Journal of Biological Chemistry最新文献

Extracellular vesicles (EVs) are cell-derived vesicles secreted by all cell types into the extracellular spaces. EVs comprise a heterogenous population of vesicles that carry bioactive molecules, such as proteins, lipids, and RNAs, which they can deliver to recipient cells. Over the past few years, EVs have been recognized for their vital role in intercellular communication, and thereby in various physiological and pathological processes. In addition, EVs are increasingly being studied as potential drug delivery vehicles. It is therefore crucial to understand the mechanisms and molecular players underlying EV uptake and functional cargo delivery. Several studies have investigated various EV uptake pathways; nonetheless, molecular mechanisms governing uptake and cargo transfer remain largely lacking. Here, we show, using a CRISPR/Cas9-mediated reporter system, that integrin β1 on recipient cells plays an important role in EV uptake and EV-mediated RNA delivery. Additionally, using both RNA interference and blocking antibodies, we show that association of integrin β1 with integrin α4 is essential for this process. We demonstrate that α4β1 on recipient cells interacts with EVs through surface localized fibronectin via binding to its Leucine-Aspartic acid-Valine (LDV) motif, and that blocking of this interaction reduces both EV uptake and RNA delivery. Thus, we identify a key mechanism in EV uptake and cargo delivery which could potentially facilitate research into EV biology and pave the way for the development of novel therapeutic approaches by targeting pathways that lead to functional cargo delivery.

Hypoxia, a hallmark of solid tumors, is associated with increased lipid droplet (LD) accumulation. However, the mechanisms underlying this remain elusive. Here, we identify Mediator complex subunit 15 (MED15) as a critical regulator of hypoxia-inducible factor (HIF) signaling, potentially impacting LD accumulation. In mammalian cells, we elucidated that MED15, as a HIF target gene, participates in promoting HIF transcriptional activity without affecting HIFα protein levels, creating a positive feedback loop. Furthermore, zebrafish deficiency in med15 displayed decreased HIF activity and impaired tolerance to hypoxic stress. Functionally, MED15 deficiency attenuated the proliferation of colon and renal cancer cells in vitro and tumor growth in vivo. Mechanistically, MED15 acts upstream of carnitine palmitoyltransferase 1A (CPT1A), a key enzyme in fatty acid oxidation, ultimately promoting HIF-mediated LD accumulation. Disrupting the MED15-CPT1A axis impairs this process. These findings reveal a novel MED15-HIF-CPT1A axis that promotes LD formation, potentially contributing to hypoxic tumor progression.

The regulation of inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) activity is thought to define the spatiotemporal patterns of Ca²⁺ signals necessary for the appropriate activation of downstream effectors. The binding of both IP₃ and Ca²⁺ are obligatory for IP₃R channel opening. Ca²⁺, however regulates IP₃R activity in a biphasic manner. Ca²⁺ binding to a high-affinity pocket formed by the ARM3 domain and linker domain promotes IP₃R channel opening without altering the Ca²⁺ dependency for channel inactivation. These data suggest a distinct low-affinity Ca²⁺ binding site is responsible for the reduction in IP₃R activity at higher [Ca²⁺]. We mutated a cluster of acidic residues in the ARM2 and central linker domain of IP₃R type-1, reported to coordinate Ca²⁺ in cryo-EM structures of the IP₃R type 3. This "CD Ca²⁺ binding site" is well-conserved in all IP₃R sub-types. CD site Ca²⁺ binding mutants where the negatively charged glutamic acid residues were mutated to alanine exhibited enhanced sensitivity to IP₃-generating agonists. Ca²⁺ binding mutants displayed spontaneous elemental Ca²⁺ puffs and the number of IP₃-induced Ca²⁺ puffs were augmented in cells stably expressing Ca²⁺ binding site mutants. The inhibitory effect of high [Ca²⁺] on single channel-open probability (P_o) was reduced in mutant channels and this effect was dependent on [ATP]. This indicates that Ca²⁺ binding to the putative CD Ca²⁺ inhibitory site facilitates the reduction in IP₃R channel activation at subsaturating, likely physiological cytosolic [ATP], and suggest that at higher [ATP], additional Ca²⁺ binding motifs may contribute to the biphasic regulation of IP₃-induced Ca²⁺ release.

Extracellular vesicles (EVs) are nano-sized particles secreted by many cell types, including tumor cells, and play key roles in cellular communication by transporting functional RNAs. This study aims to elucidate the role of lncRNA BANCR in EVs derived from breast cancer (BC) cells in trastuzumab resistance. Differentially expressed lncRNAs and downstream targets in BC-resistant samples were identified. SKBR-3 cells were treated with trastuzumab to generate resistant cells (SKBR-3TR), and EVs from these cells (SKBR-3TR-EVs) were isolated and characterized. Functional studies of BANCR were performed in SKBR-3 and SKBR-3TR cells. Co-culturing SKBR-3 cells with SKBR-3TR-EVs assessed changes in cell behavior. A xenograft model in nude mice examined in vivo tumorigenicity and trastuzumab resistance. BANCR was highly expressed in SKBR-3TR cells and EVs, linked to trastuzumab resistance. SKBR-3TR-EVs transferred BANCR to SKBR-3 cells, where BANCR inhibited miR-34a-5p, reducing its expression. HMGA1 was identified as a miR-34a-5p target. BANCR activated the HMGA1/Wnt/β-catenin pathway by inhibiting miR-34a-5p, promoting resistance. In vivo experiments showed that BANCR inhibition delayed tumorigenesis and reversed trastuzumab resistance. BC cell-derived EVs containing BANCR may enhance resistance to trastuzumab by regulating the miR-34a-5p/HMGA1/Wnt/β-catenin axis, presenting a potential target for BC therapy.

Genomes are blueprints of life essential for an organism's survival, propagation, and evolutionary adaptation. Eukaryotic genomes comprises DNA, core histones and several other nonhistone proteins packaged into chromatin in the tiny nucleus. Chromatin structural organization restricts transcription protein and DNA access, permitting binding only after specific chromatin remodelling events. The fundamental processes in living cells, including transcription, replication, repair, and recombination, are thus regulated by chromatin structure through ATP-dependent remodelling, histone variant incorporation, and various covalent histone modifications including phosphorylation, acetylation, and ubiquitination. These modifications, particularly involving histone variant H2AX, furthermore play crucial roles in DNA damage responses by enabling repair protein access to damage. Chromatin also stabilizes the genome by regulating DNA repair mechanisms while suppressing damage from endogenous and exogenous sources. Environmental factors such as ionizing radiations induce DNA damage, and if repair is compromised, can lead to chromosomal abnormalities and gene amplifications as observed in several tumor types. Consequently chromatin architecture controls the genome blueprint fidelity and activity: it orchestrates correct gene expression, genomic integrity, DNA repair, transcription, replication, and recombination. This review considers connecting chromatin organization to functional outcomes impacting transcription, DNA repair and genomic integrity as an emerging grand challenge for predictive molecular cell biology.

Disintegrins are cysteine-rich proteins found in snake venoms. These proteins selectively bind to integrins, which play a key role in the regulation of many physiopathological processes. They are coreless proteins that display almost all hydrophobic residues on the protein surface. The exposed hydrophobic residues form surface clusters stabilized by the interaction with the hydrophilic residues in the vicinity and the hydration shell. In the present work, we aimed to determine the stability of surface hydrophobic clusters (SHCs) and their role in protein folding and biological activity. We used urea denaturation curves followed by ¹H and ¹⁵N chemical shifts to determine the free energy of unfolding (ΔG_F-U) and CLEANEX experiments to measure the water exchange rates of the surface amides (k_ex). The amides with higher local stability and protected from water exchange are those near or at the SHCs, which form a hydrophobic face. SHCs act as foldons, guiding oxidative folding and contributing to the formation of the disulfide bond framework, which is essential for establishing the concave shape and, ultimately, the binding cleft. On the opposite side of the protein are the residues with lower local stability and amides that exchange fast with water. This face coincides with the binding cleft of the protein to the αVβ3 integrin. Taken together, the present work established a correlation between protein hydration and the binding surface.

After 70% partial hepatectomy (PHx), the metabolic pathways leading to hepatocyte lipid droplet accumulation during liver regeneration remain unclear. Aquaporin 5 (Aqp5) is an aquaporin that facilitates the transport of both water and hydrogen peroxide (H₂O₂). In this study, we observed a delayed liver regeneration following PHx in Aqp5 knockout (Aqp5^-/-) mice. Considering the role of Aqp5 in H₂O₂ transport, we hypothesized that deficiency in Aqp5 may induce oxidative stress and hepatocyte injury. Through the measurement of reactive oxygen species (ROS) and redox-related indices, we observed significant alterations in ROS levels as well as malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) concentrations in regenerating livers lacking Aqp5 compared to wild-type controls. Oil Red O and 4-hydroxynonenal (4-HNE) staining results indicated that Aqp5 deficiency caused lipid accumulation during liver regeneration. The transcriptome sequencing results showed that the PPAR pathway is inhibited during the liver regeneration process in Aqp5 gene-knockout mice. The administration of the WY-14643 agonist, which targets the PPAR pathway, significantly mitigated delayed liver regeneration by enhancing hepatocyte proliferation and reducing lipid accumulation caused by Aqp5 deficiency. Our findings highlight the crucial role of Aqp5 in regulating H₂O₂ levels and lipid metabolism through the PPAR pathway during liver regeneration.

Many hematophagous organisms secrete inhibitors of the coagulation and complement systems as constituents of their salivary fluid. Whereas previous studies on salivary gland extracts from the sandfly Lutzomyia longipalpis identified SALO (Salivary Anticomplement from Lutzomyia longipalpis) as a potent inhibitor of the classical complement pathway (CP), its precise mechanism of action has remained elusive. Here we show that SALO inhibits the CP by binding selectively to the C1r zymogen. Using surface plasmon resonance, we found that SALO expressed by HEK293(T) cells (eSALO-WT) bound with nanomolar affinity to the zymogen of complement protease C1r (pro-C1r), but that it did not bind the enzymatically-active form of C1r. To gain insight into the structural basis for CP inhibition by SALO, we solved a 3.3 Å resolution crystal structure of eSALO-WT bound to a recombinant form of C1r that was engineered to remain in a zymogen-like state (zC1r-12SP). eSALO-WT formed extensive interactions with the zymogen activation loop of zC1r-12SP, including groups derived from residues R463 and I464, which compose its scissile peptide bond. Although the interactions with R463 and I464 were mediated by sidechain sulfation of eSALO-WT at position Y51, we found that this modification enhanced the potency of SALO but was not required for its activity. Consistent with our structural observations, subsequent studies showed that eSALO-WT binding to pro-C1r blocked its activation and thereby inhibited the CP in hemolytic assays of complement function. Together, our results define a new mode of inhibiting complement by blocking the farthest upstream enzymatic reaction of the CP.

Huntington's disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in exon 1 of the HTT gene. Mutant huntingtin (mHTT) associates with mitochondria, resulting in mitochondrial dysfunction and neuronal cell death. However, the underlying molecular mechanisms remain unknown. In this study, we investigate the role of N-terminal first 17 amino acids (N17) of mHTT in regulating its mitochondrial localization. Specifically, we demonstrate that the mutation at leucine 7 of N17 domain suppresses the association of mHTT with mitochondria. Blocking mitochondrial localization of HTT exon 1 with 73 glutamine repeats (HTT-Q73) strongly ameliorates polyQ induced reduction of mitochondrial membrane potential, increase of reactive oxygen species (ROS) production and decrease in NAD⁺/NADH ratio. We observe that HTT-Q73 mediated abnormal mitochondrial morphology, mitochondrial DNA deletion and cell death are abolished by HTT-Q73-L7A mutation. Finally, overexpression of HTT-Q73-L7A do not cause neurodegeneration and motor dysfunction in vivo. These findings highlight the pivotal role of the L7 residue which contributes to mHTT caused HD pathology. Targeting the L7 residue of N17 domain may be a novel therapeutic strategy to alleviate mitochondrial dysfunction and neurodegeneration in HD.

Mammals adaptively regulate energy metabolism in response to environmental changes such as starvation and cold circumstances. Thioredoxin-interacting protein (Txnip), known as a redox regulator, serves as a nutrient sensor regulating energy homeostasis. Txnip is essential for mice to adapt to starvation, but its role in adapting to cold circumstances remains unclear. Here, we identified Txnip as a pivotal factor for maintaining non-shivering thermogenesis in mice. Txnip protein levels in brown adipose tissue (BAT) were upregulated by the acute cold exposure. Txnip-deficient (Txnip^-/-) mice acclimated to thermoneutrality (30°C) exhibited significant BAT enlargement and triglyceride accumulation with downregulation of BAT signature and metabolic gene expression. Upon acute cold exposure (5°C), Txnip^-/- mice showed a rapid decline in BAT surface temperatures with the failure of increasing metabolic respiration, developing lethal hypothermia. The BAT dysfunction and cold susceptibility in Txnip^-/- mice were corrected by acclimation to 16°C, protecting the mice from life-threatening hypothermia. Transcriptomic and metabolomic analysis using dissected BAT revealed that despite preserving glycolysis, the BAT of Txnip^-/- mice failed to activate the catabolism of branched-chain amino acids and fatty acids in response to acute cold stress. These findings illustrate that Txnip is required for maintaining basal BAT function and ensuring cold-induced thermogenesis.