Journal of Biological Chemistry最新文献

Bone metastasis-induced pain is a debilitating condition that remains a pervasive clinical challenge, with effective treatments still lacking. Although ClC-3 chloride channels are known to play an important role in synaptic transmission within the central nervous system, their expression and function in peripheral sensory neurons are poorly understood. Here, we found that the downregulation of ClC-3 in dorsal root ganglion (DRG) neurons sensitized nociceptive sensory neurons and contributed to bone metastasis-induced pain. Overexpressing Clc-3 in DRG neurons attenuated tumor-induced neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats, whereas knocking down Clc-3 induced neuronal hyperexcitability and pain hypersensitivity in naïve rats. Mechanistically, tumor-associated production of insulin-like growth factor 1 (IGF1) activated the receptor IGF1R on DRGs, leading to an upregulation of histone deacetylase 2 (HDAC2), thereby suppressing the transcription of Clc-3 gene. Activation of the IGF1/IGF1R-AKT signaling pathway promotes HDAC2-mediated epigenetic silencing of Clc-3, thereby enhancing neuronal excitability and pain hypersensitivity in tumor-bearing rats. Our findings reveal a new and targetable mechanism underlying bone metastasis-induced pain, offering promising therapeutic avenues for pain management in cancer patients.

Excitatory peptide (EP) and CCHamide (CCHa) are protostome neuropeptides originally identified in lophotrochozoans (including annelids and mollusks) and arthropods, respectively, and are homologous to the deuterostome endothelin (ET) and gastrin-releasing peptide (GRP)/neuromedin-B (NMB) systems. These peptides are brain-gut peptides: in vertebrates, GRP/NMB function as satiety peptides, whereas arthropod CCHa displays species-specific actions, either inhibiting or promoting feeding. However, the mechanisms by which these peptides modulate feeding circuits remain unknown. Here, we investigated the EP/CCHa signaling pathway in Aplysia, a mollusk with a well-defined feeding circuit. We identified a single precursor encoding Aplysia EP/CCHa (apEP/CCHa). Mass spectrometry demonstrated that an apEP/CCHa peptide is present in the central ganglia. In situ hybridization and immunohistochemistry revealed apEP/CCHa-positive neurons in the CNS, immunopositive cells in the gut, and immunopositive fibers in the gut-innervating esophageal nerve. To identify potential targets, we cloned two novel apEP/CCHa receptors. Phylogenetically, one receptor clusters with lophotrochozoan EP/CCHa receptors, whereas the other unexpectedly clusters with arthropod receptors, suggesting independent lineages for the two receptors. Single-cell RNA sequencing showed that both receptors are expressed in the key feeding central pattern generator (CPG) interneurons B20 and B34. Functionally, apEP/CCHa inhibited food intake in vivo and converted ingestive motor programs to egestive ones in vitro. At the circuit level, apEP/CCHa modulated excitability of B20 and B34, and two additional interneurons (B40, B64). In summary, we demonstrate that apEP/CCHa is a brain-gut peptide that functions as a satiation signal, and identify specific feeding CPG elements through which apEP/CCHa regulates motivational state transitions.

The Chaperonin containing tailless complex polypeptide 1 (CCT) or TCP-1 ring complex (TRiC) plays a central role in maintaining cellular homeostasis by supporting protein folding and damping protein aggregation. Besides the abundant cytoskeletal proteins, actin and tubulin, CCT/TRiC is emerging as an obligate chaperone for the β-propeller domain of WD40 proteins. To date, only WD40 proteins consisting of a single β-propeller domain have been described as CCT/TRiC substrates. Using a combination of biotin proximity ligation, co-immunoprecipitation, and knockdown studies, we here identify the tandem β-propeller protein, Coronin 7 (Coro7), as a novel substrate of CCT/TRiC. This raised the question how CCT/TRiC can fold a protein that is too large to fit into its folding chamber, but consists of two domains that require its folding. Surprisingly, co-immunoprecipitation of truncated Coro7 proteins or cleaved full length Coro7 demonstrated that CCT/TRiC only interacts with the first β-propeller domain (PropA) of Coro7. Further experiments showed that CCT/TRiC preferentially binds to PropA, independently of whether this domain is situated at the N- or C-terminus of Coro7. This strongly suggests that CCT/TRiC does not identify β-propeller substrates by their topology, but instead developed specific ways to recognize β-propeller sequences that require folding.

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.

Lipoxygenases (ALOX) are non-heme iron containing dioxygenases that catalyze the oxygenation of polyenoic fatty acid containing lipids to their corresponding hydroperoxy derivatives. These enzymes are widely distributed in highly developed plants and animals. In bacteria they rarely occur but they have not been detected in archaea and viruses. The human genome involves six functional ALOX genes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) encoding for six different isoenzymes. The mouse genome carries an orthologous gene for each human ALOX gene but in addition an Aloxe12 gene has been identified in this species. The application of isoenzyme-specific loss-of-function strategies suggested that the coding multiplicity may not be interpreted as sign of functional redundancy. In fact, the different isoenzymes apparently fulfill different biological functions. Mammalian ALOX15 orthologs are allosteric enzymes but the molecular basis for their allosteric properties remains controversial. In fact, two alternative hypotheses (presence of allosteric binding sites at enzyme monomers vs. ALOX15 dimers consist of an allosteric and a catalytic monomer) have been introduced and this review is aimed at critically evaluating the pros and conts of these two mechanistic scenarios.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are attached to the cell surface via a glycolipid anchor, GPI, whose conserved core is synthesized from phosphatidylinositol (PI) in the endoplasmic reticulum through a series of enzymatic reactions. Most PI species in mammalian cells contain diacylglycerol, whereas GPI-APs predominantly possess 1-alkyl-2-acylglycerol. The basis for this characteristic lipid structure has remained unclear. Lipidomic analysis revealed that 1-alkyl-2-acyl PIs, although minor components of cellular PI, are preferentially used by GPI-N-acetylglucosaminyltransferase, which catalyzes the first step of GPI biosynthesis. GPI intermediates containing 1-alkyl-2-acylglycerol were further enriched in subsequent biosynthetic steps, resulting in mature GPIs primarily harboring this lipid species. We demonstrate that a 1-alkyl-containing precursor lipid derived from peroxisomes, likely 1-alkyl-glyceronephosphate, contributes to the formation of 1-alkyl-2-acyl PIs. Disruption of glyceronephosphate O-acyltransferase (GNPAT) or alkylglycerone phosphate synthase (AGPS), the first two enzymes of the peroxisomal ether-lipid pathway, abolished 1-alkyl-2-acyl PI, yielding GPI-APs containing only diacylglycerol. Lipidomic profiling of GPI biosynthetic intermediates in GPI-defective cells revealed accumulation of defective-step-specific intermediates, enabling the use of this approach for diagnosing inherited GPI deficiency (IGD).

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.