Biology subjects

Simon, J. J.

Publications and source records attributed to Simon, J. J..

6 recordsLinked to original sources

bioRxiv2026

Massively scaled variant effect mapping across the MAPK pathway reveals the importance of molecular and cellular context

Variation alters inherent protein properties like thermodynamic stability and catalytic activity, and these intrinsic effects are shaped by the complex cellular environment. Signaling cascades typify this complexity, with tens or hundreds of proteins acting in concert through regulated states and interactions to transduce signals. Here, we measure the effect of 186,676 variants across 17 proteins in the RAS-MAPK pathway on protein abundance and pathway activity in human cells under diverse conditions, for a total of 503,099 variant effect measurements. We discovered 14,412 dominant negative variants across 12 proteins, nearly all previously unknown, that disrupt specific domains or interaction interfaces while leaving other functions intact and that are depleted from the human population. Measuring the abundance of 106,642 variants across 10 proteins under HSP90 inhibition showed that HSP90 shapes the effect of 67% of missense variants across the pathway, buffering destabilizing variants in both strong and weak clients. Our variant effect data, collected in the context of an intact proteome, enabled us to assess the functional importance of 395 experimentally and computationally modeled MAPK protein interactions. Using additional mutational scans, we resolved how the promiscuous switch II helix binding surface of KRAS accommodates overlapping interactions with LZTR1 and SOS1, revealing distinct allosteric networks that support each interaction. Thus, we establish a framework for measuring variant effects at unprecedented scale in human cells, illuminating fundamental MAPK pathway signaling mechanisms and demonstrating that cellular context profoundly shapes the effect of most variants.

genomics↗

bioRxiv2026

High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity

High-resolution measurements of pathway activity make it possible to characterize how signaling responds to changing cellular conditions, to evaluate pathway-targeted inhibitors, and to dissect how sequence variation shapes signaling output. Here, we describe a sequencing-based assay that uses engineered, phospho-state-selective affinity reagents to quantify ERK activation state as a high-resolution, ratiometric readout of RAS/ERK MAPK pathway activity. Profiling ~1,600 BRAF variants at basal and elevated RAS-GTP levels allowed us to assign activity scores to somatic cancer and germline RASopathy variants and to distinguish residues that stabilize autoinhibition from those required for active dimer formation. We identified a large class of dominant negative variants that suppress signaling below endogenous levels through enhanced RAS engagement and an inability to form productive RAF dimers. Measuring the intracellular potency of the RAF inhibitor LXH254 against ~1,000 variants in parallel revealed hinge and activation-loop positions conferring resistance and showed that disrupting the BRAF-MEK interface is broadly sensitizing.

cell biology↗

bioRxiv2025

Multiplex design and discovery of proximity handles for programmable proteome editing

Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs1) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector "handles". We asked whether coupling de novo protein design to a multiplex screening framework could address this gap by accelerating the discovery of effector handles for intracellular protein degradation, stabilization, or relocalization. Using LABEL-seq2, a sequencing-based assay that enables multiplex, quantitative measurement of protein abundance, we screened 9,715 de novo designed candidate effector handles for their ability to recruit a target protein to components of the ubiquitin-proteasome system3 (UPS) (FBXL12, TRAF2, UCHL1, USP38) or the autophagy pathway4 (GABARAP, GABARAPL2, MAP1LC3A). In a single experiment, we discovered hundreds of de novo designed effector handles that reproducibly drove either intracellular degradation (n = 277) or stabilization (n = 204) of a reporter protein. Validation of a subset of these hits in an orthogonal assay confirmed that sequencing-based measurements from the primary screen reliably reflected changes in intracellular abundance of the target protein. Successful effector handles were discovered for both the UPS (n = 194) and autophagy (n = 287) pathways, which provide complementary routes for programmable proteome editing. Autophagy-recruiting effector handles generalized to endogenous targets, as substituting the reporter-specific target handle with a high-affinity MCL1 binder5 reduced endogenous levels of this intracellular oncoprotein6. Moreover, directing autophagy-recruiting effector handles to the outer mitochondrial membrane dramatically perturbed mitochondrial networks in a manner consistent with synthetic tethering and sequestration7,8. Beyond generating a diverse repertoire of protein abundance or localization effector handles, our results establish a scalable, low-cost platform that links deep learning-guided protein design to functional cellular readouts, and chart a course toward a general framework for programmable proteome editing.

synthetic biology↗

bioRxiv2024

Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

Raf kinases are crucial effectors in the Ras-Raf-Mek-Erk signaling pathway, making them important targets for the development of cancer therapeutics. This study investigates the variable potency of DFG-out-stabilizing Raf inhibitors in mutant KRas-expressing cell lines. We demonstrate that inhibitor potency correlates with basal Raf activity, with more active Raf being more susceptible to inhibition. We further show that DFG-out-stabilizing inhibitors disrupt high-affinity Raf-Mek interactions, promoting the formation of inhibited Raf dimers. Furthermore, we identify cobimetinib as a Mek inhibitor that uniquely sensitizes Raf to DFG-out inhibitors by disrupting autoinhibited Raf-Mek complexes. Building on this insight, we developed cobimetinib analogs with enhanced sensitization properties. Our findings provide a mechanistic framework for understanding the cellular determinants of DFG-out-stabilizing inhibitor sensitivity and offer strategies for optimizing synergistic Raf-Mek inhibitor combinations.

cell biology↗

bioRxiv2024

Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq

Multiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of [~]20,000 variant effects for [~]1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRafs signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.

molecular biology↗

bioRxiv2019

Shared and dissociable features of apathy and reward system dysfunction in bipolar I disorder and schizophrenia

BackgroundBipolar disorder I (BD-I) is defined by episodes of mania, depression, and euthymic states. These episodes are among other symptoms characterized by altered reward processing and negative symptoms (NS), in particular apathy. However, the neural correlates of these deficits are not well understood. MethodsWe first assessed the severity of negative symptoms in 25 euthymic BD-I patients compared to 25 healthy controls (HC) and 27 patients with schizophrenia (SZ). Then, we investigated ventral and dorsal striatal activation during reward anticipation in a Monetary Incentive Delayed Task and its association with NS. ResultsIn BD-I patients NS were clearly present and the severity of apathy was comparable to SZ patients. Apathy scores in the BD-I group but not in the SZ group correlated with sub-syndromal depression scores. At the neural level, we found significant ventral and dorsal striatal activation in BD-I patients and no group differences with HC or SZ patients. In contrast to patients with SZ, apathy did not correlate with striatal activation during reward anticipation. Explorative whole brain analyses revealed reduced extra-striatal activation in BD-I patients compared to HC and an association between reduced activation of the inferior frontal gyrus and apathy. ConclusionThis study found that in BD-I patients apathy is present to an extent comparable to schizophrenia, but is more strongly related to sub-syndromal depressive symptoms. The findings support the view of different pathophysiological mechanisms underlying apathy in the two disorders and suggest that extra-striatal dysfunction may contribute to impaired reward processing and apathy in BD-I.

neuroscience↗