Biology subjects

Meili, F.

Publications and source records attributed to Meili, F..

6 recordsLinked to original sources

bioRxiv2026

Single-Cell Analytics for Dose Response (SCADR) discriminates PTEN missense variants by lipid and protein phosphatase dysfunction

The proliferation of sequencing efforts has revealed a vast and expanding catalog of single nucleotide gene variants, many associated to, but with unclear roles in disease. Fully charactering variant impacts and linking specific protein dysfunctions to disease are challenging due to the multi-functional nature of many proteins and varying degree of variant effects on these functions. Lagging are sensitive approaches to empirically assess the impact of missense variant-induced single amino acid changes on a wide range of protein functions. To address these issues, we have developed an open-source computational analysis tool called SCADR (Single-Cell Analytics for Dose Response) for simultaneously measuring and comparing impacts of exogenously-expressed variants on multiple signaling pathways using multiplex phospho-antibody spectral flow cytometry in human cell lines. SCADR retains and correlates single-cell measures of signal protein activity states along with expression levels of exogenously-expressed variants, providing rich characterization of multiple protein functions, signaling protein interactions, and enhanced discrimination of variant impacts on different signaling pathways, highlighting each variants unique dysfunction profile. Here, we apply SCADR for analyses of the impact of 6 variants of the tumor-suppressor protein PTEN (P38H, C124S, G129E, Y138L, D268E, 4A) expressed in HEK293 cells on the phosphorylation states of the canonical and noncanonical downstream signaling proteins Akt, S6, CREB, ERK, and p38 detected with fluorophore-conjugated phospho-antibodies, along with an antibody detecting an N-terminal HA tag on PTEN variants allowing measures of dose-response effects of each variants expression on signaling cascades. Results identify variant-specific impacts on downstream signaling cascades.

genomics↗

bioRxiv2023

Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings

Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinate de novo target proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1 E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1 E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Brutons tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

biochemistry↗

bioRxiv2020

Multi-parametric analysis of 58 SYNGAP1 variants reveal impacts on GTPase signaling, localization and protein stability

SYNGAP1 is a Ras and Rap GTPase with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia and autism spectrum disorder (ASD), there are many variants of unknown significance (VUS). In this report, we characterize 58 variants in nine assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure the impact of variants on pERK, pGSK3{beta} and pCREB and high-content imaging to examine their subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wildtype (WT)-like in their ability to regulate pERK and pGSK3{beta}, while all variants retain at least partial ability to regulate pCREB. Interestingly, our assays reveal that a high percentage of variants located within the disordered domain of unknown function that makes up the C-terminal half of SYNGAP1 exhibited LoF, compared to the more well studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction only for two missense variants both located within the catalytic domain. Using high-content imaging, we find variants with nuclear enrichment/exclusion and aberrant nuclear speckle localization. These variants are primarily located within the C2 domain known to mediate membrane lipid interactions. We find that mislocalization is distinct from altered catalytic activity, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence pathogenicity classification.

genetics↗

bioRxiv2020

Sentinel Interaction Mapping (SIM) - A generic approach for the functional analysis of human disease gene variants using yeast

Advances in sequencing technology have led to an explosion in the number of known genetic variants of human genes. A major challenge is to now determine which of these variants contribute to diseases as a result of their effect on gene function. Here we describe a generic approach using the yeast Saccharomyces cerevisiae to quickly develop gene-specific in vivo assays that can be used to quantify the level of function of a genetic variant. Using Synthetic Dosage Lethality screening, "sentinel" yeast strains are identified that are sensitive to overexpression of a human disease gene. Variants of the gene can then be functionalized in high-throughput fashion through simple growth assays using either solid or liquid media. Sentinel Interaction Mapping (SIM) has the potential to create functional assays for the large majority of human disease genes that do not have a yeast orthologue. Using the tumour suppressor gene PTEN as an example, we show that SIM assays can provide a fast and economical means to screen a large number of genetic variants.

genomics↗

bioRxiv2019

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PHTS, we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occurred across the protein, resulting in partial to complete loss of function (LoF), which was well correlated across models. However, assays were selectively sensitive to variants located in substrate binding and catalytic domains, which exhibited complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

genetics↗

bioRxiv2019

Systematic phenomics analysis of ASD-associated genes reveals shared functions and parallel networks underlying reversible impairments in habituation learning

A major challenge facing the genetics of Autism Spectrum Disorders (ASD) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 87 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of novel genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8*chd-7 and NLGN3*nlg-1 that underlie mechanosensory hyper-responsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of all conserved ASD-associated neuroligin variants revealed varied partial loss-of-function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers novel in vivo variant functional assays, and potential therapeutic targets for ASD.

neuroscience↗