Serine Protease Inhibitors - Serpins, Serpinopathies and Human Disease

Serpins, Serpinopathies and Human Disease

Serpins are vulnerable to inactivating disease-causing mutations that result in the formation of misfolded polymers or protein aggregates ("serpinopathies"). Well-characterised serpinopathies include alpha 1-antitrypsin deficiency (alpha-1), which may cause familial emphysema and sometimes liver cirrhosis, certain familial forms of thrombosis related to antithrombin deficiency, types 1 and 2 hereditary angioedema (HAE) related to deficiency of C1-inhibitor, and familial encephalopathy with neuroserpin inclusion bodies (FENIB; a rare type of dementia caused by neuroserpin polymerisation). Serpins thus belong to a large group of molecules such as the prion proteins and the glutamine repeat containing proteins that cause proteopathies or conformational diseases.

Serpin polymerisation causes disease in two ways. First, the lack of active serpin results in uncontrolled protease activity and tissue destruction; this is seen in the case of antitrypsin deficiency. Second, the polymers themselves clog up the endoplasmic reticulum of cells that synthesize serpins, eventually resulting in cell death and tissue damage. In the case of antitrypsin deficiency, antitrypsin polymers cause the death of liver cells, sometimes resulting in liver damage and cirrhosis. Within the cell, serpin polymers are removed via endoplasmic reticulum associated degradation. However, the mechanism by which serpin polymers cause cell death remains to be fully understood.

Like cleaved serpins, serpin polymers are hyperstable with respect to heating, and each serpin monomer appears to have undergone the stressed to relaxed transition. Furthermore, serpin polymers are unable to inhibit target proteases, suggesting that the RCL is unavailable and inserted into the A-sheet. In the absence of definitive structural data, it was, therefore, postulated that serpins polymerise via a mechanism known as A-sheet polymerisation. In normal function the RCL inserts into the A β-sheet to form a fourth strand (figure 4). In the A-sheet polymerisation model, it was suggested that the RCL of one serpin molecule spontaneously inserted into the A-sheet of another, to form a long-chain polymer (figure 9). In effect, it was, thus, proposed that polymerization occurred as a consequence of the requirement of the serpin scaffold to accept an additional β-strand.

Serpins were one of the first families for which disease-causing mutations were directly analyzed in reference to the available crystal structures. In support of the A-sheet polymerisation model, it was noted that many serpin mutations that cause polymerisation localise to two distinct regions of the molecule (highlighted in figure 4a) termed the shutter and the breach. The shutter and the breach contain highly conserved residues, underlie the path of RCL insertion, and are proposed to be important for conformational change.

Two structures of cleaved serpin polymers have been solved; both of which reveal RCL / A-sheet sheet linkages similar to those predicted by the A sheet polymerisation mechanism. However, in direct contrast to the known properties of physiological serpin polymers, crystals of cleaved serpin A-sheet polymers readily dissociate into monomeric forms.

A large body of data now suggest that the events associated with serpin polymerisation occur during the folding of the molecule, and that mutations that cause serpinopathies interfere with the ability of the serpin to fold to the metastable native state. In normal serpin folding, the serpin rapidly moves through a key folding intermediate to attain the native state. Many studies have shown that it is the serpin folding intermediate that has the ability to polymerise, hence it is important that this folding species rapidly moves on to adopt native state. It was shown that mutations such as the Z-antitrypsin variant (Glu 342 to Lys) somehow prevented the final stage of seprin folding and caused the accumulation of the folding intermediate. As a result, population of the folding intermediate resulted in polymer formation. Interestingly, it was noted that once folded, the Z-antitrypsin variant closely resembles wild-type material in terms of thermal stability and inhibitory activity.

Together, these data have presented an important challenge to the A-sheet model for serpin polymerisation. On the one hand, the idea that serpin polymer formation essentially takes advantage of the serpin mechanism of conformational change is an attractive one. On the other, the biophyiscal data in particular suggest that it is a folding intermediate (rather than the native form) that polymerises, and it is clear that this intermediate must have different structural properties to the native, folded state.

In 2008, a key serpin crystal structure was determined that strongly suggests that physiological serpin polymers do not form via the A-sheet mechanism and instead form via a more extensive domain swapping event. The first such structure solved was of an antithrombin dimer (figure 10), and revealed that both strands s5A and the RCL can be incorporated into the A-sheet of another serpin molecule. This structure can readily be adapted to form long chain polymers. In 2011, the structure of a domain swapped antitrypsin trimer revealed that in polymers of this serpin the RCL is inserted, and that the C-terminal region of the molecule (comprising strands s1C, s4B and s5B) formed the domain swap (figure 11). In support of the physiological relevance of the latter structure, it was shown that antitrypsin polymers formed via a C-terminal domain swap were recognised by a monocloncal antibody specific for pathogenic antitrypsin polymers.

The new "domain swapped" model for serpin polymerisation begins to reconcile the available biophysical and biochemical data. Together, these data suggest that domain swapping events occur when mutations or environmental factors somehow interfere with the final stages of serpin folding to the native state. These data also reveal that different serpins can apparently polymerise via different types of domain swaps. Finally, while these data shed light on the final polymeric form, it is important to note that the precise toxic species of intermediate and / or polymer that causes cell death in, for example, antitrypsin deficiency, remains to be identified.