Journal of Biological Chemistry最新文献

The ectonucleotidase ENPP1 is the major extracellular hydrolase of the innate immune system activator cGAMP (2'3'-cyclic GMP-AMP). Tumors that overexpress ENPP1 and rapidly degrade cGAMP avoid immune surveillance in the tumor microenvironment and are highly resistant to cancer immunotherapy. Inhibition of cGAMP degradation by ENPP1 has emerged as a promising strategy to improve cancer therapies. A direct, real-time assay of ENPP1 enzymatic activity would benefit quantitative evaluation of candidate ENPP1 inhibitors. The non-physiological substrate p-nitrophenyl 5'-thymidine monophosphate (pNP-TMP) is commonly used for this purpose, as it offers a readily detectable colorimetric readout that can be evaluated in real time. However, compounds that potently inhibit pNP-TMP hydrolysis can weakly inhibit ENPP1 with physiological nucleotide substrates, highlighting the importance of testing ENPP1 inhibitors with native substrates (i.e., cGAMP). No direct, real-time assays for cGAMP hydrolysis are established. Here, we present a real-time, spectrophotometric assay to monitor ENPP1-catalyzed cGAMP hydrolysis. The increase in extinction coefficient associated with conversion of substrate(s) to AMP and GMP products is used to convert time courses of absorbance change to rates of product formation. Time courses of GMP product formation generated from the absorbance change superimpose with those generated by direct measurement of GMP product concentration via chemical quench-flow and HPLC analysis. ENPP1 inhibition by the non-hydrolyzable ATP analog, α,β-methylene-ATP, yields an inhibition constant (K_I) comparable to the independently determined binding affinity. This spectroscopic assay can be performed using a standard, laboratory UV-vis spectrophotometer and has the potential to be scaled up to a high-throughput, multi-well plate setup.

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants, and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78%-100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes, and obtained key insights into Mb0564, identified as a novel adhesin. The 192-287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.

We have shown that extracellular ATP promotes senescence through the activation of the P2Y11 receptor (P2Y11R). The underlying molecular mechanisms remain to be fully established. Synthesis of tryptophan (Trp)-derived indole metabolites is mediated mostly by the gut microbiota. Tryptophan metabolites can activate the aryl hydrocarbon receptor (AhR). Whether eukaryotic cells can generate Trp-derived indoles and their functional significance remain to be fully established. Here, we investigated the role of tryptophan metabolites and AhR activation in purinergic-mediated senescence of human fibroblasts. We find that ATP activated AhR in a P2Y11R-dependent manner and that AhR activation was necessary for ATP-induced senescence. Stimulation with an AhR agonist was sufficient to induce senescence. Interestingly, depletion of tryptophan in the conditioned medium inhibited ATP-induced senescence. We show that ATP stimulation upregulated the expression of the L-amino acid oxidase interleukin-4-induced-1 (IL4I1), which has been shown to metabolize Trp into indole-3-pyruvate (I3P), in a P2Y11R-dependent fashion. We find that I3P-derived Trp metabolites are upregulated in ATP-induced senescent human fibroblasts and that stimulation with I3P and I3P-derived Trp metabolites was sufficient to promote senescence in these cells. In addition, I3P stimulation activated AhR, and AhR inhibition impaired I3P-induced senescence. Downregulation of IL4I1 inhibited ATP-induced AhR activation and senescence. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce by I3P treatment, promoted the proliferation of breast cancer cells and their tumorigenic potential. Our study identifies the existence of a novel purinergic dependent signaling pathway that functionally couples tryptophan metabolism to the development of a premature senescent phenotype in human fibroblasts.

Stress is a key contributor to gastrointestinal (GI) dysmotility, particularly in patients with disorders of gut-brain interactions (DGBI). Since GI motility is governed by the enteric nervous system (ENS), stress may act by altering ENS function. While stress activates glucocorticoid signaling via the hypothalamic-pituitary-adrenal axis, the impact of stress-mediated glucocorticoid signaling on ENS biology remains poorly understood. In the central nervous system, glucocorticoids reduce specific isoforms of brain-derived neurotrophic factor (BDNF), impairing signaling through its receptor, TrkB, and contributing to behavioral dysfunction. However, the identity of ENS-specific Bdnf isoforms, their glucocorticoid sensitivity, and the effect of enhanced TrkB signaling on GI motility in stressed animals has not been characterized. Here, using male and female mice, we show that >85% of post-natal ENS Bdnf transcripts are glucocorticoid-responsive isoforms. We also demonstrate that both BDNF and its receptor TrkB (Ntrk2) are expressed by enteric neurons. Stress, in male mice, and administration of dexamethasone, a synthetic glucocorticoid receptor (GR) agonist, in both male and female mice, cause GI dysmotility, which we demonstrate is associated with significantly reduced Bdnf transcripts in the longitudinal muscle - myenteric plexus (LM-MP) tissue in vivo. Dexamethasone exposure also represses Bdnf transcript and mature protein levels in LM-MP tissue in vitro. Notably, treatment with HIOC, a selective TrkB agonist, rescues GI transit defects in dexamethasone-treated animals. These findings identify BDNF-TrkB signaling as a key modulator of stress-induced ENS dysfunction and highlight TrkB as a promising therapeutic target for GI dysmotility in DGBI.

LRIT3 is a leucine-rich repeat (LRR) protein that is expressed in the retina, and its absence causes complete congenital stationary night blindness (cCSNB), a genetically diverse disorder characterized by impaired low-light vision, myopia, and nystagmus. LRIT3 is expressed in rod and cone photoreceptors, and it trans-synaptically organizes the assembly of the glutamate signaling complex, the signalplex, on depolarizing bipolar cells (DBCs). LRIT3 is a single-pass membrane protein with extracellular LRR, IG, and FN3 domains. We express domain deletion constructs using rAAV and examine the impact on LRIT3 trafficking, as well as the structural and functional recovery of the signalplex in DBCs. We show the LRR domain may be required for trafficking LRIT3 to the synapse in cones, but not rods, and it is needed for reassembly and function of the rod BC signalplex. The IG domain is required for the localization of TRPM1 to the signalplex and thus its function. The FN3 domain is not necessary for either DBC signalplex assembly or function. Our data demonstrates that the LRR and IG domains of LRIT3 are crucial for TRPM1 localization and retinal function, and that restoring Nyctalopin localization to the DBC signalplex alone is insufficient to restore TRPM1 expression. Based on our findings, we propose a model in which the LRR domain trans-synaptically binds with Nyctalopin, while the IG domain interacts with TRPM1.

Osteocytes play critical roles in bone, making them attractive targets for therapeutics to improve bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin β3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61^high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify a role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.

Oxidative stress represents a central challenge to cellular survival. Although multiple antioxidant enzymes participate in oxidative defense, peroxiredoxins (Prxs) have long been regarded as key determinants of oxidative stress tolerance. However, this view is largely based on the oxidative sensitivity of Prx-deficient mutants and lacks direct experimental evidence demonstrating that Prxs function as terminal antioxidant effectors determining cellular tolerance to oxidative stress. In this study, through transcriptomic screening combined with systematic genetic and functional analyses, we define the key effector within the oxidative defense system and identify the mitochondrial cytochrome c peroxidase Ccp1 as a core determinant of oxidative stress tolerance. In Aspergillus nidulans, the peroxiredoxin PrxA activates the oxidative-stress transcription factor NapA, mediating Ccp1 induction. Our results indicate that the apparent requirement for Prx in oxidative stress tolerance does not arise from its role as a terminal antioxidant effector, but instead reflects its function as an upstream redox signaling factor regulating activation of the key effector enzyme Ccp1. Further functional analyses show that loss of Ccp1 or catalytic inactivation leads to dissipation of mitochondrial membrane potential, compromised mitochondrial DNA integrity, and reduced iron-sulfur enzyme activity, thereby impairing cellular tolerance to oxidative stress. Redirecting other peroxidases to mitochondria functionally substitutes for Ccp1 and restores oxidative stress tolerance. Together, these findings demonstrate that mitochondria-targeted antioxidant protection mediated by Ccp1 acts as a key defensive process for oxidative stress tolerance, while mechanistically clarifying the functional role of PrxA as an upstream redox signaling factor within the oxidative defense network of this fungus.

Conventional genetic approaches, including global gene knockout and conditional knockout strategies such as the Cre-loxP system, have some limitations arising from systemic effects or insufficient temporal resolution. The recently developed photoactivatable Cre (PA-Cre) system may have a potential to improve spatiotemporal control of gene manipulation. In this study, we established and validated the feasibility of the PA-Cre system using taste buds as a model. We generated TRE-PA-Cre:R26-rtTA/tdTomato mice to evaluate blue-light-induced Cre recombinase activity. Through systematic optimization of illumination parameters, we found that a single session of blue-light-illumination resulted in limited recombination efficiency, whereas a multi-session illumination strategy markedly increased recombination efficiency. To further assess the utility of the PA-Cre system for gene knockout, we generated TRE-PA-Cre:R26-rtTA:Tas1r3-flox mice and targeted a taste-related gene Tas1r3. Genomic DNA qPCR and RT-qPCR both showed partial reductions in Tas1r3 at the DNA and mRNA levels, respectively. Behavioral assays further revealed a selective decrease in sensitivity to sweet and umami stimuli. Together, these findings demonstrate PA-Cre-mediated gene manipulation in taste buds and establish a practical optical activation paradigm, providing a high-spatiotemporal-resolution tool for investigating gene function in optically targeted regions.

Lipomannan (LM) and lipoarabinomannan (LAM) are important components of the cell envelope of all mycobacteria that have been extensively studied for their roles in mycobacterial physiology and host-pathogen interactions. Despite the considerable progress made in deciphering the structure and biosynthesis of these lipoglycans over the last few decades, some of the key steps leading to their assembly and export to the cell surface remain ill-defined. We report on the characterization of a conserved and essential polyprenyl phosphate mannose-dependent mannosyltransferase named MptB, involved in the initial steps of the elongation of the mannan domain of LM and LAM from a phosphatidylinositol mannoside (PIM) anchor. Genetic silencing of mptB in Mycobacterium smegmatis led to the arrest of LM, LAM and PIM synthesis beyond di-mannosylated forms of these glycolipids. In cell-free assays, mptB overexpression led to the increased production of tetra-mannosylated forms of PIMs by M. smegmatis membranes, whereas reduced mptB expression resulted in the dramatically decreased synthesis of phosphatidylinositol tri-, tetra- and hexa-mannosides. Together with structural modeling predictions, the results of these assays support MptB as the α-(1,6)-mannosyltransferase elongating the mannan backbone of LM from a di- and/or tri-mannosylated PIM primer.

Histone cell cycle regulator A (HIRA) confers chromatin accessibility and regulates developmental hematopoiesis. Previously, we showed that HIRA expression is higher in patient samples from chronic myeloid leukemia (CML) compared to samples from healthy individuals. However, the underlying mechanism that connects HIRA with chromatin reorganization and pathogenesis of leukemia associated with abnormal hematopoiesis remains unexplained. We developed a HIRA-knockdown K562 CML cell line model for this study, as this cell line showed a maximal expression of HIRA in the myeloid lineage. A proteome-wide analysis demonstrated the association of HIRA with components of chromatin organization in K562 cells. FRAP and FLIM-FRET microscopy and molecular interaction studies revealed increased chromatin compaction and altered spatial distribution of chromatin towards the nuclear periphery upon downregulation of HIRA in K562 cells. Mechanistically, enhanced chromatin compaction was attributed to increased histone H3K9me3 and HP1α levels mediated by histone methyltransferase SETDB1. The enrichment of histone H3.3 and the reduction in H3K27me3 levels, resulting from the loss of EZH2 recruitment at the SETDB1 and HP1α promoters in HIRA-knockdown cells, led to an increase in their expression. This HIRA-SETDB1-H3K9me3 axis contributed to restricted cell proliferation along with loss in expression of the BCR-ABL fusion protein that causes CML. Thus, loss of HIRA promotes global chromatin condensation and redistribution, thereby regulating the BCR-ABL expression and cell proliferation. Our findings highlight how elevated HIRA expression contributes to the pathogenesis of CML and establish a regulatory axis that could be further explored for therapeutic interventions.