Abstract
Alpha-1-antitrypsin (AAT) is a protein that protects our lungs against damage from proteases released during inflammation (neutrophil elastase and proteinase-3). Lung disease in alpha1-antitrypsin deficiency (AATD) occurs when we don’t have enough of this protective activity due to lower levels of AAT in the circulation. Therefore, standard clinical tests generally measure the amount of AAT that is present in the blood. However, different lines of evidence indicate that a considerable proportion of circulating AAT mutants may be present in non-functional forms such as polymers, bound to other proteins or have reduced anti-protease activity, as demonstrated for the Z (E342K), F (R223C) and Pittsburgh (M358R) AAT variants. In addition, it has been shown that oxidation of AAT from smoking reduces its inhibitory function.
These results provide a strong rationale for including functional measurements of plasma AAT activity in AATD diagnostic protocols both for carriers of the most common AAT genotypes, MZ, SZ and ZZ, and for the increasing number of cases with rare genotypes associated with the disease. As a striking example, lack of application of a functional assay would fail to identify dysfunctional variants that are secreted normally by cells but lacks anti-elastase activity, such as the Iners AAT variant (G349R) identified by genome-wide population screenings. Reduced AAT activity could also help explain the higher risk of lung disease observed in a subgroup of MZ individuals and the more severe progression in some SZ and ZZ patients.
In this project, multiple experimental strategies will be used to investigate dysfunction of circulating AAT. The available methodologies to test AAT anti-protease function will be optimized and then applied on plasma samples from a cohort of MM, MS, MZ, SZ and ZZ subjects, in collaboration with two Italian referral centers for AATD. To further understand which AAT modifications contribute to AAT dysfunction, using these samples we will: (1) measure the levels of AAT in the inactive polymeric form; (2) measure the formation of AAT heteropolymers in heterozygotes; (3) detect the presence of oxidized AAT; (4) investigate the formation of stable complexes between specific AAT mutants (Z, F) and other plasma proteins; (4) analyze the intrinsic dysfunction of the F variant (R223C) and of amino acid variations localized in the AAT region (RCL) that mediates the interaction with target proteases.
This project will evaluate a parameter that is well-known to be associated with lung pathology in AATD but is not investigated in most diagnostic flowcharts. It is anticipated that these analyses will significantly increase our understanding of the molecular bases of AAT dysfunction, help to understand the individual variability in AATD clinical presentation and lead to wider application of AAT activity assays in clinical laboratories.
Finanziamento assegnato ad UniBS: € 128.562,00