GSDMA, the primary member of the gasdermin family found in the skin, is critical for pathogen-induced pyroptosis during infection. Recent studies revealed that another gasdermin, GSDMD, undergoes palmitoylation during pyroptosis. However, whether and how the other gasdermin members undergo lipid modification remain poorly understood. Here, we demonstrate that GSDMA is S-acylated at the conserved cysteine residues in its N-terminal domain. We show that the S-acylation of GSDMA promotes pyroptosis by facilitating its membrane anchoring and protein oligomerization, a mechanism distinct from the palmitoylation of GSDMD at the N-terminal C191 residue. In addition, we present evidence that recombinant proteins of GSDMA and GSDMD can undergo S-acylation in vitro independent of palmitoyl transferases via direct interaction with palmitoyl-CoA. Furthermore, we identify ABHD17A as one of the deacylating enzymes that regulate the dynamic fatty acylation cycle of GSDMA. Overall, our studies reveal new molecular mechanisms underlying GSDMA function through S-acylation and underscore its important role in regulating pyroptosis mediated by GSDMA.

Ufmylation is a newly identified ubiquitin-like modification of histones and plays important roles in DNA-related processes. Dissecting histone ufmylation pathways necessitates the use of chemically defined proteins to assign their structural and functional consequences; however, the preparation of ufmylated histones has not yet been reported. Here, we report the chemical synthesis of ufmylated histones and their analogs through semisynthetic strategies integrating chemoenzymatic C-terminal hydrazinolysis of ubiquitin-fold modifier 1 (UFM1) and auxiliary-mediated formation of an isopeptide bond. The results indicated that the E1-mediated activation of UFM1 can be hijacked by nucleophilic reagents, forming the full-length UFM1 hydrazide that can be readily installed onto histones via auxiliary-mediated ligations. The synthetic histones enabled us to reveal that the two known UFM1-specific proteases 1 and 2 (UfSP1 and UfSP2) cannot efficiently cleave H4 ufmylation at Lys31 (H4K31UFM1) in the nucleosome context. Furthermore, cryo-electron microscopy (cryo-EM) analysis of the H4K31UFM1-nucleosome suggested that the steric hindrance of the nucleosome around the isopeptide bond might be one of the potential reasons for the weak activities of UfSPs. Collectively, we developed practical strategies for the efficient generation of ufmylated histones and exemplified their use in biochemical and structural studies related to histone ufmylation.

Allosteric modulators offer a promising approach for targeting receptor function in pathological contexts. Although numerous orthosteric ligands have been developed, the discovery of allosteric inhibitors remains limited, hindered by challenges with identifying modulators and the perceived lower druggability of allosteric sites. In this study, we employed affinity selection-mass spectrometry (AS-MS) with a chemically diverse library to identify novel allosteric modulators of the human A2A adenosine receptor (A2AAR). Subsequent competition binding and orthogonal biophysical assays confirmed the allosteric nature of multiple initial hits, underscoring the sensitivity and utility of the AS-MS approach. Despite exhibiting relatively weak affinity, these compounds modulated cAMP production, supporting the idea that allosteric modulators can exert functional effects without requiring high potency to outcompete endogenous adenosine. Experiments in the presence of an A2AAR agonist further supported the classification of these compounds as negative allosteric modulators (NAMs), with distinct pharmacological profiles indicative of diverse mechanisms of action. An integrated computational workflow designed to predict allosteric sites and model ligand interactions provided insight into potential binding poses, yielding both intracellular and extracellular allosteric sites that aligned with the experimentally observed pharmacological properties. The identification of these previously uncharacterized NAMs represents an important step toward developing alternative A2AAR-targeted therapies, enabling pharmacological intervention through receptor sites beyond the orthosteric binding pocket.

Chemoproteomic strategies have revolutionized proteome annotation by targeting nucleophilic and redox-active side chains. However, the primary amides of asparagine (Asn) and glutamine (Gln) have long lacked robust chemical tools for proteome-wide interrogation. We report a chemoselective palladium-mediated dehydration that converts Asn/Gln amides to nitriles under mild aqueous conditions. This transformation enables the first proteome-wide mapping of chemically addressable Asn/Gln sites in lysates and living cells. Leveraging this reactivity, we establish an inverse chemoproteomic framework in which reduced nitrile formation reports PTM-mediated protection of Asn/Gln sites, including those impacted by deamidation and N-glycosylation. This approach reveals sites masked by post-translational modifications (PTMs), specifically those associated with deamidation and N-glycosylation. In yeast, this framework expanded the known N-glycoproteome, identifying numerous candidates missed by traditional glycopeptide enrichment due to low abundance or noncanonical motifs. Furthermore, comparative profiling in Candida albicans captured the dynamic remodeling of glycosylation patterns during morphogenesis. This dehydration-to-nitrile platform establishes a scalable handle on the amide proteome to map residue accessibility and PTM-linked site dynamics across biological states.

Targeted protein degradation (TPD) destroys proteins of interest (POIs) by hijacking the cellular proteolytic machinery. Most proteins in cells exist and function as part of multiprotein or macromolecular complexes, thereby allowing a single protein to control multiple biological processes. Therefore, when a small molecule degrader induces the proximity between an E3 ligase and the POI, the macromolecular context of the POI potentially influences the degradation outcomes of the POI and of the complex components. Here, we explore the degradation of the eight CK1α-SACK1 (formerly known as FAM83A-H) complexes initiated by molecular glue degraders primarily designed to target Ser/Thr kinase CK1α. We demonstrate that lenalidomide-derived degraders DEG-77 and SJ3149, which selectively target the CK1α isoform, codegrade multiple SACK1 proteins. We show that the degradation of SACK1 proteins by DEG-77 and SJ3149 requires CK1α, the CUL4ACRBN E3 ligase complex, and the proteasome. In cells derived from palmoplantar keratoderma patients harboring the CK1α-binding-deficient SACK1GR265P mutation, DEG-77 targets CK1α and mitotic SACK1D but not SACK1GR265P, highlighting the requirement for CK1α-SACK1 interaction to achieve codegradation. Our study underscores the importance of POI context in TPD and reinforces the potential for selectively targeting specific protein complexes for degradation.

Central kinases of the Hippo tumor suppressor pathway phosphorylate the transcriptional coactivators YAP and TAZ to sequester them in the cytoplasm. In cancer, Hippo pathway kinases have reduced activity, leading to translocation of YAP and TAZ into the nucleus, where they engage TEADs and other transcription factors. Here, we explore whether heterobifunctional small molecules that bind to the TEAD allosteric lipid-binding pocket can degrade the TEAD·YAP/TAZ complex. We design and synthesize heterobifunctional molecules that consist of flufenamic acid analogs that bind to the allosteric TEAD lipid pocket, a long and flexible linker, and thalidomide to engage E3 ubiquitin ligase component cereblon. Proteasomal degradation of TEAD, YAP, and TAZ for the carboxylic acid compounds was modest, but methyl ester analogs led to substantial degradation of these proteins in cancer cells. This work provides a strategy for depletion of YAP and TAZ and for exploration of their TEAD-dependent and TEAD-independent activities in vivo.

Protein arginine deiminase 4 (PAD4) is a crucial regulator of human neutrophils, playing a central role in the innate immune response as well as the initiation and progression of several immune diseases, including rheumatoid arthritis, cancer, and neurodegeneration. Current approaches for detecting PAD4 activity employ methods that cannot be used in living cells, such as immunodetection or chemical probes that need fixed material or harsh experimental conditions. As a result, they provide limited information on the real-time dynamics of PAD4 activation and localization during neutrophil activation. Here, we present a turn-on activity-based probe that fluoresces exclusively upon binding to active PAD4. This probe allows for the real-time imaging of intracellular PAD4 activity in live human neutrophils during NETosis, a specialized form of cell death, under wash-free conditions. Our results indicate that PAD4 is active in NETosis triggered by both NADPH oxidase-dependent and NADPH oxidase-independent mechanisms; however, the activation dynamics and localization vary depending on the NETosis stimulus. Our probes are valuable tools to further provide insight into the mechanistic differences in NETosis induction and progression, as well as the role of PAD4 in the innate immune system.

FtsH, an AAA + metalloprotease that is essential in bacteria and eukaryotic organelles, maintains cellular homeostasis by degrading misfolded and membrane-associated proteins. Here, we report cryo-EM structures of the Escherichia coli FtsH periplasmic domain (FtsH-PD) revealing insights into its intrinsic conformational flexibility. Our analysis resolved two distinct states: a 4.9 Å structure exhibiting the conserved α + β fold and a 7.3 Å map representing distinct rotated-helix conformation characterized by 20° clockwise rotation of two alpha helices. These findings support a model where conformational changes are present not only in the FtsH cytosolic domain but also in the periplasmic domain. This flexibility potentially facilitates substrate translocation through a combination of mechanisms involving both the FtsH-PD and the HflKC complexed with FtsH, along with lipid-scramblase activity, to assist in membrane protein extraction. This study offers new perspectives on how conformational changes in the periplasmic domain contribute to FtsH substrate degradation mechanisms.

Fucosylated glycans on glycoproteins and glycolipids play critical roles in functional regulation, significantly impacting human health and disease. Fucosylated glycans in humans are categorized into three types: core fucose, terminal fucose, and O-linked fucose, with each type contributing uniquely to physiological and pathological processes. Fucosylation is associated with various conditions, including cancer, autoimmune diseases, and developmental disorders, and it can also affect the efficacy of therapeutic antibodies. To investigate aberrant fucosylation in disease progression, azido- and alkynylfucose analogs have been utilized as orthogonal clickable probes, though mostly in nonhuman species. However, it remains unclear whether all human fucosyltransferases (FUTs) can accept these probes. In this study, we evaluated the utilization of GDP-fucose analogs, including GDP-6-azidofucose (GDP-6-Az-Fuc), GDP-6-alkynylfucose (GDP-6-Alk-Fuc), and GDP-7-alkynylfucose (GDP-7-Alk-Fuc), as donor substrates and natural N-glycans as acceptors, and compared their specificity with GDP-fucose for nine human FUTs (FUT1-9) that are involved in the biosynthesis of glycoproteins. We determined key kinetic parameters and catalytic efficiencies of individual FUTs for their fucosylation of specific biantennary N-glycan acceptors to assess their incorporation of these analogs into glycoprotein N-glycans. Compared to GDP-fucose, all analogs were much weaker substrates for FUTs except FUT4, which exhibited better tolerance toward the analogs, especially GDP-7-Alk-Fuc. Notably, GDP-7-Alk-Fuc was accepted better than GDP-6-Alk-Fuc and GDP-6-Az-Fuc as a substrate for these nine human FUTs. These findings reveal the variability in the acceptance specificity and catalytic efficiency of the human FUT family toward the probes and emphasize the potential bias in identifying fucosylated glycans as therapeutic targets.