Functional characterization of the KEOPS complex and its involvement in Galloway-Mowat syndrome – KeoGamo

The threonylcarbamoylation of the N6 nitrogen of the adenosine at position 37 (t6A) of ANN-accepting tRNAs is necessary for translation initiation and translational efficiency. The biosynthesis proceeds in two steps: YRDC/Sua5 first synthesizes a threonylcarbamoyl (TC)-AMP intermediate, and then the TC moiety is transferred onto tRNA by the ubiquitous and essential multi-protein KEOPS complex. Human KEOPS contains at least 4 subunits LAGE3, OSGEP, TP53RK, and TPRKB, with OSGEP acting as the catalytic subunit of the complex, whereas the role of the three other subunits in this enzymatic reaction is still unknown. Recently, Partner 2 identified twenty-two mutations in the four KEOPS subunit-encoding genes in patients with Galloway-Mowat syndrome (GAMOS), a rare autosomal recessive neuro-renal disorder associating nephrotic syndrome with microcephaly and neurological impairment. Inactivation of OSGEP, TP53RK or TPRKB in immortalized human podocytes led to the identification of alterations in multiple cellular processes such as proliferation, apoptosis or protein translation. Fungal KEOPS contains a 5th subunit called Gon7, which is essential for cell life. Lately, C14orf142, a small Intrinsically Disordered Protein (IDP) of unknown function, was identified as being a very remote homolog of the yeast Gon7 protein and shown to be the 5th element of human KEOPS complex and thus named GON7. Seeking additional genes of GAMOS, Partner 2 identified two mutations in the GON7 gene in 11 patients from 5 families with a more attenuated form of the disease, as well as two mutations in the YRDC gene, in 2 unrelated patients with an even more severe phenotype than that of mutated patients for the other KEOPS subunits.

The aim of the KeoGamo project is to characterize the mechanism of the highly conserved KEOPS complex and to examine its involvement in GAMOS pathophysiology. This proposal combines cellular biology, genetics, structural biochemistry (1) to understand the contribution of all subunits to the enzymatic t6A reaction mechanism, (2) to correlate structural and biochemical data with the physiopathological profile of the GAMOS mutations and (3) to obtain insight into the molecular and cellular physiopathological processes underlying the kidney and brain defects of GAMOS and elucidate the role of the newly identified GON7 subunit within the KEOPS complex. The fulfilment of these objectives will be instrumental for the understanding of the biochemical and cellular mechanisms underlying GAMOS. The scientific programme consists of three work packages:
1. Dissection of the role of all 5 subunits of human KEOPS in the t6A reaction mechanism and determination of the structure of the KEOPS-tRNA complex using crystallography and cryo-EM,
2. In vivo and in vitro characterization of the t6A activity of GAMOS mutations,
3. Pathophysiological role of t6A modification in GAMOS at the organ level.

This proposal is based on the preliminary results and the complementary skills and expertise of the three members of the consortium: H. van Tilbeurgh (coordinator, Partner 1) is a structural biologist specialized in the crystallographic analysis of protein complexes focusing over the past years on the biochemical, structural and cellular analysis of the t6A system in archaea, bacteria and eukaryotes. G. Mollet (Partner 2) is a molecular and cellular biologist working in the field of hereditary kidney disorders with an expertise in podocyte biology and pathobiology as well as in mouse models. C. Vénien-Bryan (Partner 3) is a structural biologist and a biophysicist specialized in structural studies of macromolecules using cryo-electron microscopy of single particles or 2D crystals combined to image analysis.