Abstract
Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive AlrC131S mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr C131S is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.
Highlights
Augmenter of Liver Regeneration (ALR), known as Hepatopoietin (HPO) and growth factor ERV1-like (GFER), is a protein highly up-regulated during liver regeneration and stimulates hepatocyte proliferation
In this study, we provide several experimental evidences revealing the role of alr in vertebrate liver organogenesis
Using knockdown and overexpression approaches, we demonstrated its positive function in promoting liver growth
Summary
Augmenter of Liver Regeneration (ALR), known as Hepatopoietin (HPO) and growth factor ERV1-like (GFER), is a protein highly up-regulated during liver regeneration and stimulates hepatocyte proliferation. Mammalian ALR contains a conserved sulfhydryl oxidase enzymatic domain (ERV1 domain) at the C-terminus and functions as a sulfhydryl oxidase facilitating disulfide bond formation in proteins [5]. While the enzymatic domain at the C-terminus is conserved, the N-terminal region is highly variable among ALRs in different species, implicating potentially distinct functions of this protein in different species. In both yeast and human, the mitochondria protein Mia and cytochrome c have been identified as direct in vivo substrates of Erv1/ALR [6,7,8]. Whether ALR has additional in vivo substrates inside mitochondria or at other subcellular locations is still a mystery
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