Defining the Cellular and Molecular Basis of Spondyloepiphyseal dysplasia tarda

Zappa, Francesca (2017). Defining the Cellular and Molecular Basis of Spondyloepiphyseal dysplasia tarda. PhD thesis The Open University.



One third of the proteome is transported in membrane-encapsulated vesicles from the endoplasmic reticulum (ER) to the Golgi apparatus en route to the plasma membrane, other intracellular organelles and the extracellular matrix. After folding, newly synthesized proteins reach the Golgi apparatus through COPII carriers. COPII vesicles bud from sub-domains of the ER, named ER exit sites (ERES) and are composed of five rapidly cycling proteins: the small GTPase Sar1, the inner coat (Sec23/Sec24) and the outer layer (Sec13/31). The efficiency of COPII cycling is dispensable for the transport of small soluble cargoes or trans- membrane cargoes, yet is mandatory for the exit of “extra-size” proteins, such as procollagens. To facilitate the incorporation of procollagens into nascent COPII carriers, several adaptors work in cooperation at ERES. One of them is sedlin, a small protein that acts as a co-GAP for Sar1, thus increasing its kinetics. The idea that sedlin is important for collagen transport is enforced by the evidence that a rare Mendelian disorder, Spondyloepiphyseal dysplasia tarda (SEDT), is caused by sedlin mutations in which the main clinical manifestation is an alteration in collagen deposition.

With the aim to clarify sedlin functions in membrane trafficking, I followed two different approaches: (1) biochemical analysis of sedlin interactors and (2) the generation of a sedlin KO medaka model. Using mass spectroscopy approaches, several proteins were shown to interact with sedlin of which one of the most significant was CLIC1. Subsequent experiments showed that deletion of CLIC1 delayed protein secretion. Furthermore, this work showed that CLIC1 and sedlin, as well as the COPII inner coat, take part in the integrated stress response (IRS). Upon perturbations that change cellular homeostasis such as heat shock, redox stress or unfolded protein accumulation, cells activate signalling pathways that result in translation inhibition by sequestering free mRNAs and ribonucleoproteins in cytosolic aggregates named stress granules (SGs).

I demonstrated that SGs control secretion by recruiting key components of ER-to- Golgi trafficking and thus inhibit anterograde transport. This suggested that the ISR regulates protein metabolism on two levels: translationally and post- translationally, by sequestering mRNAs and inhibiting protein export from the ER, respectively. Additionally, by using TALEN technology, I edited the sedlin gene in medaka to create a SEDLIN KO stable line (Sedl-/-). Sedl-/- fish have severe skeletal defects, which are similar to those observed in human SEDT patients, and die soon after birth suggesting that sedlin has an essential role in early postnatal medaka growth.

To investigate the molecular pathways altered in sedlin-/- vertebrates I performed a Transcriptomic analysis on whole larvae highlighting that eye and cartilage are the two most affected organs and led to the hypothesis of a common pathogenetic mechanism.

These findings provide evidence that sedlin is involved in different molecular pathways and when mutated can contribute to the pathogenesis of SEDT. Thus, my thesis work has shed light on a new control mechanism that involves ER-to-Golgi transport and establishes the first SEDT animal model, a suitable tool to find SEDT correctors.

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