Principal component analysis of the conformational freedom within the EF-hand superfamily

A database of nonredundant structures of EF-hand domains--i.e., pairs of helix-loop-helix motifs--has been assembled, and the six angles among the four helices re-determined. A principal component analysis of these angles allows us to use two such components (PC1 and PC2) to describe the system retaining 80% of the total variance. A PC2 against PC1 plot representation allows us to represent in a compact way the full range of structural diversity of EF-hand domains, their grouping into protein families, and the variation for each family upon calcium and peptide binding.     

Evidence that increases of mitochondrial immunoreactive IL-1beta by HIV-1 gp120 implicate in situ cleavage of pro-IL-1beta in the neocortex of rat

Immunoelectron microscopy analysis of brain tissue sections and rat-specific sandwich ELISA allowed the localization of interleukin-1beta (IL-1beta) immunoreactivity in the mitochondria and cytosol of neocortical tissue preparations from the brain of naive, untreated, rats and rats receiving a single daily injection into one lateral cerebral ventricle (i.c.v.) of bovine serum albumin (BSA; 100 ng/day) for seven consecutive days. Interestingly, seven days i.c.v. treatment with the HIV-1 coat protein gp120 (100 ng/day) enhances IL-1beta immunoreactivity in the cellular fractions studied. Elevation of mitochondrial immunoreactive IL-1beta levels seems to originate from the conversion operated by the interleukin converting enzyme (ICE) of mitochondrial pro-IL-1beta; in fact, IL-1beta increases reported in the ELISA experiments were paralleled by a decrease of the mitochondrial pro-IL-1beta 31-kDa band in conjunction with enhanced expression of the p20 component of activated ICE. In conclusion, the present results demonstrate that gp120-enhanced neocortical expression of IL-1beta originates, at least in part, from in situ cleavage of mitochondrial pro-IL-1beta and suggest that this, together with the central role of the mitochondrion in the expression of programmed cell death, may be important for apoptosis induced by the viral coat protein in the brain of rats.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

The anti-apoptotic Bcl-x(L) protein, a new piece in the puzzle of cytochrome c interactome

A structural model of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-x(L), which defines the protein-protein interaction surface, was obtained from solution NMR chemical shift perturbation data. The atomic level information reveals key intermolecular contacts identifying new potentially druggable areas on cytochrome c and Bcl-x(L). Involvement of residues on cytochrome c other than those in its complexes with electron transfer partners is apparent. Key differences in the contact area also exist between the Bcl-x(L) adduct with the Bak peptide and that with cytochrome c. The present model provides insights to the mechanism by which cytochrome c translocated to cytosol can be intercepted, so that the apoptosome is not assembled.     

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose-regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress-response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non-coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non-coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress-response genes.     

Cytochrome c and organic molecules: solution structure of the p-aminophenol adduct

Protein-protein interactions are driven by specific properties of the molecular surfaces. Cytochrome c, a small electron transfer protein, is involved in a number of biologically relevant interactions with macromolecular partners. Small molecules may interfere with such interactions by binding to the surface of cytochrome c. Here we investigated the possibility of weak intermolecular interactions between reduced cytochrome c and a library of 325 small molecules, using WaterLOGSY NMR spectroscopy. Specific binding was found for p-aminophenol. The solution structure of the p-aminophenol-cytochrome c adduct was determined using a combination of in silico tools and NMR-based restraints. The ligand interacts in a specific binding site on the protein surface through a combination of stacking and H-bond interactions. Small but meaningful rearrangements of the solvent-exposed side chains are observed upon ligand binding and contribute to the stabilization of the complex.     

Metalation of the amyotrophic lateral sclerosis mutant glycine 37 to arginine superoxide dismutase (SOD1) apoprotein restores its structural and dynamical properties in solution to those of metalated wild-type SOD1

The G37R copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis by an unknown mechanism. This particular mutation occurs in the beta barrel plug, a region proposed to be critical for the structural stability of the protein. The behavior of G37R asSOD1 was studied in solution where it was observed that, when the protein is fully metalated, its global structure, mobility, and stability are virtually indistinguishable from those of the nonmutated protein. By contrast, although the presence of the G37R mutation does not result in a substantial change of the overall structure of the metal-free apoprotein in solution, it does affect the key conformational features of the beta-barrel plug such that (i) apo G37R asSOD1 melts at a temperature approximately 10 degrees C lower than apo asSOD1, (ii) it aggregates more rapidly than apo asSOD1, and (iii) interaction with trifluoroethanol (TFE) can deform it into a structure with a much higher degree of alpha-helical content. The increased plasticity of the apo G37R asSOD1 mutant protein is likely responsible for its enhanced tendency to aggregate in concentrated solutions. These results suggest further that it is the metal-free apo forms of the mutant SOD1 protein that are the agents of its toxicity.     

Profiling metabolites and lipoproteins in COMETA,an Italian cohort of COVID-19 patients

Metabolomics and lipidomics have been used in several studies to define the biochemical alterations induced by COVID-19 in comparison with healthy controls. Those studies highlighted the presence of a strong signature, attributable to both metabolites and lipoproteins/lipids. Here, ¹H NMR spectra were acquired on EDTA-plasma from three groups of subjects: i) hospitalized COVID-19 positive patients (≤21 days from the first positive nasopharyngeal swab); ii) hospitalized COVID-19 positive patients (>21 days from the first positive nasopharyngeal swab); iii) subjects after 2–6 months from SARS-CoV-2 eradication. A Random Forest model built using the EDTA-plasma spectra of COVID-19 patients ≤21 days and Post COVID-19 subjects, provided a high discrimination accuracy (93.6%), indicating both the presence of a strong fingerprint of the acute infection and the substantial metabolic healing of Post COVID-19 subjects. The differences originate from significant alterations in the concentrations of 16 metabolites and 74 lipoprotein components. The model was then used to predict the spectra of COVID-19>21 days subjects. In this group, the metabolite levels are closer to those of the Post COVID-19 subjects than to those of the COVID-19≤21 days; the opposite occurs for the lipoproteins. Within the acute phase patients, characteristic trends in metabolite levels are observed as a function of the disease severity. The metabolites found altered in COVID-19≤21 days patients with respect to Post COVID-19 individuals overlap with acute infection biomarkers identified previously in comparison with healthy subjects. Along the trajectory towards healing, the metabolome reverts back to the “healthy” state faster than the lipoproteome.     

Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.