Biology subjects

Katanaev, V. L.

Publications and source records attributed to Katanaev, V. L..

5 recordsLinked to original sources

bioRxiv2025

Visualizing the Dominant GPCR Coupling of Pathogenic Gαo Mutants in GNAO1-Related Disorders

Heterozygous mutations in the GNAO1 gene, which encodes the Go subunit of heterotrimeric G proteins, cause a spectrum of rare neurodevelopmental disorders ranging from early-onset epileptic encephalopathy to milder dystonia phenotypes. Disease dominance of Go mutants appears to arise from multiple functional disruptions, including impaired guanine nucleotide handling, failure to adopt the active conformation, and neomorphic interactions with Ric8A/B chaperones. In parallel, several Go variants have been independently reported to dominantly engage G protein-coupled receptors (GPCRs) or to sequester G{beta}{gamma}, two mechanistically distinct behaviors. These conclusions were inferred from different indirect biosensor assays, likely contributing to the apparent contradiction in their proposed mechanisms. To overcome this, we developed a split-YFP-based bimolecular fluorescence complementation (BiFC) assay to visualize receptor-Go protein complexes at the plasma membrane. Using this system, we found that severe Go variants (S47G, G203R, R209C, E246K) fail to disengage from activated Gi/o-coupled GPCRs, thereby preventing downstream receptor phosphorylation and endocytosis. By contrast, milder dystonia-linked mutants (C215Y and T241_N242insPQ) showed near-normal receptor internalization and only minor phosphorylation defects. These findings establish dominant GPCR coupling as a molecular hallmark of severe GNAO1-related disorders and point to split-YFP BiFC as a robust platform for probing mutant G protein behavior in genetic disease.

cell biology↗

bioRxiv2023

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic adaptation to malnutrition in Drosophila

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. We show that experimental populations adapted during >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. This divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, which was originally present in a sample from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, suggesting that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored because of its role in suppressing growth in response to nutrient shortage. However, fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, such as in nutrient acquisition and metabolism, which ensure that the organism can actually sustain the faster growth promoted by fiz downregulation.

evolutionary biology↗

bioRxiv2023

Ric8 proteins as the neomorphic partners of G alpha o in GNAO1 encephalopathies

GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G-protein Go. Of >40 pathogenic mutations, most are single amino acid substitutions spreading across Go sequence. We perform extensive characterization of Go mutants showing abnormal GTP uptake and hydrolysis, and deficiencies to bind G{beta}{gamma} and RGS19. Plasma membrane localization of Go is decreased for a subset of mutations that leads to epileptic manifestations. Pathogenic mutants massively gain interaction with Ric8A/B proteins, delocalizing them from cytoplasm to Golgi. Being general G-subunit chaperones and binding multiple other proteins, Ric8A/B likely mediate the disease dominance when engaging in neomorphic interactions with pathogenic Go. As the strength of Go-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. One-Sentence SummaryNeomorphic mutations in Go gain dominant interactions with Ric8A/B, correlating with severity in pediatric encephalopathies.

cell biology↗

bioRxiv2020

Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo

Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Go - -subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Go N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

cell biology↗

bioRxiv2020

Humanization of Drosophila Gαo to model GNAO1 paediatric encephalopathies

Several hundred genes have been identified to contribute to epilepsy - the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Go, the major neuronal -subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering in addition to epilepsy from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only poorly been analysed at the biochemical or neuronal levels. Adequate animal models to study molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modelling the disease in Drosophila, we here describe humanization of the Go locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of Go with respective human GNAO1 sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in-between, as well as the non-coding exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight and longevity are also normal. Human Go-specific antibodies confirm the endogenous-level expression of the humanized Go, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.

genetics↗