino

Epicatechin, catechin, and dimeric procyanidins inhibit PMA-induced NF-κB activation at multiple steps in Jurkat T cells

The capacity of the flavan‐3‐ols [(–)‐epicatechin (EC) and (+)‐catechin (CT)] and a B dimeric procyanidin (DP‐B) to modulate phorbol 12‐myristate 13‐acetate (PMA)‐induced NF‐κB activation in Jurkat T cells was investigated. The classic PMA‐triggered increase in cell oxidants was prevented when cells were preincubated for 24 h with EC, CT, or DP‐B (1.7–17.2 μM). PMA induced the phosphorylation of IKKβ and the subsequent degradation of IκBα: These events were inhibited in cells pretreated with the flavonoids. PMA induced a 4.6‐fold increase in NF‐κB nuclear binding activity in control cells. Pretreatment with EC, CT, or DP‐B decreased PMA‐induced NF‐κB binding activity and the transactivation of the NF‐κB‐driven gene IL‐2. EC, CT, and DP‐B inhibited, in vitro, NF‐κB binding to its DNA consensus sequence, but they had no effect on the binding activity of CREB or OCT‐1. Thus, EC, CT, or DP‐B can influence the immune response by modulating NF‐κB activation. This modulation can occur at early (regulation of oxidant levels, IKK activation) as well as late (binding of NF‐κB to DNA) stages of the NF‐κB activation cascade. A model is presented for possible interactions between DP‐B and NF‐κB proteins, which could lead to the inhibition of NF‐κB binding to κB sites.

Correction for phylogeny, small number of observations and data redundancy improves the identification of coevolving amino acid pairs using mutual information

MOTIVATION Mutual information (MI) theory is often applied to predict positional correlations in a multiple sequence alignment (MSA) to make possible the analysis of those positions structurally or functionally important in a given fold or protein family. Accurate identification of coevolving positions in protein sequences is difficult due to the high background signal imposed by phylogeny and noise. Several methods have been proposed using MI to identify coevolving amino acids in protein families. RESULTS After evaluating two current methods, we demonstrate how the use of sequence-weighting techniques to reduce sequence redundancy and low-count corrections to account for small number of observations in limited size sequence families, can significantly improve the predictability of MI. The evaluation is made on large sets of both in silico-generated alignments as well as on biological sequence data. The methods included in the analysis are the APC (average product correction) and RCW (row-column weighting) methods. The best performing method was APC including sequence-weighting and low-count corrections. The use of sequence-permutations to calculate a MI rescaling is shown to significantly improve the prediction accuracy and allows for direct comparison of information values across protein families. Finally, we demonstrate how a lower bound of 400 sequences <62% identical is needed in an MSA in order to achieve meaningful predictive performances. With our contribution, we achieve a noteworthy improvement on the current procedures to determine coevolution and residue contacts, and we believe that this will have potential impacts on the understanding of protein structure, function and folding.

Dimeric procyanidins are inhibitors of NF-κB–DNA binding

Given the central role of the transcription factor NF-kappaB in inflammation, molecules that can inhibit NF-kappaB are being actively investigated. The present work characterize potential interactions between dimeric procyanidins [B-type (B1 and B2) and A-type (A1 and A2)] and NF-kappaB proteins. B1 and B2, inhibited tumor necrosis factor alpha (TNFalpha)- and phorbol 12-myristate 13-acetate (PMA)-induced transactivation of NF-kappaB-driven genes and the increase of NF-kappaB-DNA nuclear binding in Jurkat T cells. B1 and B2, added in vitro to nuclear fractions, inhibited NF-kappaB binding to its DNA consensus sequence. B1 and B2 prevented the binding of RelA and p50 recombinant proteins to its DNA consensus sequence. All these effects were not observed with A1 and A2. Putative molecular models for possible interactions of B1, B2, A1 and A2, with NF-kappaB proteins were constructed, indicating that B-type dimeric procyanidins have higher possibilities of chemical interactions with NF-kappaB than A-type dimeric procyanidins. The results support the concept that B-type dimeric procyanidins can provide anti-inflammatory benefits due to their ability to reduce NF-kappaB binding to the DNA.

Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification.

Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.

The Structure of Calreticulin C-terminal Domain Is Modulated by Physiological Variations of Calcium Concentration

Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.

Probing protein conformation with a minimal photochemical reagent.

3H‐diazirine (3H‐DZN), a photoreactive gas similar in size to water, was used to probe the topography of the surface and inner space of proteins. On photolysis 3H‐DZN generates 3H‐methylene carbene, which reacts unselectively with its molecular cage, inserting even into C‐H bonds. Labeling of bovine α‐lactalbumin (α‐LA, MW: 14,200) with 1 mM 3H‐DZN yielded 0.0041 mol CH2/mol of protein, in agreement with the expectation for an unspecific surface‐labeling phenomenon. The cooperative urea‐induced unfolding of α‐LA, as monitored by the extent of 3H‐methylene labeling, agrees with that measured by circular dichroism spectroscopy in the far and near ultraviolet regions. At 8 M urea, the unfolded state U was labeled 25–30% more than the native state N primarily because of the increase in the accessible surface area (ASA) of the protein occurring upon unfolding. However, this result lies below the ∼100% increment expected from theoretical estimates of ASA of state U. Among other factors, most likely the existence of a residual structure in U, that involves helices H2 and H4 of the α subdomain, might account for this fact, as shown by a comparative analysis of peptide labeling patterns of N and U samples. In this paper, we demonstrate the usefulness of the 3H‐methylene labeling method to monitor conformational transitions and map solvent accessibility along the polypeptide sequence, thus opening the possibility of outlining structural features of nonnative states (i.e., denatured states, molten globule). We anticipate that this technique also would help to identify ligand binding and oligomerization sites in proteins.

Exploring protein interfaces with a general photochemical reagent

Protein folding, natural conformational changes, or interaction between partners involved in recognition phenomena brings about differences in the solvent‐accessible surface area (SASA) of the polypeptide chain. This primary event can be monitored by the differential chemical reactivity of functional groups along the protein sequence. Diazirine (DZN), a photoreactive gas similar in size to water, generates methylene carbene (:CH2). The extreme chemical reactivity of this species allows the almost instantaneous and indiscriminate modification of its immediate molecular cage. 3H‐DZN was successfully used in our laboratory for studying protein structure and folding. Here we address for the first time the usefulness of this probe to examine the area of interaction in protein–protein complexes. For this purpose we chose the complex formed between hen egg white lysozyme (HEWL) and the monoclonal antibody IgG1 D1.3. :CH2 labeling of free HEWL or complexed with IgG1 D1.3 yields 2.76 and 2.32 mmol CH2 per mole protein at 1 mM DZN concentration, respectively. This reduction (15%) becomes consistent with the expected decrement in the SASA of HEWL occurring upon complexation derived from crystallographic data (11%), in agreement with the known unspecific surface labeling reaction of :CH2. Further comparative analysis at the level of tryptic peptides led to the identification of the sites involved in the interaction. Remarkably, those peptides implicated in the contact area show the highest differential labeling: H15GLDNYR21, G117TDVQAWIR125, andG22YSLGNWVCAAK33. Thus, protein footprinting with DZN emerges as a feasible methodology useful for mapping contact regions of protein domains involved in macromolecular assemblies.

Stoichiometry of lipid–protein interaction assessed by hydrophobic photolabeling

Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P‐type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K‐ATPase. The photoactivatable phosphatidylcholine analogue [125I]TID‐PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C12E10. After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K‐ATPase (α‐subunit) and 5.6 mol PC/mol protein for the Na,K‐ATPase (β‐subunit).

Experimentally Approaching the Solvent-Accessible Surface Area of a Protein: Insights into the Acid Molten Globule of Bovine α-Lactalbumin

Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein alpha-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH(2)) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of alpha-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH(2) labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH(2) labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states.

Amyloidogenic propensity of a natural variant of human apolipoprotein A-I: stability and interaction with ligands.

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.