Journal of Biological Chemistry最新文献

As an integral part of the innate immune system, the complement system is a tightly regulated proteolytic cascade, playing a critical role in microbial defense, inflammation activation, and dying host cell clearance. Complement proteins are now emerging as subjects of intense research and drug development, since dysregulation of the complement system plays a critical role in several diseases and disorders, such as Paroxysmal Nocturnal Hemoglobinuria (PNH) and Geographic Atrophy (GA). Within the complement cascade, complement C3 is the central component, situated at the convergence of all complement activation pathways, rendering it an attractive target for complement-related diseases. However, due to the complicated Structure-Activity Relationship (SAR) of C3, elucidating the mechanisms of C3 inhibition on diverse epitopes is the basis for the rational design of C3-targeted therapeutics. Here, we have developed a set of comprehensive biochemical assays that are tailored to the specific steps within the complement cascade, allowing for a thorough understanding of the pharmacological consequences of different C3 inhibitors at each stage. Utilizing three Model Inhibitors (MIs) with different epitopes, we found that inhibition of MG4/MG5 domains has potent inhibition efficacy across all the complement activation pathways by interrupting C3-C3 convertase interaction, while inhibition of C345C domain displays a bias over the Alternative Pathway (AP) inhibition by impairing AP C3 proconvertase formation. This study elucidates the intricate impact of C3 inhibition by targeting different epitopes, offering valuable insights into understanding the mechanism and facilitating the rational design of C3-targeted therapeutics.

Clear cell renal cell carcinoma (ccRCC) is a disease rooted in metabolic disorders, distinguished by abnormally high activity of glucose 6-phosphate dehydrogenase (G6PD). G6PD serves as a key rate-limiting enzyme in the pentose phosphate pathway. Meanwhile, BRAF-activated non-coding RNA (BANCR) has emerged as a crucial regulatory factor linked to various cancers. The expression pattern of BANCR varies across different cancer types, exhibiting apparent duality in its function. However, the precise role and underlying mechanisms of BANCR in ccRCC tumorigenesis remain incompletely understood. Our study indicated that BANCR was downregulated in ccRCC and influenced cell survival by modulating cell proliferation, apoptosis, and G6PD enzyme activity. The underlying mechanism was that BANCR could directly bind to G6PD through a lncRNA-protein interaction, ultimately inhibiting G6PD activity by impeding its dimer formation. Moreover, BANCR exhibited the capability to modulate the glucose metabolic flow in ccRCC cells. Subsequent experiments demonstrated a significant inhibition of tumor growth in vivo upon overexpression of BANCR, and G6PD played a pivotal role in mediating the tumor suppressive effect of BANCR in ccRCC cells. In conclusion, this study provides novel insights into the molecular pathogenesis of ccRCC, highlights a distinct and new regulatory mechanism responsible for the ectopic overactivation of G6PD in ccRCC progression, and suggests that BANCR-mediated suppression of G6PD activity could emerge as a potential therapeutic strategy for ccRCC treatment.

Nicotinamide mononucleotide (NMN) is a widely investigated metabolic precursor to the prominent enzyme cofactor nicotinamide adenine dinucleotide (NAD⁺), where it is assumed that delivery of this compound results in its direct incorporation into NAD⁺ via the canonical salvage / recycling pathway. Surprisingly, treatment with this salvage pathway intermediate leads to increases in nicotinic acid mononucleotide (NaMN) and nicotinic acid adenine dinucleotide (NaAD), two members of the Preiss-Handler / de novo pathways. In mammals, these pathways are not known to intersect prior to the production of NAD⁺. Here, we show that the cell surface enzyme CD38 can mediate a base exchange reaction on NMN, whereby the nicotinamide ring is exchanged with a free nicotinic acid to yield the Preiss-Handler / de novo pathway intermediate NaMN, with in vivo small molecule inhibition of CD38 abolishing the NMN-induced increase in NaMN and NaAD. Together, these data demonstrate a new mechanism by which the salvage pathway and Preiss-Handler / de novo pathways can exchange intermediates in mammalian NAD⁺ biosynthesis.

Siphonaxanthin is a carotenoid found in green algae that exhibits potent anti-inflammatory activities. We previously reported that ingested siphonaxanthin accumulates in various organs of mice; however, its metabolic conversion remains largely unknown. In this study, we isolated three siphonaxanthin dehydrometabolites and determined their chemical structures. Two of these metabolites were obtained using the post-mitochondrial supernatant prepared from mouse liver, while the third was obtained using the post-mitochondrial supernatant prepared from rat liver. The human liver S9 fraction also generated two metabolites: one was identical to one of the rat metabolites, and the other was identical to one of the mouse metabolites. ¹H-NMR revealed that all three metabolites had one or two additional α,β-unsaturated carbonyl groups (compared with siphonaxanthin). We also evaluated their anti-inflammatory activities and found that these three metabolites suppressed Toll-like receptor 1/2-mediated interferon regulatory factor (IRF) activation more potently than siphonaxanthin. Pharmacological inhibition studies revealed that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is crucial for the inhibition of IRF activation by these metabolites. The Nrf2-mediated decrease in the mRNA expression of the stimulator of interferon genes (STING) was determined to be one of the molecular mechanisms underlying this suppression. Thus, the hepatic metabolic conversion of siphonaxanthin generates an α,β-unsaturated carbonyl group, which boosts its IRF-inhibitory effect by activating Nrf2.

Repeat expansion disorders are caused by abnormal expansion of microsatellite repeats. Repeat-associated non-AUG (RAN) translation is one of the pathogenic mechanisms underlying repeat expansion disorders, but the exact molecular mechanism underlying RAN translation remains unclear. Polyamines are ubiquitous biogenic amines that are essential for cell proliferation and cellular functions. They are predominantly found in cells in complexes with RNA and influence many cellular events, but the relationship between polyamines and RAN translation is yet to be explored. Here, we show that, in both a cell-free protein synthesis system and cell culture, polyamines promote RAN translation of RNA containing CCUG repeats. The CCUG-dependent RAN translation is suppressed when cells are depleted of polyamines but can be recovered by the addition of polyamines. Thermal stability analysis revealed that the tertiary structure of the CCUG-repeat RNA is stabilized by the polyamines. Spermine was the most effective polyamine for stabilizing CCUG-repeat RNA and enhancing RAN translation. These results suggest that polyamines, particularly spermine, modulate RAN translation of CCUG-repeat RNA by stabilizing the tertiary structure of the repeat RNA.

Given the pivotal role of the Eph-Ephrin signaling pathway in tumor progression, agonists or antagonists targeting Eph-Ephrin have emerged as promising anticancer strategies. However, the implications of glycosylation modifications within Eph-Ephrin and their targeted protein therapeutics remain elusive. Here, we identify that N-glycosylation within the receptor-binding domain (RBD) of ephrin B1 (EFNB1) is indispensable for its functional repertoire. Notably, compared with wildtype EFNB1, the glycosylation-deficient N139D mutant drastically diminishes the sensitivity of tumor cells with chemotherapeutic agents, suggesting the existence of both glycosylation-dependent and -independent effects mediated by EFNB1. Transcriptomic analysis highlights immune response and oxidative phosphorylation as the primary signaling pathways modulated by glycosylation modifications. In coculture systems, the EFNB1-RBD-Fc recombinant protein, while inhibiting B-lymphoma cells, also exerts differential impacts on stromal cells depending on their glycosylation status. Furthermore, the efficacy of both glycosylated and nonglycosylated EFNB1-RBD-Fc is influenced by the endogenous EFNB1 levels within tumor cells. Taking together, this study demonstrates the complexity and multifaceted roles of glycosylation in modulating EFNB1 function. These findings underscore the need for a nuanced understanding of glycosylation patterns in Eph-Ephrin-targeted therapies to optimize their therapeutic potential.

Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via a direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.

Increasing evidence supports the role of an augmented immune response in the early development and progression of renal complications caused by diabetes. We recently demonstrated that podocyte-specific expression of stress response protein regulated in development and DNA damage response 1 (REDD1) contributes to activation of the pro-inflammatory transcription factor NF-κB in the kidney of diabetic mice. The studies here were designed to define the specific signaling events whereby REDD1 promotes NF-κB activation in the context of diabetic nephropathy. Streptozotocin (STZ)-induced diabetes promoted activation of glycogen synthase kinase 3β (GSK3β) in the kidney, which was prevented by REDD1 ablation. REDD1 was necessary and sufficient to enhance GSK3β activity in human podocyte cultures exposed to hyperglycemic conditions. GSK3β suppression prevented NF-κB activation and normalized the expression of pro-inflammatory factors in podocytes exposed to hyperglycemic conditions. In the kidneys of diabetic mice and in podocytes exposed to hyperglycemic conditions, REDD1-dependent GSK3β signaling promoted activation of the inhibitor of κB (IκB) kinase (IKK) complex upstream of NF-κB. GSK3β knockdown in podocytes exposed to hyperglycemic conditions reduced macrophage chemotaxis. Similarly, in diabetic mice treated with a GSK3 inhibitor, immune cell infiltration in the kidneys was reduced. Overall, the data support a model wherein hyperglycemia amplifies the activation of GSK3β in a REDD1-dependent manner, leading to canonical NF-κB signaling and an augmented renal immune response in diabetic nephropathy.

One of the key events in DNA damage response (DDR) is activation of checkpoint kinases leading to activation of ribonucleotide reductase (RNR) and increased synthesis of deoxyribonucleotide triphosphates (dNTPs), required for DNA repair. Among other mechanisms, the activation of dNTP synthesis is driven by derepression of genes encoding RNR subunits RNR2, RNR3, and RNR4, following checkpoint activation and checkpoint kinase Dun1p-mediated phosphorylation and inactivation of transcriptional repressor Crt1p. We report here that in the absence of genotoxic stress during respiratory growth on nonfermentable carbon source acetate, inactivation of checkpoint kinases results in significant growth defect and alters transcriptional regulation of RNR2-4 genes and genes encoding enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles and gluconeogenesis. Dun1p, independently of its kinase activity or signaling from the upstream checkpoint kinase Rad53p, represses RNR2, RNR3, and RNR4 genes by maintaining Crt1p occupancy in the corresponding promoters. Consistently with the role of dNTPs in the regulation of mitochondrial DNA (mtDNA) copy number, DUN1 inactivation elevates mtDNA copy number in acetate-grown cells. Together, our data reveal an unexpected role for Dun1p in transcriptional regulation of RNR2-4 and metabolic genes during growth on nonfermentable carbon source and suggest that Dun1p contributes to transcription regulation independently of its kinase activity as a structural component by binding to protein(s) involved in gene regulation.