Journal of Biological Chemistry最新文献

Glycosylation is an evolutionarily conserved post-translational modification of most proteins that are either secreted from cells or remain embedded within membranes as transmembrane proteins. It controls protein stability, plasma half-life, intracellular trafficking and can contribute to the actual biological function of the protein. Protein glycosylation can be divided into N-linked glycosylation that refers to the linkage of an oligosaccharide to the amide nitrogen of an asparagine residue, O-glycosylation that describes attachment of an oligosaccharide to the hydroxyl oxygen of a serine or threonine residue, and C-mannosylation, a rare modification in which a mannose residue is bound to the indole of a tryptophan residue via a carbon-carbon linkage. In this review, we summarize current knowledge about C-mannosylation. We describe how C-mannosylation was initially discovered and on which types of proteins it usually occurs. We explain the operation of the C-mannosyltransferases, the enzymes that attach the mannose to the substrate proteins, and which conformations the C-mannose adopt. Furthermore, we summarize what is known so far about the influence of the C-mannosylation on the function of the actual protein. Our review highlights an often overlooked post-translational modification as important regulator of protein function.

Subcellular compartmentalization may be an effective way of controlling the abundance and activity of miRNAs in mammalian cells. Exploring the regulatory processes that control miRNA activity, we found that specific miRNAs are reversibly localized to the mitochondrial matrix in a context-dependent manner. Our data suggest a de novo role of mitochondria as miRNA storage site in mammalian cells. miR-122 is a key hepatic miRNA regulating metabolic processes in the mammalian liver. In this study, we observed increased mitochondrial targeting of miR-122 in amino acid-starved hepatic cells. Interestingly, when cells are refed with amino acids, mitochondrial miR-122 is relocalized to the cytosol and reused for translational repression. Moreover, this phenomenon is not limited to miR-122, as other mitochondrial miRNAs (mito-miRs) follow similar transient storage inside mitochondria in stressed cells. Bioinformatic analysis revealed that mitochondria-localized mito-miRs preferentially target mRNAs encoding crucial mitochondrial components related to apoptosis. Hence, hepatic cells regulate apoptosis pathways during the starvation-refeeding cycle by shuttling a specific set of miRNAs to and from mitochondria, thereby balancing cytosolic miRNA content. Stress response miRNA binder ELAVL1 or HuR protein was found to be both necessary and sufficient for transporting the mito-miRs to the mitochondrial matrix - a process also controlled by the interaction between mitochondria and the endoplasmic reticulum.

EphA2, a receptor tyrosine kinase, is overexpressed in various cancers. Its ligand-independent non-canonical signaling is pro-tumorigenic, and elevated EphA2 expression is associated with poor prognosis in patients. Although preclinical and clinical studies targeting EphA2 have been conducted as cancer therapeutics, its role in the DNA damage response remains elusive. This study examined the role of EphA2 in cell cycle progression in Adriamycin (ADR)-treated cells. ADR treatment transcriptionally upregulated EphA2 expression in a p53-independent manner. Suppression of EphA2 upregulation abrogated G2 arrest, as evidenced by reductions in both cyclin B1 accumulation and Wee1 inhibition-driven cell division. However, the 2N-G1 cell population remained low, with increased tetraploid cells. Time-lapse imaging revealed that tetraploid formation resulted from mitotic bypass rather than mitotic slippage or cytokinesis failure. EphA2 knockdown upregulated p21 expression together with p53, and p21 knockdown suppressed EphA2 knockdown-induced mitotic bypass. Monitoring fluorescence from a GFP fusion with the cyclin B1 destruction box demonstrated degradation in interphase without cell division, suggesting premature activation of APC/C^Cdh1 in interphase. Notably, p21 upregulation following EphA2 knockdown was observed specifically in cervical cancer cell lines. Finally, ADR-induced suppression of cell proliferation was further enhanced by EphA2 knockdown and partially reversed by p21 knockdown. In conclusion, EphA2 suppression induces p21-dependent mitotic bypass and tetraploidization, leading to reduced cell proliferation. EphA2 upregulation following DNA damage may be pro-tumorigenic by maintaining G2 arrest to keep DNA damage at tolerable levels. These findings provide a rationale for combining EphA2 inhibition with DNA-damaging agents in certain cancer types.

TET2 is an epigenetic modifier whose canonical activity leads to the removal of cytosine methylation in the genome, which in essence results in the activation of gene expression. This function is particularly well described in the context of hematopoiesis and its alterations that lead to leukemia. However, in recent years, it has become evident that the non-canonical functions of TET2 also play a vital role in its activity. Rather than depending on its catalytic activity, these functions arise from TET2 interactions with other epigenetic modifiers. This review summarizes the structure, regulation, and functions of TET2 in immune cells. We describe how TET2 controls gene expression at both the DNA and RNA levels. In addition, we discuss the role of TET2 in hematopoietic stem cell fate and in clonal hematopoiesis of indeterminate potential (CHIP). Finally, we highlight the impact of TET2 mutations on age-related inflammatory diseases, including cardiovascular and neurodegenerative disorders. Collectively, available evidence positions TET2 as a key integrator of epigenetic state and immune signaling, with context-dependent effects on inflammation and tissue homeostasis, and underscores the therapeutic potential of targeting TET2-dependent pathways in clonal hematopoiesis and inflammatory diseases.

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.

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).

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.