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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.

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 formation of ordered Z α1‐antitrypsin polymers is central to liver disease in α1‐antitrypsin deficiency. The nascent α1‐antitrypsin folds via the M* intermediate to native monomer or becomes incorporated into a soluble polymer that can be secreted, degraded or precipitate as insoluble inclusions. We describe the kinetics of the clearance of Z α1‐antitrypsin polymers and show that they can be abolished with a small molecule preventing the formation of M*., 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.

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., A monoclonal antibody developed as a single-molecule probe has revealed pathological aggregates of α-1-antitrypsin capture the WT variant.

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.

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.

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.

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.

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.

Pathological polymers of Z alpha1-AT can be secreted from cells through the canonical secretory pathway http://ow.ly/WHxZh

Previous studies on IgM secretion demonstrated a role for the mu chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of mu and alpha heavy chains prompted us to investigate the role of cysteine-mediated, retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, alpha 2L2 subunits, but not mu2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the alpha chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.

Proteins that are not secreted or otherwise released by cells must be eventually degraded. Cytosolic degradation cannot cope with proteins that have been delivered unidirectionally to membrane-bound compartments. Hence, proteolytic systems are likely to be present in all compartments where proteins tend to accumulate (e.g., mitochondria, peroxisomes). In this respect, the endoplasmic reticulum (ER) appears to be no exception, since an independent degradative system has been identified in this organelle (Lippincott-Schwartz et al., 1988; Klausner and Sitia, 1990).