Biology subjects

Larsen-Ledet, S.

Publications and source records attributed to Larsen-Ledet, S..

5 recordsLinked to original sources

bioRxiv2026

An Atlas of Short Linear Motif-Mediated Human Protein-Protein Interactions

Short linear motifs (SLiMs) within intrinsically disordered protein regions mediate transient interactions crucial for cell physiology1. However, the global interaction landscape of human SLiMs remains largely uncharted. Here we present the Atlas of SLiM-mediated Human protein-protein Interactions (ASHI), which maps more than 20,000 interactions by screening over 800 human protein domains against a library of one million peptides tiling the human disordered proteome. ASHI expands the SLiM interactome, uncovers novel binding modes for known peptide-binding domains, and reveals unexpected peptide-binding activities in enzymes, chaperones, RNA-binding proteins, and modification-reader domains. Furthermore, intrinsically disordered regions emerge as densely encoded interaction platforms where interaction specificity is governed by diverse mechanisms, including key motif determinants, flanking residues, competition, and multivalency. These data provide an unprecedented foundation for modeling dynamic interaction networks, interpreting disease-associated variants, and decoding the dark proteome.

biochemistry↗

bioRxiv2025

Blocking protein quality control degradation leads to structural stabilization of DHFR indel variants

Gene variants leading to insertions or deletions of amino acid residues (indels) often have detrimental consequences for the folding of the encoded protein. Yet at some positions indels are tolerated or result merely in partial unfolding and hypomorphic phenotypes. Typically unfolded proteins are targeted for protein quality control (PQC) degradation via the ubiquitin-proteasome system, which in yeast is mediated by specific E3 ubiquitin-protein ligases, including Ubr1 and San1. Here we systematically probed the folding of a library of indel variants in the DHFR protein using a sensitive yeast-based protein folding reporter. We show that deletion of Ubr1 or San1 leads to a greater fraction of folded DHFR indel variants, primarily positioned towards the N- and C-terminal regions in DHFR. Intriguingly, most of the DHFR indels that are structurally stabilized in the E3 knockout strains, are also stabilized at lowered temperatures and upon binding the DHFR inhibitor methotrexate. This suggests that blocking PQC degradation can restore function to partially unfolded hypomorph variants, thus providing a potential therapeutic avenue for protein misfolding diseases.

biochemistry↗

bioRxiv2024

Disentangling the mutational effects on protein stability and interaction of human MLH1

Missense mutations can have diverse effects on proteins, depending on their location within the protein and the specific amino acid substitution. Mutations in the DNA mismatch repair gene MLH1 are associated with Lynch syndrome, yet the underlying mechanism of most disease-causing mutations remains elusive. To address this gap, we aim to disentangle the mutational effects on two essential properties for MLH1 function: protein stability and protein-protein interaction. We systematically examine the cellular abundance and interaction with PMS2 of 4839 (94%) MLH1 variants in the C-terminal domain. Our combined data shows that most MLH1 variants lose interaction with PMS2 due to reduced cellular abundance. However, substitutions to charged residues in the canonical interface lead to reduced interaction with PMS2. Unexpectedly, we also identify a distal region in the C-terminal domain of MLH1 where substitutions cause both decreased and increased binding with PMS2, and propose a region in PMS2 as the binding site. Our data successfully distinguish benign from pathogenic MLH1 variants and correlate with thermodynamic stability predictions and evolutionary conservation. This work provides mechanistic insights into variant consequences and may help interpret MLH1 variants. Author SummaryMutations in proteins, including single amino acid substitutions, can disrupt normal cellular function and lead to diseases. Hence, there is great interest in understanding their consequences from both basic science and clinical genetics perspectives. Loss-of-function variants in the human mismatch repair protein MLH1 are associated with Lynch syndrome, a hereditary condition that significantly increases the risk of certain cancers. Detailed insights into the molecular consequences of MLH1 variants are essential for understanding the mechanisms underlying Lynch syndrome. In this study, we use high-throughput experimental approaches to test how missense mutations affect the cellular abundance of MLH1 and the interaction with its partner, PMS2. We find that many variants disrupting the interaction with PMS2 also lead to reduced cellular abundance levels, suggesting a strong relationship between protein stability and interaction in MLH1. Additionally, we identify a potential novel interaction site between MLH1 and PMS2, where mutations in MLH1 either increase or decrease interaction without affecting abundance. Our datasets are in line with the classification of clinically annotated MLH1 variants and may aid in interpreting variants of uncertain significance, while also providing new insights into the molecular details of the interaction with PMS2.

molecular biology↗

bioRxiv2024

Systematic characterization of indel variants using a yeast-based protein folding sensor

Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet compared to missense variants the effects of indels are poorly understood and predicted. To approach this issue, we developed a sensitive protein folding sensor based on complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor accurately reports on the folding of disease-linked missense variants and de novo designed proteins. Applying the folding sensor to a saturated library of single amino acid indel variants in human DHFR revealed that most regions which tolerate indels are confined to internal loops and the N- and C-termini. Surprisingly, indels are also allowed at a central -helix. Several indels are temperature-sensitive and the folding of most of these indels is rescued upon binding to the competitive DHFR inhibitor methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels operate by destabilizing the native fold and that these computational tools may be useful for classification of indels observed in population sequencing.

biochemistry↗

bioRxiv2021

Disease-linked mutations trigger exposure of a protein quality control degron in the DHFR protein

Degrons are short stretches of amino acids or structural motifs that are embedded in proteins. They mediate recognition by E3 ubiquitin-protein ligases and thus confer protein degradation via the ubiquitin-proteasome system. Well-described degrons include the N-degrons, destruction boxes, and the PIP degrons, which mediate the controlled degradation of various proteins including signaling components and cell cycle regulators. In comparison, the so-called protein quality control (PQC) degrons that mediate the degradation of structurally destabilized or misfolded proteins are not well described. Here, we show that disease-linked DHFR missense variants are structurally destabilized and chaperone-dependent proteasome targets. We systematically mapped regions within DHFR to assess those that act as cytosolic PQC degrons in yeast cells. Two regions, DHFR-Deg13-36 (here Deg1) and DHFR-Deg61-84 (here Deg2), act as degrons and conferred degradation to unrelated fusion partners. The proteasomal turnover of Deg2 was dependent on the molecular chaperone Hsp70. Structural analyses by NMR and hydrogen/deuterium exchange revealed that Deg2 is buried in wild-type DHFR, but becomes transiently exposed in the disease-linked missense variants.

biochemistry↗