Journal of Biological Chemistry最新文献

Obesity and type 2 diabetes (T2D)-linked hyperglycemia, along with their associated complications, have reached pandemic proportions, constituting a major public health issue. Genetic deletion or pharmacological inhibition of purine nucleotide-metabolizing enzymes has emerged as a potential strategy for treating diseases. We previously showed that cytosolic 5'-nucleotidase II (NT5C2)-deficient mice were protected against high-fat diet (HFD)-induced insulin resistance. This study investigated effects of dual deletion of cytosolic 5'-nucleotidases IA (NT5C1A) and II (NT5C2) in mice. We found that NT5C1A/NT5C2 double-knockout (NT5C-dKO) mice exhibited mild hypoglycemia, associated with enhanced skeletal muscle insulin action and reduced hepatic glucose production. This phenotype was accompanied by liver and skeletal muscle proteomic alterations notably related to amino acid metabolism, besides potential involvement of adenosine monophosphate (AMP)-activated protein kinase (AMPK). Our findings support the development of novel anti-diabetic treatments using small-molecule cytosolic 5'-nucleotidase inhibitors.

Female reproductive health is troubled by oocyte maturation disorder. In mammals, granulosa cells (GCs) mediate LH action on oocyte maturation and ovulation. However, the pathogenesis of disordered GCs in oocyte maturation arrest is rarely studied. Our previous study has showed that HDAC3 in GCs was decreased by LH at physiological condition. Here, we observed significantly elevated HDAC3 levels in GCs from patients with oocyte maturation disorder following LH treatment compared to those with normal oocyte maturation. To clarify whether abnormally high levels of HDAC3 in ovulatory GCs resulted in female infertility, a mice model of GC-conditional overexpression of Hdac3 was constructed. The results showed that abnormally high levels of HDAC3 in ovulatory GCs inhibited LH induction on oocyte maturation and ovulation, resulting in female infertility. Further, in GCs with abnormal high levels of HDAC3, the upregulation of oocyte maturation-related genes induced by LH was attenuated by HDAC3 through a reduction in H3K14ac levels in the promoter regions, implying that the action of LH in GCs was largely negatively controlled by HDAC3. Applying HDAC3 inhibitors enhanced the expression of multiple genes associated with oocyte maturation in GCs from clinical patients, ultimately improving both the oocyte maturation rate and developmental quality, as demonstrated by a higher blastocyst development rate. The findings contribute to both enrich understanding upon the pathological mechanisms and supply optimal treatment strategies for patients with oocyte maturation disorder.

The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors that bind via an LxCxE motif. The pocket domain is the most highly-conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rb's pocket domain, we used fluorescence polarization and differential scanning fluorimetry to quantify changes caused by 75 cancer-associated missense variants to E2F transactivation domain (E2F^TD) binding, LxCxE binding, and the thermostability of the pocket domain. We find that 43% of the missense variants tested reduce Rb-E2F^TD binding. Many of these variants are not located at the E2F^TD binding site, yet they destabilize the fold of the protein and show temperature-sensitive binding effects. We also find that 21% of tested mutations reduce LxCxE binding, and several mutations selectively disrupt either E2F^TD or LxCxE binding. Protein X-ray crystallography of four missense variants reveals how mutations destabilize the protein fold and inhibit E2F^TD or LxCxE binding. Taken together, this work provides the first understanding of the multiple ways through which stability, structure and function of Rb's pocket domain are altered by common missense mutations seen in cancer.

The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that contain the incorrect cofactor type cannot reduce N₂ to support diazotrophic growth there must be a way to prevent misincorporation of an incorrect cofactor when different nitrogenase isozyme systems are produced at the same time. Here, we show that fidelity of the Fe-only nitrogenase is preserved by blocking the misincorporation of either FeMo-cofactor or FeV-cofactor during its maturation. This protection is accomplished by a two-domain protein, designated AnfO. It is shown that the N-terminal domain of AnfO binds to an immature form of the Fe-only nitrogenase and the C-terminal domain, tethered to the N-terminal domain by a flexible linker, has the capacity to capture FeMo- and FeV-cofactor. AnfO does not prevent the normal activation of Fe-only nitrogenase because completion of FeFe-cofactor assembly occurs within its catalytic partner and, therefore, is never available for capture by AnfO. These results support a post-translational mechanism involving the molecular sorting of structurally similar metallocofactors that involve both protein-protein interactions and metallocofactor binding while exploiting differential pathways for nitrogenase associated catalytic cofactor assembly.

Promoter-promoter and enhancer-promoter interactions are enriched in histone acetylation and central to chromatin organization in active genetic regions. Bromodomains are epigenetic 'readers' that recognize and bind histone acetylation. Bromodomains often exist in tandem or with other reader domains. Cellular knockdown of the bromodomain and extraterminal domain (BET) protein family disrupts chromatin organization, but the mechanisms through which BET proteins preserve chromatin structure are largely unknown. We hypothesize that BET proteins maintain overall chromatin structure by employing their tandem bromodomains to multivalently scaffold acetylated nucleosomes in an intra- or internucleosomal manner. To test this hypothesis biophysically, we used small-angle X-ray scattering, electron paramagnetic resonance, and Rosetta protein modeling to show that a disordered linker separates BET tandem bromodomain acetylation binding sites by 15-157 Å. Most of these modeled distances are sufficient to span the length of a nucleosome (>57 Å). Focusing on the BET family member BRD4, we employed bioluminescence resonance energy transfer and isothermal titration calorimetry to show that BRD4 bromodomain binding of multiple acetylation sites on a histone tail does not increase BRD4-histone tail affinity, suggesting that BET bromodomain intranucleosome binding is not biologically relevant. Using sucrose gradients and amplified luminescent proximity homogeneous (AlphaScreen) assays, we provide the first direct biophysical evidence that BET bromodomains can scaffold multiple acetylated nucleosomes. Taken together, our results demonstrate that BET bromodomains are capable of multivalent internucleosome scaffolding in vitro. The knowledge gained provides implications for how BET bromodomain-mediated acetylated internucleosome scaffolding may maintain cellular chromatin interactions in active genetic regions.

Metabolic dysfunction-associated steatohepatitis (MASH) is a complicated process that contributes to end-stage liver disease and, eventually, hepatocellular carcinoma (HCC). Hepatocyte apoptosis, a well-defined form of cell death in MASH, is considered the primary cause of liver inflammation and fibrosis. However, the mechanisms underlying the regulation of hepatocyte apoptosis in MASH remain largely unclear. We explored the pro-apoptotic effect of hepatocyte amyloid precursor protein (APP) in MASH. C57BL/6J mice were fed a western diet plus sugar water, a high-fat high-fructose diet or a methionine and choline deficiency diet induce MASH. APP expression was analyzed in murine MASH specimens. App^-/- mice and mice with adeno-associated virus-mediated APP overexpression were established to study the role of APP in MASH. Palmitic acid was used to mimic lipotoxicity induced MASH in AML12 cells. We identified a dramatic increase in APP expression in hepatocytes of patients with MASH and three different mouse models. Suppression of APP attenuated hepatic steatosis, inflammation, and fibrosis in MASH mice, whereas its restoration activated MASH pathogenesis. Furthermore, increased death receptor 6 (DR6) was observed in MASH mouse livers. Mechanistically, APP interacted with DR6, a tumor necrosis factor receptor, to facilitate DR6 expression and activation. Activated DR6 increased apoptosis in hepatocytes, which was associated with an increase in pro-apoptotic effectors (cleaved-caspase 3/7). Our results highlight the role of the APP/DR6 axis in hepatocyte apoptosis, inflammation activation and fibrosis formation in murine MASH model, providing new insights into therapeutic strategies for MASH.

The success of modern metabolomics analysis depends on the separation of metabolites in complex samples using methods such as liquid chromatography and mass spectrometry. Herein, we present a protocol for resolving a broad range of polar metabolites, based on hydrophilic interaction liquid chromatography with a zwitterionic bonded phase (HILICz). In optimising this protocol, we encountered pressure fluctuations, a widespread problem that impacts metabolite analysis, restricts batch sizes, and imposes instrument downtime, ultimately incurring substantial time and financial expense. Thus, we use this opportunity as a case study to demonstrate the steps taken to overcome such pressure fluctuations, resulting in a protocol that robustly and consistently resolves polar metabolites in large batches of samples (>100 samples, equating to >40 hours of run-time). This consistency is essential to address the growing demand for repeatable in-depth metabolomics analysis of complex samples.

ASICs (acid-sensing ion channels) are proton-evoked sodium ion channels, highly distributed in the peripheral and central nervous system. ASICs are involved in pain perception and ASIC3 channel is presumed as the target of promising analgesics. Peptide drugs have attracted the attention of pharmaceutical developers due to their advantages such as low toxic side effects and targeted specificity. Although numbers of chemicals acting on ASICs are emerging, there are limited reports on peptide inhibitor acting on ASIC3 channel. Here, we found that spider-derived peptide LCTx-F2 suppressed the activity of ASIC3 channel in concentration-dependent manner. By performing peptide mutation and molecular docking, we revealed the molecular mechanism of LCTx-F2 inhibiting ASIC3 channel, in which β-hairpin of LCTx-F2 penetrated the acidic pocket of the channel. Similarly, LCTx-F2 also inhibited ASIC1a channel by occupying the acidic pocket, but N-terminal of the peptide sticked into the region. The bonds relationship between critical residues of LCTx-F2 and the channels were uncovered by molecular docking and dynamic simulation. Thus, our findings indicated the molecular mechanism by which LCTx-F2 acting on ASIC3 and ASIC1a channels and provided a novel temple of analgesic drug targeting the channels.

Plasmodium vivax is emerging as the most prevalent species causing malaria outside Africa. Most P. vivax infections are relapses due to the reactivation of the dormant liver stage parasites (hypnozoites). Hypnozoites are a major reservoir for transmission but undetectable by commercial diagnostic tests. Antibodies against P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) are among the most reliable serological biomarkers for recent P. vivax infections in the prior nine months and act as indirect biomarkers for risk of relapse. We sought to design stabilized variants of PvRBP2b, under stringent conditions of minimally perturbing the solvent-accessible surfaces to maintain its antigenicity profile. Furthermore, for some of the designs, due to limited diversity of natural PvRBP2b homologs, we combined AI-based ProteinMPNN and PROSS atomistic design calculations. The best, bearing 19 core mutations relative to PvRBP2b, expressed 16-fold greater amounts (up to 11 mg per L), and had 14 °C higher thermal tolerance than the parental protein. Critically, the stabilized designs retained binding to naturally acquired human monoclonal antibodies with nanomolar affinities, suggesting that the immunologically competent surfaces were retained as was confirmed by crystallographic analyses. Using longitudinal observational cohorts from malaria endemic regions of Thailand, Brazil and the Solomon Islands, we show that antibody responses against the designs are highly correlated with those against the parental protein and can classify individuals as recently infected with P. vivax. This efficient computational stability design methodology can be used to enhance the biophysical properties of other recalcitrant proteins for use as diagnostics or vaccine immunogens.

Mycobacterium tuberculosis (Mtb) is one of the world's successful pathogens that flexibly adapts its metabolic nature during infection of the host, and in response to drugs. Here we used genome scale metabolic modelling coupled with differential producibility analysis (DPA) to translate RNA seq datasets into metabolite signals and identified drug-associated metabolic response profiles. We tested four TB drugs bedaquiline (BDQ), isoniazid (INH), rifampicin (RIF) and clarithromycin (CLA); conducted RNA seq experiments of Mtb exposed to the individual drugs at subinhibitory concentrations, followed by DPA of gene expression data to map up and downregulated metabolites. Here we highlight those metabolic pathways that were flexibly used by Mtb to tolerate stress generated upon drug exposure. BDQ and INH upregulated maximum number of central carbon metabolites in glycolysis, pentose phosphate pathway and tri-carboxylic acid cycle with concomitant downregulation of lipid and amino acid metabolite classes. Oxaloacetate was significantly upregulated in all four drug-treated Mtb cells highlighting it as an important metabolite in Mtb's metabolism. Amino acid metabolism was selectively induced by different drugs. We have enhanced our knowledge on Mtb's carbon and nitrogen metabolic adaptations in the presence of drugs and identify metabolic nodes for therapeutic development against TB. Our work also provides DPA omics platform to interrogate RNA seq datasets of any organism that can be reconstructed as a genome scale metabolic network.