Linear Motifs Research Articles

The conformation of FOXM1 homodimers in vivo is crucial for regulating transcriptional activities.

Conformational changes in a transcription factor can significantly affect its transcriptional activity. The activated form of the FOXM1 transcription factor regulates the transcriptional network of genes essential for cell cycle progression and carcinogenesis. However, the mechanism and impact of FOXM1 conformational change on its transcriptional activity in vivo throughout the cell cycle progression remain unexplored. Here, we demonstrate that FOXM1 proteins form novel intermolecular homodimerizations in vivo, and these conformational changes in FOXM1 homodimers impact activity during the cell cycle. Specifically, during the G1 phase, FOXM1 undergoes autorepressive homodimerization, wherein the αβα motif in the C-terminal transcriptional activation domain interacts with the ββαβ motif in the N-terminal repression domain, as evidenced by FRET imaging. Phosphorylation of the αβα motif by PLK1 at S715/S724 disrupts ββαβ-αβα hydrophobic interactions, thereby facilitating a conserved αβα motif switch binding partner to the novel intrinsically disordered regions, leading to FOXM1 autostimulatory homodimerization persisting from the S phase to the G2/M phase in vivo. Furthermore, we identified a minimal ββαβ motif peptide that effectively inhibits cancer cell proliferation both in cell culture and in a mouse tumor model, suggesting a promising autorepression approach for targeting FOXM1 in cancer therapy.

Characterization and organization of telomeric-linked helicase (tlh) gene families in Fusarium oxysporum.

Telomere-linked RecQ helicase (tlh) genes have been reported in several fungi and a choanoflagellate in the regions adjacent to the terminal telomere repeats. In this study, we explored the Telomere-linked RecQ helicase (tlh) genes in four strains of Fusarium oxysporum, offering new insights into their genomic structure, functional motifs, and impact on chromosomal ends. We conducted a comprehensive analysis, comparing the tlh genes of F. oxysporum with those previously identified in other organisms and uncovering significant similarities. Through comparative genomics, we identified conserved protein motifs across these genes, including a TLH domain, C2H2, and RecQ helicase motifs. Our phylogenetic analysis positions the F. oxysporum tlh genes in a cluster with other known tlhs, suggesting a shared evolutionary origin. Mutation analysis revealed a relatively low level of deleterious mutations in tlh gene paralogs, with a notable proportion of full-size structures maintained across strains. Analysis of subtelomeric sequences indicates that a region with almost identical sequences flanks the majority of chromosome ends, termed tlh-containing region (TLHcr), across these strains. The presence of TLHcrs at chromosome ends, either as single entities or in arrays, underscores their potential role in telomere function and genome stability. Our findings provide a detailed examination of tlh genes in four strains of F. oxysporum, laying the groundwork for future studies on their biological significance and evolutionary history in fungal genomes.

Conformational plasticity links structural instability of NAA10F128I and NAA10F128L mutants to their catalytic deregulation

The acetylation of proteins' N-terminal amino groups by the N-acetyltransferase complexes plays a crucial role in modulating the spatial stability and functional activities of diverse human proteins. Mutations disrupting the stability and function of NAA10 result in X-linked rare genetic disorders. In this study, we conducted a global analysis of the impact of fifteen disease-associated missense mutations in NAA10. The analyses revealed that mutations in specific residues, such as Y43, V107, V111, and F128, predictably disrupted interactions essential for NAA10 stability, while most mutations (except R79C, A111W, Q129P, and N178K) expectedly led to structural destabilization. Mutations in many conserved residues within short linear motifs and post-translational modification sites were predicted to affect NAA10 functionality and regulation. All mutations were classified as pathogenic, with F128I and F128L identified as the most destabilizing mutations. The findings show that the F128L and F128I mutations employ different mechanisms for the loss of catalytic activities of NAA10F128L and NAA10F128I due to their structural instability. These two mutations induce distinct folding energy states that differentially modulate the structures of different regions of NAA10F128L and NAA10F128I. Specifically, the predicted instability caused by the F128I mutation results in decreased flexibility within the substrate-binding region, impairing the substrate peptide binding ability of NAA10F128I. Conversely, F128L is predicted to reduce the flexibility of the region containing the acetyl-CoA binding residues in NAA10F128L. Our study provides insights into the mechanism of catalytic inactivation of mutants of NAA10, particularly elucidating the mechanistic features of the structural and functional pathogenicity of the F128L and F128I mutations.

Analysis of the structure and interactions of the SARS-CoV-2 ORF7b accessory protein

SARS-CoV-2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus-host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV-2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly α-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR. We also show that ORF7b forms heterogeneous higher-order multimers. We determined ORF7b interactions with cellular TM leucine zipper proteins using both biochemical and NMR approaches, providing evidence for ORF7b interaction with the TM domains of E-cadherin, as well as phospholamban. Our results place ORF7b as a hypothetical interferer in cellular processes that utilize leucine zipper motifs in transmembrane multimerization domains.

Molecular cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos.

Embryonic genome activation (EGA) is a critical step in early embryonic development, as it marks the transition from relying on maternal factors to the initiation of transcription from embryo's own genome. The factors associated with EGA are not well understood and need further investigation. PRD-like (PRDL) homeodomain transcription factors (TFs) are considered to play crucial roles in this early event during development but these TFs have evolved differently, even within mammalian lineages. Different numbers of PRDL TFs have been predicted in bovine (Bos taurus); however, their divergent evolution requires species-specific confirmation and functional investigations. In this study, we conducted molecular cloning of mRNAs for the PRDL TFs ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 in bovine oocytes or in vitro fertilized (IVF) preimplantation embryos. Our results confirmed the expression of PRDL TF genes in early bovine development at the cDNA level and uncovered their structures. For each investigated PRDL TF gene, we isolated at least one homeodomain-encoding cDNA fragment, indicative of DNA binding and thus potential role in transcriptional regulation in developing bovine embryos. Additionally, our cDNA cloning approach allowed us to reveal breed-related differences in bovine, as evidenced by the identification of a high number of single nucleotide variants (SNVs) across the PRDL class homeobox genes. Subsequently, we observed the prediction of the 9aa transactivation domain (9aaTAD) motif in the putative protein sequence of TPRX3 leading us to conduct functional analysis of this gene. We demonstrated that the TPRX3 overexpression in bovine fibroblast induces not only protein-coding genes but also short noncoding RNAs involved in splicing and RNA editing. We supported this finding by identifying a shared set of genes between our and published bovine early embryo development datasets. Providing full-length cDNA evidence for previously predicted homeobox genes that belong to PRDL class improves the annotation of the bovine genome. Updating the annotation with seven developmentally-important genes will enhance the accuracy of RNAseq analysis with datasets derived from bovine preimplantation embryos. In addition, the absence of TPRX3 in humans highlights the species-specific and TF-specific regulation of biological processes during early embryo development.

Structure-aware annotation of leucine-rich repeat domains.

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains.

Upregulation vs. loss of function of NTRK2 in 44 affected individuals leads to two distinct neurodevelopmental disorders

IntroductionHeterozygous pathogenic variants in NTRK2 (HGNC: 8032) have been associated with global developmental delay. However, only scattered cases have been described in small or general studies. The aim of our work was to consolidate our understanding of NTRK2-related disorders and to delineate the clinical presentation MethodsWe report extended cohort of 44 affected individuals, of whom 19 are from the literature and 25 were previously unreported. ResultsOur analysis led to splitting the cohort into two entities. DiscussionOne group had variants in the cholesterol binding motif of the transmembrane domain, with most of these being the recurrent variant c.1301A>G p.(Tyr434Cys). These variants probably lead to upregulation of TRKB activity and to a severe phenotype of developmental delay/intellectual disability, muscular hypotonia, therapy-refractory epilepsy, visual impairment and blindness, and feeding difficulties. The second group had truncating variants or variants that presumably disturb the 3D structure of the protein leading to loss of function. These individuals had a remarkably milder phenotype of developmental delay, obesity and hyperphagia.

Assembly of the human multi-tRNA synthetase complex through leucine zipper motifs.

Aminoacyl-tRNA synthetases (ARSs) are responsible for the ligation of amino acids to their cognate tRNAs. In human, nine ARSs form a multi-tRNA synthetase complex (MSC) with three ARS-interacting multifunctional proteins (AIMPs). Among the components of MSC, arginyl-tRNA synthetase 1 (RARS1) and two AIMPs (AIMP1 and AIMP2) have leucine zipper (LZ) motifs, which they utilize for their assembly in an MSC. RARS1 and AIMP1 have two LZ motifs (LZ1 and LZ2) in their N-terminus, respectively, while AIMP2 has one LZ motif between its lysyl-tRNA synthetase 1 (KARS1)-binding motif and glutathione transferase-homology domain, which links aspartyl-tRNA synthetase 1 (DARS1). Although the interaction mode between AIMP1 and RARS1, which also binds glutaminyl-tRNA synthetase 1 (QARS1), has been revealed, the mode in the presence of AIMP2 is still ambiguous since AIMP2 is known to not only bind to AIMP1 but also form a homodimer through its LZ. Here, we determined a crystal structure of the LZ complex of AIMP1 and AIMP2 and revealed the interaction mode of a heterotrimeric complex of RARS1, AIMP1, and AIMP2. The complex is established by a three-stranded coiled-coil structure with RARS1 LZ1, AIMP1 LZ1, and AIMP2 LZ and is completed with a two-stranded coiled-coil structure of RARS1 LZ2 and AIMP1 LZ2. In the human MSC, this heterotrimeric complex of RARS1, AIMP1, and AIMP2 allows for a subcomplex of fourteen protein molecules, in which two QARS1-RARS1-AIMP1-AIMP2-2×KARS1 complexes are linked separately to a dimeric DARS1.

LkERF6 enhances drought and salt tolerance in transgenic tobacco by regulating ROS homeostasis

The transcription factor Ethylene Responsive Factor (ERF) is crucial for responding to various environmental stressors. Proteins containing the ERF-associated amphiphilic repression (EAR) motif often inhibit gene expression. However, the functions of LkERF, an EAR motif-containing protein from Larix kaempferi, especially in reactive oxygen species (ROS) homeostasis, are not well understood. In the present research, we introduce a novel transcription factor, LkERF6, which contains an EAR motif and positively regulates gene expression, thereby enhancing drought and salt tolerance in tobacco. LkERF6 is classified within the ERF-B1 subfamily due to its conserved AP2/ERF domain and EAR motif. Subcellular localization assays demonstrated LkERF6 is primarily localized in the nucleus. Further analysis revealed that LkERF6 interacts with GCC and DRE elements and is significantly induced by NaCl and PEG6000. Moreover, LkERF6 transgenic tobacco plants exhibit lower ROS accumulation and higher levels of antioxidant enzyme activities. Additionally, correlation analysis identified a strong association between LkERF6 and three genes: LkSOD, LkCCS, and LkCAT. Y1H, EMAS, and DLR assays confirmed that LkERF6 directly interacts with the promoters of these genes through GCC-box and DRE-box to activate their expression. These findings shed new light on the function of EAR motif-containing transcription factors and highlight LkERF6’s crucial role in enhancing abiotic stress resistance by activating multiple ROS clearance genes.

Systematic screening of enhancer-blocking insulators in Drosophila identifies their DNA sequence determinants.

Long-range transcriptional activation of gene promoters by abundant enhancers in animal genomes calls for mechanisms to limit inappropriate regulation. DNA elements called insulators serve this purpose by shielding promoters from an enhancer when interposed. Unlike promoters and enhancers, insulators have not been systematically characterized due to lacking high-throughput screening assays, and questions regarding how insulators are distributed and encoded in the genome remain. Here, we establish "insulator-seq" as a plasmid-based massively parallel reporter assay in Drosophila cultured cells to perform a systematic insulator screen of selected genomic loci. Screening developmental gene loci showed that not all insulator protein binding sites effectively block enhancer-promoter communication. Deep insulator mutagenesis identified sequences flexibly positioned around the CTCF insulator protein binding motif that are critical for functionality. The ability to screen millions of DNA sequences without positional effect has enabled functional mapping of insulators and provided further insights into the determinants of insulators.

Genome-wide analysis of phytochrome-interacting factor (PIF) families and their potential roles in light and gibberellin signaling in Chinese pine

Phytochrome-interacting factors (PIFs) are a subgroup of transcription factors within the basic helix-loop-helix (bHLH) family, playing a crucial role in integrating various environmental signals to regulate plant growth and development. Despite the significance of PIFs in these processes, a comprehensive genome-wide analysis of PIFs in conifers has yet to be conducted. In this investigation, three PtPIF genes were identified in Chinese pine, categorized into three subgroups, with conserved motifs indicating the presence of the APA/APB motif and bHLH domain in the PtPIF1 and PtPIF3 proteins. Phylogenetic analysis revealed that the PtPIF1 and PtPIF3 proteins belong to the PIF7/8 and PIF3 groups, respectively, and were relatively conserved among gymnosperms. Additionally, a class of PIF lacking APA/APB motif was identified in conifers, suggesting its function may differ from that of traditional PIFs. The cis-elements of the PtPIF genes were systematically examined, and analysis of PtPIF gene expression across various tissues and under different light, temperature, and plant hormone conditions demonstrated similar expression profiles for PtPIF1 and PtPIF3. Investigations into protein-protein interactions and co-expression networks speculated the involvement of PtPIFs and PtPHYA/Bs in circadian rhythms and hormone signal transduction. Further analysis of transcriptome data and experimental validation indicated an interaction between PtPIF3 and PtPHYB1, potentially linked to diurnal rhythms. Notably, the study revealed that PtPIF3 may be involved in gibberellic acid (GA) signaling through its interaction with PtDELLAs, suggesting a potential role for PtPIF3 in mediating both light and GA responses. Overall, this research provides a foundation for future studies investigating the functions of PIFs in conifer growth and development.

CompariPSSM: a PSSM-PSSM comparison tool for motif binding determinant analysis.

Short Linear Motifs (SLiMs) are compact functional modules that mediate low-affinity protein-protein interactions. SLiMs direct the function of many dynamic signalling and regulatory complexes playing a central role in most biological processes of the cell. Motif binding determinants describe the contribution of each residue in a motif-containing peptide to the affinity and specificity of binding to the motif-binding partner. Motif binding determinants are generally defined as a motif consensus pattern or a position-specific scoring matrix (PSSM) encoding quantitative preferences. Motif binding determinant comparison is an important motif analysis task and can be applied to motif annotation, classification, clustering, discovery and benchmarking. Currently, binding determinant comparison is generally performed by analysing consensus similarity, however, this ignores important quantitative information in both the consensus and non-consensus positions. We have created a new tool, CompariPSSM, that quantifies the similarity between motif binding determinants using sliding window PSSM-PSSM comparison and scores PSSM similarity using a randomisation-based probabilistic framework. The tool has been benchmarked on curated data from the Eukaryotic Linear Motif (ELM) database and experimental data from Proteomic Phage Display (ProP-PD). CompariPSSM can be used for peptide classification to validate motif classes, peptide clustering to group functionally related conserved disordered regions, and benchmarking experimental motif discovery methods. CompariPSSM is available at https://slim.icr.ac.uk/projects/comparipssm. Supplementary data are available at Bioinformatics online.

Mechanistic insights into uptake, transfer and exchange of metal ions by the three-metal clusters of a metalloprotein.

Metallothioneins (MTs) are small proteins that coordinate d-block metal ions in sulfur-metal clusters to control metal ion concentrations within the cell. Here we study metal cluster formation in the MT of the periwinkle Littorina littorea (LlMT) by nuclear magnetic resonance (NMR). We demonstrate that the three Cd2+ ions in each domain are taken up highly cooperatively, that is, in an all-or-none fashion, with a four- to six-fold higher affinity for the C-terminal domain. During the transfer of metal ions from Cd2+-loaded MT to apo MT, Cd2+ is most efficiently transferred from the metalated protein to the apo C-terminal domain. This behavior might be connected to unique structural motifs in the C-terminal domain, such as two double-CXC motifs and an increased proportion of positively charged residues. In Cd2+/Zn2+ metal exchange experiments, the N-terminal domain displayed the most efficient inter-molecular metal exchange. Amide hydrogen exchange reveals fewer protected amides for the N-terminal domain, suggesting the structure might more easily "open up" to facilitate metal exchange. Experiments with a physical separation of donor and acceptor species demonstrate that metal exchange and transfer require protein-protein contacts. These findings provide insights into the mechanism of metal uptake and metal transfer, which are important processes during metal detoxification in snail MTs.

LRRTM2 controls presynapse nano-organization and AMPA receptor sub-positioning through Neurexin-binding interface

Synapses are organized into nanocolumns that control synaptic transmission efficacy through precise alignment of postsynaptic neurotransmitter receptors and presynaptic release sites. Recent evidence show that Leucine-Rich Repeat Transmembrane protein LRRTM2, highly enriched and confined at synapses, interacts with Neurexins through its C-terminal cap, but the role of this binding interface has not been explored in synapse formation and function. Here, we develop a conditional knock-out mouse model (cKO) to address the molecular mechanisms of LRRTM2 regulation, and its role in synapse organization and function. We show that LRRTM2 cKO specifically impairs excitatory synapse formation and function in mice. Surface expression, synaptic clustering, and membrane dynamics of LRRTM2 are tightly controlled by selective motifs in the C-terminal domain. Conversely, the N-terminal domain controls presynapse nano-organization and postsynapse AMPAR sub-positioning and stabilization through the recently identified Neurexin-binding interface. Thus, we identify LRRTM2 as a central organizer of pre- and post- excitatory synapse nanostructure through interaction with presynaptic Neurexins.

Disordered regions of human eIF4B orchestrate a dynamic self-association landscape

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

BIT: Bayesian Identification of Transcriptional Regulators from Epigenomics-Based Query Region Sets.

Transcriptional regulators (TRs) are master controllers of gene expression and play a critical role in both normal tissue development and disease progression. However, existing computational methods for identification of TRs regulating specific biological processes have significant limitations, such as relying on distance on a linear chromosome or binding motifs that have low specificity. Many also use statistical tests in ways that lack interpretability and rigorous confidence measures. We introduce BIT, a novel Bayesian hierarchical model for in-silico TR identification. Leveraging a comprehensive library of TR ChIP-seq data, BIT offers a fully integrated Bayesian approach to assess genome-wide consistency between user-provided epigenomic profiling data and the TR binding library, enabling the identification of critical TRs while quantifying uncertainty. It avoids estimation and inference in a sequential manner or numerous isolated statistical tests, thereby enhancing accuracy and interpretability. BIT successfully identified critical TRs in perturbation experiments, functionally essential TRs in various cancer types, and cell-type-specific TRs within heterogeneous cell populations, offering deeper biological insights into transcriptional regulation.

Structural insights into supramolecular interactions in isostructural salts of 2,4,6-triaminopyrimidinium with various heterocyclic carboxylates.

2,4,6-Triaminopyrimidine is an interesting and challenging molecule due to the presence of multiple hydrogen-bond donors and acceptors. Its noncovalent interactions with a variety of carboxylic acids provide several supramolecular aggregates with frequently occurring molecular synthons. The present work focuses on the supramolecular interactions of 2,4,6-triaminopyrimidinium 3-(indol-3-yl)propionate-3-(indol-3-yl)propionic acid (1/1), C4H8N5+·C11H10NO2-·C11H11NO2, (I), 2,4,6-triaminopyrimidinium 2-(indol-3-yl)acetate, C4H8N5+·C10H8NO2-, (II), 2,4,6-triaminopyrimidinium 5-bromothiophene-2-carboxylate, C4H8N5+·C5H2BrO2S-, (III), and 2,4,6-triaminopyrimidinium 5-chlorothiophene-2-carboxylate, C4H8N5+·C5H2ClO2S-, (IV). All four salts exhibit robust homomeric and heteromeric R22(8) ring motifs. Salts (I) and (II) develop sextuple [in (I)] and quadruple [in (I) and (II)] hydrogen-bonded arrays through fused-ring motifs. Salt (II) exhibits a rosette-like architecture. Salt (IV) is isostructural and isomorphous with salt (III), exhibiting an identical crystal structure with a different composition and an identical supramolecular architecture. In salts (III) and (IV), a linear hetero-tetrameric motif is formed and, in addition, both salts exhibit halogen-π interactions which enhance the crystal stability. All four salts develop a supramolecular hydrogen-bonded pattern facilitated by several N-H...O and N-H...N hydrogen bonds with multiple furcated donors and acceptors.

Genomic and Transcriptional Analysis of the Necroptosis Pathway Elements RIPK and MLKL in Sea Cucumber, Holothuria leucospilota.

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms.

Appendage- and Scaffold-Diverse Electrophilic and Photoreactive Probes for Integrated Phenotypic Screening-Target Identification Campaigns via a Minimalist Bifunctional Isocyanide.

One of the grand challenges in chemical biology is identifying a small-molecule modulator for all proteins within a proteome. To expand the variety and number of ligandable proteins for drug discovery, the objective of this study was to synthesize and evaluate the protein target profiles of electrophilic and photoreactive fully functionalized small-molecule probes (FFSMPs) featuring increased scaffold-, appendage-, and protein-reactive functional group (PRFG) diversity. FFSMPs contain: (1) a protein-binding motif, (2) an electrophilic or photoreactive PRFG for target protein capture, and (3) a terminal alkyne for click chemistry-based proteomic applications. These compounds can be directly applied in phenotypic screening programs to identify ligand-protein pairs in cells unbiasedly. Herein, we highlight 17 examples from 34 structurally diverse FFSMPs featuring five electrophiles, three photoreactive groups, and 15 chemical scaffolds. Essential to the synthesis of the FFSMPs was a new minimalist bifunctional isocyanide in an "isocyanide-based multicomponent reaction-Boc deprotection-arming" synthetic sequence. To the best of our knowledge, this is the first report concerning the preparation of appendage- and scaffold-diverse FFSMPs for integrated phenotypic screening-target identification campaigns with the ability to examine either electrophilic or photoreactive PRFGs. In contrast, the status quo for such studies has been appendage-diverse FFSMPs comprised of a single chemical scaffold and a single PRFG, which limits efficient target protein capture and/or chemical space sampling significantly in the quest for discovering new drug targets and/or compounds with novel mechanisms of action. Phenotypic screening of the electrophilic members of our library identified several FFSMPs with potent antiproliferative activity against MCF10CA1a breast cancer cells. One of these FFSMPs (Compound 4a) covalently targeted and potently inhibited protein disulfide isomerase A1 (PDIA1). This study supports the continued use of minimalist bifunctional isocyanides as valuable building blocks for preparing structurally diverse FFSMPs for integrated phenotypic screening-target identification campaigns.

An "R-spondin code" for multimodal signaling ON-OFF states.

R-spondins (RSPOs) are a family of secreted proteins and stem cell growth factors that are potent co-activators of Wnt signaling. Recently, RSPO2 and RSPO3 were shown to be multifunctional, not only amplifying Wnt- but also binding BMP- and FGF receptors to downregulate signaling. The common mechanism underlying these diverse functions is that RSPO2 and RSPO3 act as "endocytosers" that link transmembrane proteins to ZNRF3/RNF43 E3 ligases and trigger target internalization. Thus, RSPOs are natural protein targeting chimeras for cell surface proteins. Conducting data mining and cell surface binding assays we report additional candidate RSPO targets, including SMO, PTC1,2, LGI1, ROBO4, and PTPR(F/S). We propose that there is an "R-spondin code" that imparts combinatorial signaling ON-OFF states of multiple growth factors. This code involves the modular RSPO domains, notably distinct motifs in the divergent RSPO-TSP1 domains to mediate target interaction and internalization. The RSPO code offers a novel framework for the understanding how diverse signaling pathways may be coordinately regulated in development and disease.