Ugo I. Ekeowa

Defining the mechanism of polymerization in the serpinopathies

The serpinopathies result from the ordered polymerization of mutants of members of the serine proteinase inhibitor (serpin) superfamily. These polymers are retained within the cell of synthesis where they cause a toxic gain of function. The serpinopathies are exemplified by inclusions that form with the common severe Z mutant of α1-antitrypsin that are associated with liver cirrhosis. There is considerable controversy as to the pathway of serpin polymerization and the structure of pathogenic polymers that cause disease. We have used synthetic peptides, limited proteolysis, monoclonal antibodies, and ion mobility-mass spectrometry to characterize the polymerogenic intermediate and pathological polymers formed by Z α1-antitrypsin. Our data are best explained by a model in which polymers form through a single intermediate and with a reactive center loop-β-sheet A linkage. Our data are not compatible with the recent model in which polymers are linked by a β-hairpin of the reactive center loop and strand 5A. Understanding the structure of the serpin polymer is essential for rational drug design strategies that aim to block polymerization and so treat α1-antitrypsin deficiency and the serpinopathies.

A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with α1‐antitrypsin deficiency

Alpha1‐antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation‐specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by α1‐antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; α1‐antitrypsin Kings) identified in a 6‐week‐old boy who presented with prolonged jaundice. His334Asp α1‐antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild‐type M α1‐antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp α1‐antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of α1‐antitrypsin. Conclusion: Z and shutter domain mutants of α1‐antitrypsin form polymers with a shared epitope and so are likely to have a similar structure. HEPATOLOGY 2010

Mechanisms of emphysema in α1-antitrypsin deficiency: molecular and cellular insights

The severe, early onset emphysema that occurs in patients with circulating deficiency of α1-antitrypsin (α1-AT) attests to the importance of this protease inhibitor in maintaining lung parenchymal integrity. It has led to the powerful concept of protease:antiprotease balance being crucial to alveolar homeostasis. Pathogenic mutations cause α1-AT to self-associate into polymer chains that accumulate intracellularly rather than proceeding along the secretory pathway. Polymerisation of α1-AT abolishes antiprotease activity and confers toxic gain-of-function effects. Since α1-AT is predominantly synthesised in the liver, where it does not play a major homeostatic role, the directly toxic effects of polymerisation are clearest here. However, data from molecular, cellular, animal and ex vivo studies indicate that intrapulmonary polymerisation of α1-AT and inflammatory positive feedback loops may augment the destructive effects of decreased antiprotease levels in the lung. This review integrates the findings from these different approaches and highlights how multiple pathways may converge to give the severe, panacinar emphysema phenotype seen in α1-AT deficiency.

α1-Antitrypsin deficiency, chronic obstructive pulmonary disease and the serpinopathies

alpha1-Antitrypsin is the prototypical member of the serine proteinase inhibitor or serpin superfamily of proteins. The family includes alpha1-antichymotrypsin, C1 inhibitor, antithrombin and neuroserpin, which are all linked by a common molecular structure and the same suicidal mechanism for inhibiting their target enzymes. Point mutations result in an aberrant conformational transition and the formation of polymers that are retained within the cell of synthesis. The intracellular accumulation of polymers of mutant alpha1-antitrypsin and neuroserpin results in a toxic gain-of-function phenotype associated with cirrhosis and dementia respectively. The lack of important inhibitors results in overactivity of proteolytic cascades and diseases such as COPD (chronic obstructive pulmonary disease) (alpha1-antitrypsin and alpha1-antichymotrypsin), thrombosis (antithrombin) and angio-oedema (C1 inhibitor). We have grouped these conditions that share the same underlying disease mechanism together as the serpinopathies. In the present review, the molecular and pathophysiological basis of alpha1-antitrypsin deficiency and other serpinopathies are considered, and we show how understanding this unusual mechanism of disease has resulted in the development of novel therapeutic strategies.

Unravelling the twists and turns of the serpinopathies.

Members of the serine protease inhibitor (serpin) superfamily are found in all branches of life and play an important role in the regulation of enzymes involved in proteolytic cascades. Mutants of the serpins result in a delay in folding, with unstable intermediates being cleared by endoplasmic reticulum‐associated degradation. The remaining protein is either fully folded and secreted or retained as ordered polymers within the endoplasmic reticulum of the cell of synthesis. This results in a group of diseases termed the serpinopathies, which are typified by mutations of α1‐antitrypsin and neuroserpin in association with cirrhosis and the dementia familial encephalopathy with neuroserpin inclusion bodies, respectively. Current evidence strongly suggests that polymers of mutants of α1‐antitrypsin and neuroserpin are linked by the sequential insertion of the reactive loop of one molecule into β‐sheet A of another. The ordered structure of the polymers within the endoplasmic reticulum stimulates nuclear factor‐kappa B by a pathway that is independent of the unfolded protein response. This chronic activation of nuclear factor‐kappa B may contribute to the cell toxicity associated with mutations of the serpins. We review the pathobiology of the serpinopathies and the development of novel therapeutic strategies for treating the inclusions that cause disease. These include the use of small molecules to block polymerization, stimulation of autophagy to clear inclusions and stem cell technology to correct the underlying molecular defect.

Characterisation of serpin polymers in vitro and in vivo

Neuroserpin is a member of the serine protease inhibitor or serpin superfamily of proteins. It is secreted by neurones and plays an important role in the regulation of tissue plasminogen activator at the synapse. Point mutations in the neuroserpin gene cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. This is one of a group of disorders caused by mutations in the serpins that are collectively known as the serpinopathies. Others include α(1)-antitrypsin deficiency and deficiency of C1 inhibitor, antithrombin and α(1)-antichymotrypsin. The serpinopathies are characterised by delays in protein folding and the retention of ordered polymers of the mutant serpin within the cell of synthesis. The clinical phenotype results from either a toxic gain of function from the inclusions or a loss of function, as there is insufficient protease inhibitor to regulate important proteolytic cascades. We describe here the methods required to characterise the polymerisation of neuroserpin and draw parallels with the polymerisation of α(1)-antitrypsin. It is important to recognise that the conditions in which experiments are performed will have a major effect on the findings. For example, incubation of monomeric serpins with guanidine or urea will produce polymers that are not found in vivo. The characterisation of the pathological polymers requires heating of the folded protein or alternatively the assessment of ordered polymers from cell and animal models of disease or from the tissues of humans who carry the mutation.

Structural dynamics associated with intermediate formation in an archetypal conformational disease.

Summary In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α1-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α1-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

Mutant Z α1‐antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1‐antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1‐antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1‐antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single‐chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1‐antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1‐antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small‐molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1‐antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi‐Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single‐chain variable fragment intrabody prevents intracellular polymerization of Z α1‐antitrypsin while allowing its antiproteinase activity. FASEB J. 29, 2667‐2678 (2015). www.fasebj.org

α1-antitrypsin deficiency and inflammation

α1-antitrypsin deficiency is an autosomal recessive disorder that results from point mutations in the SERPINA1 gene. The Z mutation (Glu342Lys) accounts for the majority of cases of severe α1-antitrypsin deficiency. It causes the protein to misfold into ordered polymers that accumulate within the endoplasmic reticulum of hepatocytes. It is these polymers that form the periodic acid Schiff positive inclusions that are characteristic of this condition. These inclusions are associated with neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating α1-antitrypsin exposes the lungs to uncontrolled proteolytic attack and so can predispose the Z α1-antitrypsin homozygote to early-onset emphysema. α1-antitrypsin polymers can also form in extracellular tissues where they activate and sustain inflammatory cascades. This may provide an explanation for both progressive emphysema in individuals who receive adequate replacement therapy and the selective advantage associated with α1-antitrypsin deficiency. Therapeutic strategies are now being developed to block the aberrant conformational transitions of mutant α1-antitrypsin and so treat the associated disease.

Targeting serpins in high-throughput and structure-based drug design

Native, metastable serpins inherently tend to undergo stabilizing conformational transitions in mechanisms of health (e.g., enzyme inhibition) and disease (serpinopathies). This intrinsic tendency is modifiable by ligand binding, thus structure-based drug design is an attractive strategy in the serpinopathies. This can be viewed as a labor-intensive approach, and historically, its intellectual attractiveness has been tempered by relatively limited success in development of drugs reaching clinical practice. However, the increasing availability of a range of powerful experimental systems and higher-throughput techniques is causing academic and early-stage industrial pharmaceutical approaches to converge. In this review, we outline the different systems and techniques that are bridging the gap between what have traditionally been considered distinct disciplines. The individual methods are not serpin-specific. Indeed, many have only recently been applied to serpins, and thus investigators in other fields may have greater experience of their use to date. However, by presenting examples from our work and that of other investigators in the serpin field, we highlight how techniques with potential for automation and scaling can be combined to address a range of context-specific challenges in targeting the serpinopathies.