Journal of Biological Chemistry最新文献

Surfactant Protein C (SP-C), a hydrophobic protein exclusively synthesized and secreted by alveolar type II (AT2) cells, is important for reducing alveolar surface tension in the distal lung. Chronic interstitial pulmonary diseases have been associated with SFTPC mutations. However, a detailed understanding of SP-C maturation in the secretory pathway and disruptions caused by mutations has remained incomplete. The goal of this study was to comprehensively ascertain differences in trafficking and post-translational processing between wild-type and disease-associated SP-C mutants using doxycycline-inducible mouse lung epithelial (MLE-12) cell lines expressing either wildtype SP-C or the common clinical variant SP-C^I73T, validated using primary AT2 cells isolated from a murine SP-C^I73T pulmonary fibrosis model and induced pluripotent stem cell (iPSC)-derived human AT2 cells expressing the same mutant. In all 3 models SP-C^WT was highly concentrated in acidic Lysosomal Related Organelles (LROs) while SP-C^I73T accumulated on the plasma membrane, which was corroborated by inhibition of clathrin-mediated endocytosis, surface biotinylation, immunogold EM, immunofluorescent staining, and proteinase K protection assays supporting divergence of SP-C^I73T trafficking from SP-C^WT. The exclusion of SP-C^I73T from normal routing occurred early in the biosynthetic pathway as Brefeldin A blocked processing of both SP-C proproteins, while a 20˚C temperature shift caused selective accumulation of a processed proSP-C^WT intermediate, suggesting initial C-terminal cleavage of proSP-C^WT occurs in late-Golgi/trans-Golgi network (TGN). This cleavage event was sensitive to DC1, an inhibitor of furin-related subtilisin-like proprotein convertase (PPC) family members. Site-directed mutagenesis of canonical residues K160/R167 within a predicted PPC recognition site in the proSP-C COOH domain blocked its processing. Expression constructs encoding inhibitory pre-proprotein (pp) peptide fragments of Furin and ppPC7 each inhibited cleavage of proSP-C^WT by MLE-12 cells. Collectively, our data demonstrate that trafficking pathways for maturation of WT and mutant I73T SP-C diverge prior to the TGN where initial cleavage of the COOH-terminal SP-C propeptide occurs via a Furin-like proprotein convertase.

SETD2 is the primary methyltransferase responsible for generating H3K36me3, an epigenetic mark that is essential for transcriptional regulation and chromatin integrity. SETD2 mutations are frequently observed in various cancers and tend to cluster within its catalytic SET domain. Despite the clinical relevance of SETD2 missense mutations in cancer, their biochemical and structural consequences remain insufficiently characterized. Here, we present the enzymatic and structural characterization of the SETD2 L1609P mutant enzyme identified in leukemia. The L1609 residue is located in the SET domain within a conserved hydrophobic pocket that is involved in substrate H3K36 recognition. Interestingly, site-directed mutagenesis of residues within this hydrophobic pocket leads to SETD2 enzyme variants with either decreased or increased H3K36me3 methyltransferase activity, suggesting that cancer mutations affecting the L1609 residue could result in a loss- or gain-of-function enzyme variant. Using molecular and cellular approaches we show that the SETD2 L1609P mutant exhibits reduced H3K36 methyltransferase activity, decreased protein stability and poor cellular expression. Consistently, the crystal structure of the SETD2 L1609P in complex with a H3K36M peptide shows remodeling of the active site. These findings support the pivotal role of SETD2 inactivation and subsequent disruption of H3K36me3 deposition in oncogenesis, particularly in hematologic malignancies. Our study provides the first mechanistic and three-dimensional protein structure information on how SETD2-associated cancer mutations can lead to altered H3K36 methyltransferase activity.

Totipotency is the first cell fate emerged from fertilization, but remains poorly understood at the molecular level. Totipotent blastomeres are characterized by the presence of topologically associating domains (TADs) with significantly weakened structural integrity. In this study, we performed high-resolution 3D genome architecture profiling of two established mouse totipotent-like models, ciTotiSCs and TBLCs, and revealed that both largely retained TADs found in ICM/ESCs. Yet, amid the apparent TAD conservation, TAD strength was indeed considerably weakened upon the acquisition of totipotency. Integrative analysis of epigenetic and Hi-C data revealed that pluripotency genes underwent coordinated epigenetic landscape remodeling and 3D contact reorganization, which collectively drove pluripotency silencing. Epigenetic remodeling was also observed at totipotency gene loci. This work represents the first systematic effort to benchmark totipotency models at the 3D genome level and provides a framework to establish totipotency through 3D genome folding.

Mutations in the ATP-binding cassette transporter ABCA4 are responsible for recessive Stargardt disease (STGD1) a juvenile form of macular degeneration. In preclinical and clinical studies it has been shown that deficiency in ABCA4 leads to accelerated formation of the toxic bisretinoid fluorophores that form as the product of nonenzymatic reactions of retinaldehyde with phosphatidylethanolamine (PE) (2:1 ratio). Here, by comparing photoreceptor cell viability in albino versus agouti Abca4^-/- mice and by dark-rearing albino Abca4^-/- mice we show that photoreceptor cell degeneration in the Abca4^-/- mouse is at least partially driven by light. Elevated vitamin A in chow and a high fat diet reduced photoreceptor cell viability. PE and N-retinylidiene-phosphatidylethanolamine were reduced as were steady state levels of retinoid in light-adapted eyes. As expected, bisretinoids, measured as short-wavelength fundus autofluorescence, were elevated in both pigmented and albino Abca4^-/- mice. Hyperautofluorescent puncta in fundus autofluorescence images colocalized in spectral domain optical coherence tomography scans with aberrant hyperreflectivity that occupied photoreceptor-attributable bands and extended anteriorly to interrupt the ellipsoid zone and external limiting membrane. In epifluorescence images of Abca4^-/- retina, retinal pigment epithelium was autofluorescent due to bisretinoid accumulation. Occasionally AF lesions extended anteriorly from the RPE to a horizontal band exhibiting less pronounced autofluorescence at the level of photoreceptor inner and outer segments. These lesions did not co-localize with IBA1-labeled microglia. The hyperautofluorescent foci that presented as hyperreflective lesions in SD-OCT form in photoreceptor inner segments and are reminiscent of fundus flecks in STGD1.

Many GPCRs use endocytosis to promote gene transcription by prolonging signaling through the Gs-coupled cAMP / cAMP-dependent protein kinase (PKA) cascade. However, not all GPCRs efficiently internalize after agonist-induced activation and, among those that do, considerable differences have been observed in the ability of different GPCRs to stimulate endosomal cAMP production in different cell types. We asked if endocytosis distinguishes the signaling profiles of GPCRs that are naturally coexpressed in the same cells, focusing on three Gs-coupled GPCRs endogenous to human kidney-derived (HEK293) cells: the vasoactive intestinal peptide receptor-1 (VIPR1 / VPAC1) and β2-adrenergic receptor (β₂AR / ADRB2) which both rapidly internalize after activation and the adenosine-2B receptor (A_2BR / ADORA2B) which we show here does not. For VIPR1, endocytosis significantly prolongs both the global cAMP elevation and cytoplasmic PKA activity increase. For β2AR, endocytosis has little effect on the global cAMP elevation but, nonetheless, it significantly prolongs the cytoplasmic PKA activity increase. A_2BR differs still further, with endocytosis having little effect on signal duration measured at either intermediate step. We then show that further downstream steps in the cascade, nuclear PKA activation and transcriptional induction, are significantly endocytosis-dependent when stimulated through VIPR1 and β2AR but endocytosis-independent through A_2BR. We conclude that endocytosis indeed distinguishes the signaling profiles of endogenously coexpressed GPCRs. We propose that quantitative differences in GPCR internalization and activation in endosomes program in cells a GPCR-selective, spatiotemporal 'cAMP code' that is spatially 'decoded' by proximity to local cytoplasmic PKA stores and then temporally interpreted by the nucleus.

Rho GTPases are members of the Ras superfamily of small GTPases that regulate cell morphology, motility, polarization and cell cycling. Like members of the Ras subfamily, Rho subfamily GTPases dysregulation is implicated in a range of tumors and can serve as a valid drug target. In this work, we investigate the evolutionary trajectory of Rho GTPases within a region of the protein that has been exploited for cancer drug discovery within the Ras subfamily branch - the "switch II pocket". Our previous work has illustrated the role of allostery in this region of H-Ras in modulation of intrinsic hydrolysis and effector-binding capacity. Here, we report that a highly conserved salt bridge within the Rho subfamily stabilizes the RhoA GTPase active site in a catalytically favorable conformation. We probed the roles of the Rho salt bridge via X-ray crystallography, accelerated molecular dynamics simulations (aMD), and enzymatic studies. We showed that the removal of a residue within switch II of RhoA, the salt bridge residue R70, can impart catastrophic effects on active site organization and GTP hydrolysis. As expected, removal of the analogous R68 in H-Ras, which is not involved in a salt bridge interaction, results in a structure with changes in the active site and a decrease in GTP hydrolysis rate constant that are more moderate than observed for RhoA. The anionic partner of R70, E102, also modulates active site conformation and, upon removal, decreases intrinsic hydrolysis. Based on aMD simulations, we uncovered evidence of epistatic relationships between the Rho salt bridge, the distal residue K98 and P-loop residue D13 which coordinate allosteric communication from the switch regions directly to the active site. Finally, we describe the functional landscape of switch II pocket in the context of both Rho subfamily evolution and potential for drug discovery.

The function of extracellular vesicles (EVs) is determined by the molecular cargo they carry from their parent cells. Although Apoptosis-linked gene 2-interacting protein X (Alix) is known to regulate EV cargo loading and functional properties, the specific mechanisms underlying its role in mediating β-catenin sorting and function remain unclear. In this study, we first observed the co-localization of Alix and β-catenin through immunofluorescence staining. To assess whether the interaction between Alix and β-catenin affects the function of mesenchymal stem cell (MSC)-derived EVs, we generated Alix-knockdown (KD) and Alix-overexpressing (OE) MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-OE-MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-KD-MSCs suppressed angiogenesis. Mechanistically, we confirmed that the Alix-β-catenin interaction selectively enhances β-catenin enrichment within EVs. In conclusion, our findings demonstrate that Alix plays a critical role in selectively packaging β-catenin into EVs, thereby enhancing their pro-angiogenic potency. Modified.

Sialic acids are the terminal monosaccharides of the glycocalyx that critically shape cell-cell interactions, and are strongly implicated in regulating immune recognition and tissue homeostasis. In cancer, aberrant sialylation rewires the tumor microenvironment by enhancing ligands of the inhibitory Siglecs, suppressing immune effector functions, and facilitating metastatic dissemination. This review provides a comprehensive synthesis of the dual role of sialyltransferases (the "writers") and Siglecs/Selectins (the "readers") in cancer progression. We examine the structural and functional diversity of these molecules, their dysregulation in malignancy, and their impact on tumor-immune dynamics. Finally, we highlight emerging therapeutic strategies, including sialyltransferase inhibitors, sialidase conjugates, and Siglec-targeted immunotherapies, which collectively position the sialome as a tractable frontier in cancer treatment.

Biomolecular NMR spectroscopy has been a keystone in structural biology for decades. It can provide unique, atomic-level insights into protein dynamics, interactions, and conformational ensembles. However, its complex workflows and fragmented data analysis pipelines are often perceived as significant barriers to entry. This review highlights the POKY suite as a comprehensive solution that modernizes and streamlines the entire biomolecular NMR process. From spectral processing to structure calculation, POKY creates a single user-friendly cyberinfrastructure for a seamless and efficient NMR data analysis environment. A key aspect of its design is the integration of various artificial intelligence (AI) components to streamline complex tasks and reduce user burden, such as automation, unsupervised learning, and more. While recent advances within in silico AI prediction models have raised questions about the role of experimental data, POKY provides a clear answer. This ecosystem can create a powerful synergy between the experimental data with structure prediction. modernizing the experimental workflow, POKY makes NMR more accessible and powerful, reinforcing its vital role instructural biology.

Mechanistic studies have yielded novel prostaglandin analogs and acyclic products initially of interest in understanding cyclooxygenase structure-function, later found in vivo and of interest due to unique biological activities. Beyond arachidonic acid, fatty acid substrates span from 18 to 22 carbons and may contain ester/amide modification or epoxide/hydroxy moieties at the first double bond. Stereo control with the unconventional substrates remains largely intact although cyclization may be diverted or halted altogether, and catalysis proceeds with insertion of one, two, or three molecules of oxygen into substrates. A switch in stereochemistry at the 15-carbon occurs in a natural cyclooxygenase from coral and has received attention upon aspirin treatment of COX-2. The latter produces 15R-HETE and analogs from other fatty acids that may be further oxygenated by lipoxygenases. Functional plasticity in cyclooxygenase catalysis has enabled the formation and discovery of a host of novel eicosanoids and provided mechanistic insight into the COX reaction mechanisms.