Quantification of circulating alpha-1-antitrypsin polymers associated with different SERPINA1 genotypes.

OBJECTIVES: Alpha-1-antitrypsin deficiency is a genetic disorder caused by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin (AAT), the major serine protease inhibitor in plasma. Reduced AAT levels are associated with elevated risk of developing emphysema mainly due to uncontrolled activity of neutrophil elastase in the lungs. The prevalent Z-AAT mutant and many rare pathogenic AAT variants also predispose to liver disease due to their accumulation as polymeric chains in hepatocytes. Part of these polymers are secreted into the bloodstream and could represent biomarkers of intra-hepatic accumulation. Moreover, being inactive, they further lower lung protection against proteases. Aim of our study is to accurately quantify the percentage of circulating polymers (CP) in a cohort of subjects with different SERPINA1 genotypes. METHODS: CP concentration was measured in plasma or Dried Blood Spot (DBS) by a sensitive sandwich ELISA based on capture by the polymer-specific 2C1 monoclonal antibody. RESULTS: CP were significantly elevated in patients with the prevalent PI*SZ and PI*ZZ genotypes, with considerable intra-genotype variability. Notably, higher percentage of polymers was observed in association with elevated C-reactive protein. CP levels were also increased in carriers of the Mmalton variant, and of Mprocida, I, Plowell and Mherleen in heterozygosity with Z-AAT. CONCLUSIONS: These findings highlight the importance of implementing CP quantification in a clinical laboratory. Indeed, the variable amount of CP in patients with the same genotype may correlate with the variable severity of the associated lung and liver diseases. Moreover, CP can reveal the polymerogenic potential of newly discovered ultrarare AAT variants.

Characterization of Novel Alpha-1-Antitrypsin Coding Variants in a Mammalian Cellular Model.

Advances in genetic screening technologies have considerably accelerated the discovery of rare alpha-1-antitrypsin (AAT) variants. Expression in cellular models is an effective approach to evaluate the pathogenic potential of these new AAT variants, whose clinical significance would otherwise remain uncertain. Here we provide a detailed description of established methods for in vitro characterization of AAT coding variants expressed in HEK293T/17 cells. The protocols include determination of secretion efficiency, the tendency to form polymeric chains and  the anti-elastase inhibitory activity.

Probing of the reactive center loop region of alpha-1-antitrypsin by mutagenesis predicts new type-2 dysfunctional variants.

Lung disease in alpha-1-antitrypsin deficiency (AATD) mainly results from insufficient control of the serine proteases neutrophil elastase (NE) and proteinase-3 due to reduced plasma levels of alpha-1-antitrypsin (AAT) variants. Mutations in the specificity-determining reactive center loop (RCL) of AAT would be predicted to minimally affect protein folding and secretion by hepatocytes but can impair anti-protease activity or alter the target protease. These properly secreted but dysfunctional 'type-2' variants would not be identified by common diagnostic protocols that are predicated on a reduction in circulating AAT. This has potential clinical relevance: in addition to the dysfunctional Pittsburgh and Iners variants reported previously, several uncharacterized RCL variants are present in genome variation databases. To prospectively evaluate the impact of RCL variations on secretion and anti-protease activity, here we performed a systematic screening of amino acid substitutions occurring at the AAT-NE interface. Twenty-three AAT variants that can result from single nucleotide polymorphisms in this region, including 11 present in sequence variation databases, were expressed in a mammalian cell model. All demonstrated unaltered protein folding and secretion. However, when their ability to form stable complexes with NE was evaluated by western blot, enzymatic assays, and a novel ELISA developed to quantify AAT-NE complexes, substrate-like and NE-binding deficient dysfunctional variants were identified. This emphasizes the ability of the RCL to accommodate inactivating substitutions without impacting the integrity of the native molecule and demonstrates that this class of molecule violates a generally accepted paradigm that equates circulating levels with functional protection of lung tissue.

Human iPSC-Derived 3D Hepatic Organoids in a Miniaturized Dynamic Culture System.

The process of identifying and approving a new drug is a time-consuming and expensive procedure. One of the biggest issues to overcome is the risk of hepatotoxicity, which is one of the main reasons for drug withdrawal from the market. While animal models are the gold standard in preclinical drug testing, the translation of results into therapeutic intervention is often ambiguous due to interspecies differences in hepatic metabolism. The discovery of human induced pluripotent stem cells (hiPSCs) and their derivatives has opened new possibilities for drug testing. We used mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells, to miniaturize the process of generating hepatic organoids. These organoids were then cultivated in vitro using both static and dynamic cultures. Additionally, we tested spheroids solely composed by induced hepatocytes. By miniaturizing the system, we demonstrated the possibility of maintaining the organoids, but not the spheroids, in culture for up to 1 week. This timeframe may be sufficient to carry out a hypothetical pharmacological test or screening. In conclusion, we propose that the hiPSC-derived liver organoid model could complement or, in the near future, replace the pharmacological and toxicological tests conducted on animals.

Comprehensive Clinical Diagnostic Pipelines Reveal New Variants in Alpha-1 Antitrypsin Deficiency.

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.

Neuroserpin: structure, function, physiology and pathology.

Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

The molecular species responsible for α1 -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α1 -antitrypsin polymers in hepatocytes is central to liver disease in α1 -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1 -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1 -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.

Intrahepatic heteropolymerization of M and Z alpha-1-antitrypsin.

The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%-5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo. To this end, we developed a monoclonal antibody (mAb2H2) that specifically binds to M in preference to Z A1AT, localized its epitope using crystallography to a region perturbed by the Z (Glu342Lys) substitution, and used Fab fragments to label polymers isolated from an MZ heterozygote liver explant. Glu342 is critical to the affinity of mAb2H2, since it also recognized the mild S-deficiency variant (Glu264Val) present in circulating polymers from SZ heterozygotes. Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample. These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals.

Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population.

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 ß-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.

Cellular Models for the Serpinopathies.

Our current knowledge about the cellular mechanisms underlying serpin-related disorders, the serpinopathies, is predominantly based on studies in cell culture models of disease, particularly for alpha-1 antitrypsin (AAT, SERPINA1) deficiency causing emphysema and the familial encephalopathy with neuroserpin (NS, SERPINI1) inclusion bodies (FENIB). FENIB, a neurodegenerative dementia, is caused by polymerization of NS (Miranda and Lomas, Cell Mol Life Sci 63:709-722, 2006; Roussel BD et al., Epileptic Disor 18:103-110, 2016), while AAT deficiency presents as a result of several divergent mutations in the AAT gene that cause lack of protein synthesis or complete intracellular degradation (null variants) or polymer formation (polymerogenic variants) (Lomas et al., J Hepatol 65:413-424, 2016; Greene et al., Nat Rev Dis Primers 2:16051, 2016; Ferrarotti et al. Orphanet J Rare D 9:172, 2014). Both diseases have been extensively modeled in cell culture systems by expressing mutant variants in a variety of ways. Here we describe the methodologies we follow in our cell model systems used to examine serpin disorders.

Real-world clinical applicability of pathogenicity predictors assessed on SERPINA1 mutations in alpha-1-antitrypsin deficiency.

The growth of publicly available data informing upon genetic variations, mechanisms of disease, and disease subphenotypes offers great potential for personalized medicine. Computational approaches are likely required to assess a large number of novel genetic variants. However, the integration of genetic, structural, and pathophysiological data still represents a challenge for computational predictions and their clinical use. We addressed these issues for alpha-1-antitrypsin deficiency, a disease mediated by mutations in the SERPINA1 gene encoding alpha-1-antitrypsin. We compiled a comprehensive database of SERPINA1 coding mutations and assigned them apparent pathological relevance based upon available data. "Benign" and "pathogenic" variations were used to assess performance of 31 pathogenicity predictors. Well-performing algorithms clustered the subset of variants known to be severely pathogenic with high scores. Eight new mutations identified in the ExAC database and achieving high scores were selected for characterization in cell models and showed secretory deficiency and polymer formation, supporting the predictive power of our computational approach. The behavior of the pathogenic new variants and consistent outliers were rationalized by considering the protein structural context and residue conservation. These findings highlight the potential of computational methods to provide meaningful predictions of the pathogenic significance of novel mutations and identify areas for further investigation.

Heteropolymerization of α-1-antitrypsin mutants in cell models mimicking heterozygosity.

The most common genotype associated with severe α-1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α-1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M. However, it is unknown whether Z AAT can co-polymerize with other AAT variants in vivo. We co-expressed two AAT variants, each modified by a different tag, in cell models that replicate AAT deficiency. We used pull-down assays to investigate interactions between co-expressed variants and showed that Z AAT forms heteropolymers with S and with the rare Mmalton (Phe52del) and Mwurzburg (Pro369Ser) mutants, and to a lesser extent with the wild-type protein. Heteropolymers were recognized by the 2C1 mAb that binds to Z polymers in vivo. There was increased intracellular accumulation of AAT variants when co-expressed with Z AAT, suggesting a dominant negative effect of the Z allele. The molecular interactions between S and Z AAT were confirmed by confocal microscopy showing their colocalization within dilated ER cisternae and by positivity in Proximity Ligation Assays. These results provide the first evidence of intracellular co-polymerization of AAT mutants and contribute to understanding the risk of liver disease in SZ and MZ heterozygotes.

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight.

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress.

Cysteines as Redox Molecular Switches and Targets of Disease.

Thiol groups can undergo numerous modifications, making cysteine a unique molecular switch. Cysteine plays structural and regulatory roles as part of proteins or glutathione, contributing to maintain redox homeostasis and regulate signaling within and amongst cells. Not surprisingly therefore, cysteines are associated with many hereditary and acquired diseases. Mutations in the primary protein sequence (gain or loss of a cysteine) are most frequent in membrane and secretory proteins, correlating with the key roles of disulfide bonds. On the contrary, in the cytosol and nucleus, aberrant post-translational oxidative modifications of thiol groups, reflecting redox changes in the surrounding environment, are a more frequent cause of dysregulation of protein function. This essay highlights the regulatory functions performed by protein cysteine residues and provides a framework for understanding how mutation and/or (in)activation of this key amino acid can cause disease.

The pathological Trento variant of alpha-1-antitrypsin (E75V) shows nonclassical behaviour during polymerization.

Severe alpha-1-antitrypsin deficiency (AATD) is most frequently associated with the alpha-1-antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early-onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. Polyacrylamide gel electrophoresis (PAGE) and ELISA-based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localized between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilizing interaction between helix C and the posthelix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT.