Lung disease in alpha-1-antitrypsin deficiency (AATD) results from dysregulated proteolytic activity, mainly by neutrophil elastase (HNE), in the lung parenchyma. This is the result of a substantial reduction of circulating alpha-1-antitrypsin (AAT) and the presence in the plasma of inactive polymers of AAT. Moreover, some AAT mutants have reduced intrinsic activity toward HNE, as demonstrated for the common Z mutant, as well as for other rarer variants. Here we report the identification and characterisation of the novel AAT reactive centre loop variant Gly349Arg (p.G373R) present in the ExAC database. This AAT variant is secreted at normal levels in cellular models of AATD but shows a severe reduction in anti-HNE activity. Biochemical and molecular dynamics studies suggest it exhibits unfavourable RCL presentation to cognate proteases and compromised insertion of the RCL into β-sheet A. Identification of a fully dysfunctional AAT mutant that does not show a secretory defect underlines the importance of accurate genotyping of patients with pulmonary AATD manifestations regardless of the presence of normal levels of AAT in the circulation. This subtype of disease is reminiscent of dysfunctional phenotypes in anti-thrombin and C1-inibitor deficiencies so, accordingly, we classify this variant as the first pure functionally-deficient (type II) AATD mutant.

Mitotane is the cornerstone of medical treatment of adrenocortical carcinoma. Estrogenic-like side effects frequently occur in patients, and previous studies explored the chemical nature of the interaction between estrogen receptor-α (ER-α) and toxic compounds, including the DDD derivatives. We used molecular docking and molecular dynamics (MD) simulations to explore the possible interaction between mitotane and the ER-α receptor and the induced conformational changes. The ER-α expressing MCF-7 cells were exposed to mitotane with/without tamoxifen, and the cell viability/proliferation was evaluated by MTT assay and direct count. The transient ER-α silencing was performed using two ER-α siRNA (50 nM) and verified by Western blot. MDA-MB-231 cells were used as a negative control. Mitotane showed a similar docking configuration to 17β-estradiol and bisphenol A (BPA) and a significant binding affinity to ER-α. MD simulations showed that mitotane preserves the active conformation of ER-α more than both BPA and Bisphenol C, classifying it as an agonist. Exposure of MCF-7 cells to mitotane led to the concentration-dependent increase of cell viability and proliferation, which was reduced in the presence of tamoxifen and nullified by the transient ER-α knock-down. Integrating bioinformatics approaches with cell biology and pharmacological methods, we demonstrated that mitotane directly binds and activates ER-α.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed disorder associated with mutations in the SERPINA1 gene encoding alpha-1 antitrypsin (AAT). Severe AATD can manifest as pulmonary emphysema and progressive liver disease. Besides the most common pathogenic variants S (E264V) and Z (E342K), many rarer genetic variants of AAT have been found in patients and in the general population. Here we report a panel of new SERPINA1 variants, including 4 null and 16 missense alleles, identified among a cohort of individuals with suspected AATD whose phenotypic follow-up showed inconclusive or atypical results. Because the pathogenic significance of the missense variants was unclear purely on the basis of clinical data, the integration of computational, biochemical, and cellular studies was used to define the associated risk of disease. Established pathogenicity predictors and structural analysis identified a panel of candidate damaging mutations that were characterized by expression in mammalian cell models. Polymer formation, intracellular accumulation, and secretory efficiency were evaluated experimentally. Our results identified two AAT mutants with a Z-like polymerogenic severe deficiency profile (Smilano and Mcampolongo) and three milder variants (Xsarezzo, Pdublin, and Ctiberias). Overall, the experimentally determined behavior of the variants was in agreement with the pathogenicity scores of the REVEL (an ensemble method for predicting the pathogenicity of rare missense variants) predictor, supporting the utility of this bioinformatic tool in the initial assessment of newly identified amino acid substitutions of AAT. Our study, in addition to describing 20 new SERPINA1 variants, provides a model for a multidisciplinary approach to classification of rare AAT variants and their clinical impact on individuals with rare AATD genotypes.

In mammals, adenosine (A) to inosine (I) RNA editing is performed by adenosine deaminases acting on RNA (ADAR), ADAR1 and ADAR2 enzymes, encoded by mRNAs that might undergo splicing process. In rat, two splicing events produce several isoforms of ADAR2, called ADAR2a, ADAR2b, ADAR2e, and ADAR2f, but only ADAR2a and ADAR2b are translated into an active protein. In particular, they differ for ten amino acids located in the catalytic domain of ADAR2b. Here, we focused on these two isoforms, analyzing the splicing pattern and their different function during rat neuronal maturation. We found an increase of editing levels in cortical neurons overexpressing ADAR2a compared to those overexpressing ADAR2b. These results indicate ADAR2a isoform as the most active one, as reported for the homologous human short variant. Furthermore, we showed that the differential editing activity is not due to a different dimerization of the two isoforms; it seems to be linked to the ten amino acids loop of ADAR2b that might interfere with RNA binding, occupying the space volume in which the RNA should be present in case of binding. These data might shed light on the complexity of ADAR2 regulations.

The $H_2^{+}$ ion is the simplest example in which a chemical bond exists, created by one electron between two protons. As all chemical bonds, it is usually considered inexplicable in a classical frame. Here, in view of the extremely large velocities attained by the electron near the protons, we consider a relativistic extension of the standard classical three-body model. This has a great impact since the reference unperturbed system (clamped protons) is no more integrable, and indeed by molecular dynamics simulations, we find that the modification entails the existence of a large region of strongly chaotic motions for the unperturbed system, which lead, for the full system, to a collapse of the molecule. For motions of generic type, with the electron bouncing between the protons, there exists an open region of motions regular enough for producing a bond. Such a region is characterized by the property that the electron’s trajectories have an angular momentum $p_{\phi }$ along the inter-nuclear axis of the order of the reduced Planck’s constant $\hslash$ . Moreover, special initial data exist for which the experimental bond length and oscillation frequency of the protons (but not the dissociation energy) are well reproduced. Also, well reproduced is the quantum potential, albeit only in an extended interval about the minimum.

Neutrophil elastase (NE) is released by activated neutrophils during an inflammatory response and exerts proteolytic activity on elastin and other extracellular matrix components. This protease is rapidly inhibited by the plasma serine protease inhibitor alpha‐1‐antitrypsin (AAT), and the importance of this protective activity on lung tissue is highlighted by the development of early onset emphysema in individuals with AAT deficiency. As a serpin, AAT presents a surface‐exposed reactive centre loop (RCL) whose sequence mirrors the target protease specificity. Following binding of NE in a ‘Michaelis’ encounter complex, cleavage of the RCL results in an irreversible complex between the two molecules. Here, the structure of the AAT‐NE encounter complex was studied by molecular dynamics, mutagenesis and enzyme kinetics. Exploration of the geometry of interaction between the two molecules revealed the possibility that the interaction interface extends beyond the RCL; a persistent feature of the simulations was the interaction between a region located upstream of β‐strand 4C of AAT, comprising three acidic residues (Asp202, Glu199 and Glu204), and Arg147 of NE. Mutation of the acidic residues to either alanine or serine, or a D202R substitution, resulted in a reduced rate of association between recombinant AAT and NE. Addition of salt to the buffer had little effect for these mutants but substantially reduced the rate of interaction of the wild‐type protein. These data are consistent with a role for this acidic region on AAT as an exosite that contributes to an optimal interaction with its physiological protease target.