Kymera Therapeutics

To investigate whether MLKL crotonylation is associated with tubular autophagy-lysosome pathway homeostasis in lupus nephritis (LN) and to explore its relationship with RAB1A-mechanistic target of rapamycin (mTOR) signalling. Crotonylome proteomics was performed in peripheral blood mononuclear cells from patients with LN, patients with systemic lupus erythematosus without nephritis and healthy controls. Renal biopsy tissues were evaluated for tubulointerstitial fibrosis and autophagy-lysosome pathway-related markers. Mechanistic studies were conducted in lipopolysaccharide-stimulated HK-2 cells. Autophagic flux was assessed using bafilomycin A1. The dependency of mTOR/autophagy changes on RAB1A was tested by siRNA-mediated knockdown. MLKL was identified as a differentially crotonylated protein in LN, with increased crotonylation at K95 and K219. Kidney tissues from patients with LN showed increased fibronectin and collagen III deposition compared with controls, whereas no significant difference was observed between class IV and class V LN. LC3 signal did not differ significantly between groups, whereas LAMP1 expression and LC3-LAMP1 co-localisation were reduced in LN. In HK-2 cells, crotonylation-deficient MLKL mutants were associated with increased LC3-II and reduced p62, whereas K219Q showed the opposite pattern. Autophagic flux assays using bafilomycin A1 showed that K219R-expressing cells had higher LC3-II levels than WT cells both before and after lysosomal inhibition, with comparable BafA1-induced LC3-II accumulation, consistent with increased autophagosome formation rather than impaired lysosomal degradation. HDAC1 knockdown increased MLKL crotonylation and was accompanied by mTOR activation. MLKL crotonylation enhanced RAB1A guanriphosphat osphate (GTP) binding without altering total RAB1A abundance. RAB1A knockdown in MLKL WT-expressing cells attenuated mTOR phosphorylation and partly reversed the autophagy-suppressive marker profile. Sodium crotonate induced an autophagy-suppressive marker profile that was partly reversed by rapamycin. MLKL crotonylation is associated with activation of the RAB1A-mTOR axis and altered tubular autophagy-lysosome pathway homeostasis in LN. These findings suggest that tubular injury-related changes in LN may not be fully reflected by glomerulus-based classification alone.

Protein fragments are increasingly recognized as both biomarkers and therapeutic targets, offering important insights into disease mechanisms and potential intervention strategies. Evidence from in vitro and in vivo studies indicates that these fragments are not merely degradation products but possess distinct biological activities, actively modulating immune signaling and contributing to both acute and chronic inflammatory processes. Here, we provide an overview of proteases and their inhibitors, with a particular focus on peptide fragments generated through proteolytic cleavage. Special emphasis is placed on fragments derived from α1-antitrypsin (AAT), an acute-phase glycoprotein and major inhibitor of neutrophil elastase and other serine proteases. AAT-derived peptides of varying lengths, generated by both target and nontarget proteases, including metalloproteases, have been detected in human biological fluids and tissues. Beyond reflecting proteolytic activity, these peptides provide clinically relevant information on disease-associated inflammation and tissue remodeling. Accordingly, they are emerging as promising diagnostic, monitoring, and predictive biomarkers. Here, we summarize current knowledge on cleaved AAT fragments, their biological functions, and their potential clinical applications.

The global rise of multidrug-resistant bacteria is a major health threat, with Klebsiella pneumoniae identified by the WHO as a critical priority pathogen. Phage-derived depolymerases have emerged as promising countermeasures because they can degrade the capsular polysaccharide (CPS) that shields bacteria from the immune system, thereby increasing their susceptibility to antibiotics and host defences. However, the limited understanding of how they recognise specific CPS structures remains a major obstacle to developing effective depolymerase-based therapeutics. In this study, we provide a comprehensive NMR and mutational analysis to characterise the mechanism of action of a miniaturised depolymerase, here mKP34gp57, targeting the clinically relevant K63 CPS. We show by NMR that mKP34gp57 hydrolyses CPS with high efficiency through an endoglycosidase-retaining mechanism. During substrate recognition, our data demonstrate that the enzyme interacts predominantly with the galactose and fucose moieties, which serve as the critical recognition features and thus the principal determinants of CPS specificity. Computational studies provide structural clues for the roles of the catalytic residues E266/E300 and D151. Finally, we prove that the hexasaccharide produced upon CPS hydrolysis stimulates dendritic cell maturation and T-helper-driven lymphocyte proliferation. Identifying the binding determinants that govern CPS recognition by mKP34gp57, and using this information to generate immunogenic fragments, deepens our understanding of how minidepolymerases can be rationally engineered to achieve tailored serotype specificity, and improved therapeutic and diagnostic potential.

RNA N6-methyladenosine (m6A), as the most prevalent and abundant RNA modification on mRNAs, plays indispensable roles in biological processes and development by modulating the fate of target RNAs. However, whether other METTL proteins, besides dominant METTL3 and METTL14, participate in the intricate dynamic regulation of mRNA m6A remains elusive. Here, we reveal that METTL18 catalyzes the methylation of histidine within the YTHDF2 protein, which in turn facilitates the stability of m6A-modified RNAs. Mechanistically, METTL18 directly interacts with YTHDF2 and catalyzes the methylation of histidine at position 437 of YTHDF2, attenuating its binding to m6A-modified target RNAs, thereby safeguarding these RNAs from degradation. Our findings not only demonstrate the crucial roles of METTL family proteins but also deepen our understanding of the intricate regulatory dynamics of m6A modification.

Mucosal inflammatory disorders such as dry eye disease (DED) and inflammatory bowel disease (IBD), driven by TNF-α-mediated immune-epithelial crosstalk, remain therapeutically challenging. Conventional inhibitors often suffer from poor mucosal permeability and transient efficacy with 1:1 binding. Although extracellular protein degradation (eTPD) technologies can eliminate pathogenic proteins, most of them operate stoichiometrically, limiting dosage efficiency and durability. Here, we report a receptor-mediated endolysosomal recycling chimera (RECYC) platform that enables catalytic, recyclable degradation of TNF-α with multiple turnovers, achieving sustained suppression of mucosal inflammation. RECYC integrates a pH-responsive peptide that binds TNF-α at physiological pH and releases it in acidic endosomes, with a CI-M6PR-targeting aptamer that remains pH-stable to drive continuous recycling. The anti-TNF-α RECYC achieved efficient uptake and recyclable clearance of extracellular TNF-α across conjunctival and colonic epithelial cells and macrophages, achieving superstoichiometric TNF-α clearance with up to 7.5 catalytic turnovers. The ∼30 kDa RECYC exhibited superior tissue penetration in murine cornea and colon compared to inhibitory antibodies. In murine DED and IBD models, RECYC restored ocular surface homeostasis and reduced colon inflammation with low-dose, infrequent administration. Human corneal limbus organoids further confirmed prolonged anti-inflammatory activity. Together, RECYC establishes a modular, recyclable eTPD platform for sustained treatment of TNF-α-driven mucosal inflammation.

Sepsis-associated liver dysfunction is a life-threatening condition with a high mortality rate and no mechanism-based therapy. In this study, we identify the cross-linking enzyme transglutaminase 2 (TG2) as a driver of liver inflammation by activating macrophages in a mouse model of sepsis. Pharmacological inhibition of TG2 improves survival and reduces multiorgan inflammation, with the liver as a primary therapeutic target. Mechanistically, TG2 activity was up-regulated in macrophages, where it cross-linked vimentin to promote oligomerization and intermediate filament remodeling. Genetic ablation of TG2 or vimentin suppressed macrophage cytokine production and attenuated lipopolysaccharide-induced inflammation. Notably, vimentin-deficient macrophages exhibited enhanced proteasome recruitment to detergent-insoluble protein aggregates, accelerating the degradation of proinflammatory mediators such as Traf6, thereby dampening nuclear factor κB signaling. Proteomic profiling revealed a previously unrecognized Rab27a-positive vesicle trafficking pathway for inflammatory aggregate clearance. Together, these findings define a TG2-vimentin axis that controls macrophage activation through proteostasis regulation, linking cytoskeletal remodeling to inflammatory signaling.

Telomere shortening drives hair follicle senescence, yet successful reversal of this aging process has remained elusive. Here, we report that stem cell overexpression of Acd, the gene encoding TPP1 (telomere protection protein 1)-a shelterin component that recruits telomerase-accelerates hair regeneration in mice. Stabilization of TPP1 protein via topical application of TELODIN, a synthetic octapeptide that inhibits TPP1 degradation, similarly promotes hair regeneration. Mechanistically, increased TPP1 mobilizes bulge hair follicle stem cells (bHFSCs) by coordinating telomere capping and telomerase recruitment. By facilitating the synchronization and transition of hair follicles from the quiescent telogen phase to the proliferative anagen phase, TPP1 drives bHFSCs proliferation, differentiation, and migration. These findings identify TPP1 as a master regulator of hair regeneration through telomere capping and elongation, and suggest that topical TELODIN represents a novel therapeutic strategy for advancing hair growth. This work establishes a therapeutic framework for telomere-targeted interventions against alopecia.