Structure and function of ZMPSTE24, an integral membrane protease mutated in progeria

One of the most serious global challenges over the next 30 years will be to address the profound implications of ageing for global economies and society. A recent UN report predicted that by 2050, 22% of the world's population will be over age 60. Maintaining physical and mental health requires increasing intervention with age, with most people requiring at least three forms of routine treatment in their 80s. There is therefore a pressing need to understand the mechanisms for ageing-related diseases, such as stroke and coronary heart disease.

Premature ageing syndromes such as Hutchinson-Gilford progeria syndrome (HGPS) can help us to understand the causes of normal ageing. Children with HGPS manifest many of the features observed in physiologic ageing, including balding, thinning skin and artherosclerosis. They usually die in their teens from a stroke or heart disease. HGPS is caused by a defect in one the components of the structural scaffolding for the cell nucleus, which acts as an anchor point for nuclear proteins and DNA. When this scaffold is disrupted, the cell nucleus is misshapen; chromosomes are disorganised; and cells are unable to divide effectively. These features have been observed in cells from normal 80-year-olds, particularly in the vascular wall, where artherosclerosis takes place. This problem occurs because one of the proteins that forms part of the scaffold, prelamin A, is not correctly processed. In particular, the mutant prelamin A is not processed correctly by an enzyme called ZMPSTE24.

We recently succeeded in producing active ZMPSTE24 enzyme and went on to solve its three-dimensional structure using X-ray crystallography (Quigley et al., Science, 2013). This exciting development gives us, for the first time, a framework to understand some of the biochemical mechanisms leading to premature ageing. The structure looked like nothing ever seen before. ZMPSTE24 forms a barrel surrounding a huge water-filled chamber inside the nuclear membrane, with its enzymatic domain at one end. There are a series of side portals in the chamber wall that permit access to inside the barrel. We now plan to define how ZMPSTE24 cleaves its substrates and why mutations in ZMPSTE24 cause disease. By understanding these underlying biochemical mechanisms, we better understand diseases of ageingageing, and possibly even develop treatments for the children who suffer from premature ageing.

Premature ageing diseases provide valuable insights into the mechanisms of normal ageing. Several premature ageing diseases are caused by failure in the post-translational processing of prelamin A, by an integral membrane zinc metalloprotease, ZMPSTE24. Mutations in the gene for ZMPSTE24 have been linked to metabolic syndrome, normal ageing and cancer. Our recent crystal structure of ZMPSTE24 (Quigley et al., Science, 2013) showed a 7 transmembrane helix barrel surrounding a huge chamber, capped by a zinc metalloprotease domain. The objective of this grant is to understand the underlying molecular mechanisms of prelamin A cleavage by ZMPSTE24 and how this cleavage reaction is disrupted in disease.

There are four key components to the processing of prelamin A by ZMPSTE24: substrate entry, binding, cleavage and product release. I will investigate each step using site-directed mutagenesis, enzymology, X-ray crystallography, in silico simulations, photo-affinity labelling, protein footprinting and native mass spectrometry. I will then investigate how ZMPSTE24 achieves its exquisite substrate selectivity, cleaving only one of the 300+ isoprenylated proteins in cells, by analysing its activity on libraries on libraries of substrate peptides. I will also test the theory that role of the intramembrane chamber may be to restrict access to the active site. Once we have a clear understanding of the molecular mechanism by which ZMPSTE24 cleaves prelamin A, I will then have the tools to understand how mutations in ZMPSTE24 reduce its activity and how mutations in prelamin A prevent cleavage.

My proposed studies will give us a clear picture of the molecular mechanisms for substrate entry, recognition and cleavage, and an understanding of the effects of disease-causing mutations, potentially paving the way for future therapeutic interventions for both progeria and physiologic ageing.

The potential long-term impact of this research for the public and the economy is considerable. While the function of ZMPSTE24 and prelamin A processing were initially determined through their involvement in rare diseases, there is now emerging evidence that this system plays a critical role in normal ageing and cancer. The MRC has recognised this in the research priority theme: "Living a long and healthy life" and the research in this grant directly addresses issues associated with ageing.

Impact on health and wellbeing for the public:

Failure of prelamin A processing leads to premature ageing diseases such as restrictive dermopathy (RD), Hutchinson Gilford progeria syndrome (HGPS) and mandibuloacral dysplasia (MAD). These are all rare but severe conditions. RD leads to death around the time of birth and HGPS patients die in their teens. MAD is a less severe recessive condition caused by point mutations in ZMPSTE24. Therapies to treat HGPS or RD would be difficult to achieve but it may in future be possible to produce activators of ZMPSTE24 which could treat some cases of MAD.

More common are metabolic syndrome (20% of the population) and ageing (>90% of the population). Some patients with metabolic syndrome have mutations in either ZMPSTE24 or the prelamin A tail that could affect prelamin A processing. There may therefore be a potential to treat some cases of metabolic syndrome and this could be relevant for 0.2 % of the population. There is still much to discover about the role of prelamin A processing in physiologic ageing. HGPS patients have many of the symptoms of older individuals, including thin skin, hair loss and failure to thrive, in addition to the heart disease and strokes that are often the cause of death for both groups of patients. Modulation of prelamin A processing and ZMPSTE24 activity levels could potentially have an impact on many of the adverse physical effects of ageing, since similar symptoms are seen in HGPS patients.

In cancer there are also preliminary data in mosaic mice showing that loss of ZMPSTE24 activity and prelamin A accumulation may contribute to a reduction in metastasis in cancer cells. This may contribute to the lower rate of metastasis seen in older cancer patients. In the future modulation of ZMPSTE24 activity at the gene or protein level could be a useful tool in the fight against cancer.

In the future it will become commonplace for many individuals to have their entire genome sequenced and this will provide a considerable challenge to clinicians in interpreting the results of polymorphisms and disease mutations. Mutations in ZMPSTE24 and prelamin A could be found in many individuals and the understanding how these changes might affect function will be essential, so that clinicians can advise patients on the risks associated with these mutations.

Impact on the economy:

It is impossible to underestimate the impact of ageing on the economy. A recent UN report predicted that by 2050, 22% of the world's population will be over age 60. As the population ages the impact on society will increase relentlessly. Maintaining both physical and mental health is therefore critical to future economic success. There is therefore a pressing need to understand the mechanisms underlying the adverse effects of ageing, such as atherosclerosis, stroke and heart disease.

The ageing population combined with low replacement rates leaves relatively few individuals in employment supporting a large number of retired people. Worldwide, government economic policies are reliant on increasing the length of an individuals working life, to reduce the pensions and tax burden. Maintaining a healthy ageing workforce is therefore essential to underpin these strategies.

The development of treatments for the physical effects of ageing such as atherosclerosis, failure to thrive, hair loss and skin thinning would provide a major new direction for the pharmaceutical industry.