The differential interactomes of the KRAS splice variants identify BIRC6 as a ubiquitin ligase for KRAS4A.

Transcripts of the KRAS locus are alternatively spliced to generate two proteins, KRAS4A and KRAS4B, which differ in their membrane-targeting sequences. These splice variants have been conserved for more than 450 million years, suggesting non-overlapping functions driven by differential membrane association. Here, we use proximity labeling to map the differential interactomes of the KRAS splice variants.

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues.

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'.

Bioorthogonally activated reactive species for target identification.

A target identification platform derived from the bioorthogonal activation of reactive species is described. We explore the reactivity of halogenated enamine -oxides and report that the previously undisclosed α,γ-halogenated enamine -oxides can be reduced biooorthogonally by diboron reagents to produce highly electrophilic α,β-unsaturated haloiminium ions suitable for labeling a range of amino acid residues on proteins in a 1,2- or 1,4-fashion. Affinity labeling reagents bearing this motif enable ligand-directed protein modification and afford highly sensitive and selective target identification in unbiased chemoproteomics experiments.

Exploration of the Tunability of BRD4 Degradation by DCAF16 -labelling Covalent Glues.

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4 ligase, have been mostly discovered serendipitously.

Cryo-EM structure of a RAS/RAF recruitment complex.

RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer.

Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors.

Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy.

Assessing risk of vector transmission of Chagas disease through blood source analysis using LC-MS/MS for hemoglobin sequence identification.

Chagas disease is mainly transmitted by triatomine insect vectors that feed on vertebrate blood. The disease has complex domiciliary infestation patterns and parasite transmission dynamics, influenced by biological, ecological, and socioeconomic factors. In this context, feeding patterns have been used to understand vector movement and transmission risk.

Allosteric MEK inhibitors act on BRAF/MEK complexes to block MEK activation.

The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma.

FYN and ABL Regulate the Interaction Networks of the DCBLD Receptor Family.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs.

Defining the TLT-1 interactome from resting and activated human platelets.

The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults.

Dimerization of small integral membrane protein 1 promotes cell surface presentation of the Vel blood group epitope.

The Vel blood group antigen is carried on the short extracellular segment of the 78-amino-acid-long, type II transmembrane protein SMIM1 of unknown function. Here, using biochemical analysis and flow cytometry of cells expressing wild-type and mutant alleles of SMIM1, we demonstrate that dimerization of SMIM1 promotes cell surface display of the Vel epitope. We show that SMIM1 dimerization is mediated both by an extracellular Cys77-dependent, homomeric disulfide linkage and via a GxxxG helix-helix interaction motif in the transmembrane domain.

Protein mass spectrometry detects multiple bloodmeals for enhanced Chagas disease vector ecology.

Chagas disease, a neglected tropical disease endemic in Latin America, is caused by the protozoan parasite Trypanosoma cruzi and is responsible for significant health impacts, especially in rural communities. The parasite is transmitted by insect vectors in the Triatominae subfamily and due to lack of vaccines and limited treatment options, vector control is the main way of controlling the disease. Knowing what vectors are feeding on directly enhances our understanding of the ecology and biology of the different vector species and can potentially aid in engaging communities in active disease control, a concept known as Ecohealth management.

The DCBLD receptor family: emerging signaling roles in development, homeostasis and disease.

The iscoidin, U, and CCL omain-containing (DCBLD) receptor family are composed of the type-I transmembrane proteins DCBLD1 and DCBLD2 (also ESDN and CLCP1). These proteins are highly conserved across vertebrates and possess similar domain structure to that of neuropilins, which act as critical co-receptors in developmental processes. Although DCBLD1 remains largely uncharacterized, the functional and mechanistic roles of DCBLD2 are emerging.

Protein mass spectrometry extends temporal blood meal detection over polymerase chain reaction in mouse-fed Chagas disease vectors.

Background: Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals.

Objectives: The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs.

Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A.

The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif.

Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase.

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated.

An in silico proteomics screen to predict and prioritize protein-protein interactions dependent on post-translationally modified motifs.

Motivation: The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority.

Fyn-dependent phosphorylation of PlexinA1 and PlexinA2 at conserved tyrosines is essential for zebrafish eye development.

Plexins (Plxns) are semaphorin (Sema) receptors that play important signaling roles, particularly in the developing nervous system and vasculature. Sema-Plxn signaling regulates cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. However, the receptor-proximal signaling mechanisms driving Sema-Plxn signal transduction are only partially understood.

Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities.