Structural and cellular basis of alpha-1-antitrypsin (AT) deficiency and the serpinopathies

Antitrypsin is found at high concentrations in the bloodstream, where its main role is to protect the lungs against tissue damage from inflammation. Liver cells (hepatocytes) normally release individual molecules of antitrypsin into the circulation. Antitrypsin deficiency results when an individual inherits two genes with small changes in the antitrypsin protein. The Z variant, found in about 4% of people of North European decent, is the most common cause of severe antitrypsin deficiency. The Z variant causes antitrypsin to form long chains of linked molecules (called "polymers") that are trapped inside liver cells. The build-up of polymers damages the cell and increases the chance of developing liver cirrhosis and liver cancer. The reduced amount of properly formed protein in the circulation means the lungs are not as well protected against inflammation and so individuals develop emphysema. We have shown that a similar process occurs in mutants of other members of the protein family to which antitrypsin belongs. These include polymerisation of mutants of neuroserpin in the brain to cause dementia. We have grouped all the conditions together as a single class of disease that we have called 'the serpinopathies'. The application builds on 30 years of work by our group and has 5 interlinking projects within a programme of work. We propose to:

i) define of the linkage in pathological polymers isolated from the livers of Z antitrypsin homozygotes at atomic resolution by cryo-electron microscopy. We will define the structure of the pathological polymer isolated from tissues and so provide new opportunities for therapeutic strategies to block the abnormal protein-protein linkage that underlies antitrypsin deficiency.

(ii) determine the generality of the polymerization mechanism: structure of the pathological polymers caused by shutter domain mutants (Siiyama and Mmalton antitrypsin) and mutants of neuroserpin that cause FENIB. We will define the structure of disease causing polymers that form as a consequence of point mutations in different parts of the molecule from the 'Z mutation'.

(iii) use NMR to characterize intermediates on serpin polymerisation pathways and fingerprint and define the structure of the pathological polymer. This work will define protein intermediates that precede the formation of antitrypsin and neuroserpin polymers and provide an understanding of regions of the antitrypsin polymer structure that are not observed within cryo-electron microscopy as they are too mobile. It will support the development and optimization of polymer blockers and the imaging agents identified in aim (v).

(iv) visualise Z antitrypsin polymers in situ within the cellular environment. We will use a new high resolution technique, cryo-focused ion beam (FIB) milling, to provide unprecedented insight into the changes induced by Z antitrypsin polymers within the cell.

(v) use the pathological polymers as a biomarker and diagnostic tool for antitrypsin deficiency. We will follow a cohort of children with antitrypsin deficiency to confirm our initial observation that circulating antitrypsin polymers are a biomarker of liver disease. If confirmed this will allow us to recruit the most high risk individuals to clinical trials. Moreover, we will use NMR to identify small molecules that specifically bind to antitrypsin polymers to develop an assay that allows non-invasive measurement of intra-hepatic antitrypsin polymers/inclusions. This will allow us to address two key issues: (i) correlating intrahepatic polymer load with the severity of liver disease and (ii) the use of this imaging technique to accelerate drug development in man.

Taken together this work will increase our understanding of mechanism of antitrypsin deficiency and the serpinopathies and allow the development of new approaches to treatment.

We described the polymerisation of mutants that underlie antitrypsin deficiency and developed new paradigms for the liver and lung disease associated with this condition. We have also described the polymerisation of mutants of antichymotrypsin that have been associated with COPD and mutants of neuroserpin that cause a novel dementia that we called familial encephalopathy with neuroserpin inclusion bodies (FENIB). We grouped these diseases with others that result from polymerisation of mutants of the serpins as the serpinopathies. We have used biochemistry, biophysical analysis, crystallography, NMR, cell and induced pluripotent stem cells, monoclonal antibodies and Drosophila and C. elegans models of disease to dissect the structural basis of polymer formation and the cellular consequences. We are in the late stage of developing small molecules that block polymerisation with BioMarin. However, the current molecules also block inhibitory activity against neutrophil elastase. The specific aims are to: (i) determine the linkage in pathological polymers isolated from the livers of Z homozygotes at near-atomic resolution by cryo-electron microscopy; (ii) define the structure of the pathological polymers caused by shutter domain mutants of antitrypsin that cause liver disease and neuroserpin that cause FENIB (these are likely different from those caused by the Z mutation); (iii) use NMR to characterize intermediates on serpin polymerisation pathways and fingerprint and define the structure of the pathological polymer; (iv) use cutting-edge tomographic approaches to determine the morphology of Z antitrypsin polymers in situ within the cellular environment and (v) determine the use of pathological polymers as a biomarker and diagnostic tool for antitrypsin deficiency. This work will allow us to explore new diagnostic and therapeutic strategies for antitrypsin deficiency and the serpinopathies. The work is enabled by the platform facilities and technical support at UCL.