Article Text

PDF

Cellular studies in obstructive lung disease
S51 Reducing intracellular aggregation and improving the secretion of Z alpha-1 antitrypsin
  1. S Alam,
  2. J Wang,
  3. S Janciauskiene,
  4. R Mahadeva
  1. University of Cambridge, Cambridge, UK

Abstract

The Z variant (Glu342Lys) of α1-antitrypsin (AT) is one of the serpinopathies; it polymerises and accumulates in the hepatocyte endoplasmic reticulum (ER) resulting in neonatal hepatitis and liver cirrhosis. The secretory defect leaves the lungs vulnerable to elastolysis and early-onset emphysema. Prevention of polymerisation of Z-AT can be achieved in vitro by targeting strand 4a of the AT molecule. Here we evaluate whether an inhibitor of polymerisation; Ac-TTAI-NH2 (4M) would inhibit Z-AT polymerisation in a cell model. HEK293 cells were transfected with Z-AT (Z-AT cells) or control M-AT (M-AT cells). ELISA demonstrated significantly reduced Z-AT secretion, 242(SEM±63) ng/ml compared to M-AT, 2449±130 ng/ml (p≤0.001), due to retention of Z-AT polymers in inclusion bodies. This was confirmed by electron microscopy demonstrating distension of the rough ER, and ELISA and Immunoblot with a polymer specific antibody (ATZII). 4M significantly reduced the amount of polymers in inclusions in a dose-dependent manner; no peptide, 2468.5±μg/ml, 5 mg, 2181.7±26.2 ng/ml (p=0.006), 10 mg, 1576±164.7 ng/ml (p=0.001), and 20 and 30 mg completely prevented polymer formation in inclusions (p≤0.001). Unrelated peptides had no effect. Elastase activity of AT in the supernatant from Z-AT cells was significantly reduced compared to M-AT cells; p≤0.001, in keeping with the secretory defect due to retention of Z-AT in inclusion bodies. The elastase activity (and AT concentration) in the supernatant from Z-AT cells was restored by 20 mg 4M, O.D. 405 nm, Z-AT vs Z-AT + 20 μg 4M, 0.129±0.009 vs 0.788±0.054 respectively, (p≤0.001), where a higher O.D. represents higher elastase activity. Functional activity of secreted AT following treatment with 4M was confirmed by its ability to form an SDS-stable complex with elastase as shown by immunoblot. RT-PCR showed that the ER accumulation of Z-AT induced cell stress; NF-κB activation, expression of protein kinase RNA (PKR)-like ER kinase (PERK), and IL-6 (100.4±16 pg/ml) and IL-8 (2592.5±575 pg/ml), all of which could be abrogated effectively by 20 mg 4M (IL-6, 45.8±28 pg/ml, p≤0.001 and IL-8, 184.3±29 pg/ml, p=0.014). These findings are the first evidence that inhibitors of Z-AT polymerisation targeting s4A can prevent its cellular accumulation and deleterious effects. Importantly, this strategy was also able to improve plasma concentration of Z-AT.

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.