Linking alpha1-antitrypsin phase transition with cellular health

Alpha1-antitrypsin is a protein made in the liver that circulates in the blood to control inflammation in the lungs. Some people with a condition call alpha1-antitrypsin deficiency are at increased risk of liver and lung disease because they have inherited a mutated form of alpha1-antitrypsin that accumulates in liver cells and fails to reach the blood. This abnormal antitrypsin, builds up inside a part of the cell called the endoplasmic reticulum (or ER for short). This build-up then increases the risk of liver cirrhosis (scarring) and liver cancer. No current treatments apart from liver transplantation can help in this form of liver disease. We study the accumulation of alpha1-antitrypsin inside the ER so that we can develop new liver-protective treatments.

Many human diseases can arise when the ER malfunctions: so-called ER stress. The job of the ER is to make secreted proteins, like antitrypsin, allowing newly made proteins to "fold" into their unique shape that allows them to perform their specific roles in the body. ER stress happens when ER proteins fail to fold correctly, which can happen with some mutated proteins. This causes ER proteins to stick together randomly, damaging the cell. By contrast, the most common mutated form of alpha1-antitrypsin forms ordered chains of polymers that do not appear to cause ER stress directly. Instead, they make the cell more sensitive to other stresses, such as saturated fats or alcohol, which can trigger ER stress more easily. The reason why polymers of antitrypsin cause this increased sensitivity is unknown, but we recently discovered that antitrypsin polymers can solidify inside the ER to forming a porous material that traps large ER proteins while allowing smaller ones to pass through. This "molecular filtration" may prevent large ER proteins moving to where they are required and this may account for the increased sensitivity to ER stress. Importantly, we discovered that some proteins in the ER drive this solidification of antitrypsin and can be prevented from doing so by blocking certain signalling pathways inside the cell.

In the current project, we aim to identify which cellular proteins are responsible for the solidification of antitrypsin. We will also determine how the solidification of antitrypsin affects the health of cells. Finally, we will use this knowledge to develop therapies to protect liver cells from the toxic effects of alpha1-antitrypsin mutations.

How mutant Z-alpha-1-antitrypsin (Z-A1AT) impairs liver function is unclear. Endoplasmic reticulum (ER) stress elicits an unfolded protein response (UPR) that can cause hepatitis and cirrhosis. We discovered that A1AT can undergo a phase transition to form a porous solid in the ER. Small proteins can diffuse through this, but larger proteins are trapped to impair ER function. We showed A1AT phase transition to be driven by the UPR and a subset of ER chaperones.

Aim 1: Define the molecular machinery mediating the A1AT polymer phase transition
We will determine how chaperone binding drives polymerisation. (i) We will determine how calreticulin drives phase transition. (ii) We identified somatic mutations in cirrhotic livers that prevent A1AT polymerisation. Interactomes of these new variants will be compared with polymerisation-competent forms of A1AT. (iii) A domain swap involving A1AT's C-terminus drives polymerisation. We will identify chaperones that stabilise relevant folding-intermediates of A1AT by generating C-terminally modified mutants.

Aim 2: Elucidate impact of the A1AT phase transition on cellular homeostasis
Growth assays will reveal if solidification of A1AT affects cellular fitness. Pulsed release of fluorescent hepatocyte ER client proteins will be used to determine the impact of A1AT solidification on trafficking.

Aim 3: Manipulate the phase transition for therapeutic benefit
We will systematically manipulate components of the UPR to identify targets for pharmacological modulation of the A1AT phase transition. We will manipulate extracellular signalling pathways that also regulate ER homeostasis to identify additional druggable pathways to prevent A1AT solidification.

These experiments will reveal the mechanism by which solidification of A1AT polymers alter ER homeostasis and identify targets for drug development.