Abstract The epidermal growth factor receptor (EGFR) family serves as an archetypal model of receptor tyrosine kinase signaling and plays essential roles in development, tissue homeostasis, and cancer. Although receptor phosphorylation has been extensively studied, the mechanisms governing dephosphorylation remain comparatively obscured. A limited repertoire of protein tyrosine phosphatases (PTPs) directly regulates EGFR family receptors, yet their signaling specificity cannot be explained by intrinsic catalytic activity alone. Accumulating evidence indicates that PTP function is governed by higher-order mechanisms that control subcellular localization, receptor accessibility, and integration into dynamic signaling networks. In this Review, we summarize the PTPs that directly regulate the EGFR family and examine how scaffold proteins, membrane organization, intracellular trafficking, and organelle-specific platforms coordinate receptor dephosphorylation. We propose a functional taxonomy comprising five categories: assembly, localization, membrane-organizing, organelle, and regulatory scaffolds. This framework provides a basis for understanding signaling specificity and suggests therapeutic strategies targeting the spatiotemporal organization of phosphatase function.
Abstract To realize the continuous monitoring of insulin, the development of a regenerable engineered antibody is expected, whose binding constant is modulated by the mild in situ stimulation for future in vivo application. In this study, we aimed to develop pH-responsive anti-insulin antibodies based on a clinically established analytical antibody, the HUI-018 anti-insulin antibody. Its high-resolution co-crystal structure with human insulin (PDB ID: 6Z7W) provides a uniquely precise blueprint for molecular engineering by preparing engineered HUI-018 single-chain variable fragment (scFv). Among the engineered variants, the N93H/E101K mutant exhibited the biggest pH-responsiveness, showing a 5.2-folds increased dissociation rate at pH 6.0 while maintaining binding affinity and specificity comparable to wild-type (WT) at pH 7.4. Repeated cycles of insulin binding at pH 7.4 and regeneration at pH 6.0 demonstrated that the N93H/E101K mutant achieved substantially higher regeneration and rebinding efficiencies than WT. These results demonstrate that engineered pH-responsive antibody enables efficient binding site regeneration through mild pH change. To our knowledge, this is the first report of an anti-insulin antibody capable of binding site regeneration at pH 6.0, representing an important step toward antibody-based continuous insulin monitoring and continuous sensing of protein biomarkers.
Abstract Antibody delivery across the blood–brain barrier (BBB) remains challenging. We combined phage display with next-generation sequencing (NGS)-guided clone sequence-diverse selection to identify transferrin receptor 1 (TfR1)-binding VHH antibodies. Following alpacas immunization with human and rat TfR1, representative clones from distinct phylogenetic groups were screened for human/mouse cross-reactivity. Three clones (A1, Y11, and Z1) were reformatted as human Fc-mutant fusion proteins and characterized by ELISA, flow cytometry, and fluorescence imaging. All three clones recognized human and mouse TfR1, with ELISA EC₅₀ >100 nM. After intravenous administration in mice, clone Y11 showed sustained brain-associated fluorescence up to 48 h, whereas A1 showed an initially high signal that progressively declined. Fluorescent microscopy revealed preferential localization of Y11 to NeuN-positive neuronal regions, whereas A1 to lectin-positive vascular structures, indicating distinct brain localization profiles among the isolated clones. These findings identify cross-reactive TfR1-targeting VHHs with distinct brain-localization profiles and potential utility as platforms for compartment-selective CNS drug delivery.
Abstract Ultraviolet (UV) radiation is a major environmental factor responsible for photocarcinogenesis and photoaging. Although these conditions have traditionally been regarded as distinct consequences of chronic UV exposure, accumulating evidence suggests that they share common molecular mechanisms mediated by macrophage migration inhibitory factor (MIF) family cytokines. MIF and its homologue D-dopachrome tautomerase (also known as MIF-2) have emerged as important regulators of UV-induced skin damage. MIF family cytokines promote photocarcinogenesis by enhancing inflammatory responses and suppressing apoptosis of UV-damaged keratinocytes. They also contribute to photoaging by regulating matrix metalloproteinase expression, collagen degradation and extracellular matrix remodelling. Together, these findings indicate that MIF family cytokines coordinate inflammation and tissue remodelling underlying both photocarcinogenesis and photoaging, highlighting them as promising therapeutic targets for preventing chronic UV-induced skin damage.
We identified and characterized a homocysteine S-methyltransferase (HMT) from the lactic acid bacterium Leuconostoc suionicum LT-38, representing the first characterization of an HMT from lactic acid bacteria. The enzyme exhibited a kcat/Km value of 15.7 mM-1 s-1 toward L-homocysteine. It showed optimal activity at 30 °C and pH 7.5 and remained stable over a broad range of temperatures (30-60 °C) and pH values (5-9). The enzyme accepted both L- and D-homocysteine as methyl acceptors but displayed markedly higher catalytic efficiency toward the L-form (~30-fold). In contrast, L- and D-cysteine were not utilized. Both S-methyl-L-methionine and S-methyl-D-methionine were consumed in reactions containing racemic S-methyl-DL-methionine, and S-adenosyl-L-methionine was also accepted, whereas betaine and related compounds were not utilized. Gel filtration analysis indicated a monomeric structure (~32 kDa). Phylogenetic analysis showed that Ls-HMT clustered with bacterial HMT homologs and was distinct from mammalian BHMT/BHMT2 proteins. Inductively coupled plasma-mass spectrometry revealed approximately one Zn atom per enzyme molecule, and the Zn-depleted apo enzyme showed no detectable activity. These findings support that Ls-HMT is a Zn-dependent HMT with relatively high activity and expand the functional diversity of microbial HMTs.
