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Nature Communications Jun 2024Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for...
Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.
Topics: Humans; Ataxia Telangiectasia Mutated Proteins; Glioblastoma; Tumor Suppressor Protein p53; DNA Breaks, Double-Stranded; Temozolomide; Cell Line, Tumor; Mutation; Drug Resistance, Neoplasm; DNA Repair; Brain Neoplasms; Animals; DNA End-Joining Repair; Mice; Phosphorylation
PubMed: 38906885
DOI: 10.1038/s41467-024-49316-8 -
Biochimica Et Biophysica Acta.... Jun 2024Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ, exogenously...
Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.
PubMed: 38906315
DOI: 10.1016/j.bbabio.2024.149147 -
Cell Reports. Medicine Jun 2024Immune checkpoint inhibitors (ICIs) activate anti-cancer immunity by blocking T cell checkpoint molecules such as programmed death 1 (PD-1) and cytotoxic T... (Review)
Review
Immune checkpoint inhibitors (ICIs) activate anti-cancer immunity by blocking T cell checkpoint molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Although ICIs induce some durable responses in various cancer patients, they also have disadvantages, including low response rates, the potential for severe side effects, and high treatment costs. Therefore, selection of patients who can benefit from ICI treatment is critical, and identification of biomarkers is essential to improve the efficiency of ICIs. In this review, we provide updated information on established predictive biomarkers (tumor programmed death-ligand 1 [PD-L1] expression, DNA mismatch repair deficiency, microsatellite instability high, and tumor mutational burden) and potential biomarkers currently under investigation such as tumor-infiltrated and peripheral lymphocytes, gut microbiome, and signaling pathways related to DNA damage and antigen presentation. In particular, this review aims to summarize the current knowledge of biomarkers, discuss issues, and further explore future biomarkers.
PubMed: 38906149
DOI: 10.1016/j.xcrm.2024.101621 -
Gut Microbes 2024Gut bacteria regulate brain pathology of Alzheimer's disease (AD) patients and animal models; however, the underlying mechanism remains unclear. In this study,...
Gut bacteria regulate brain pathology of Alzheimer's disease (AD) patients and animal models; however, the underlying mechanism remains unclear. In this study, 3-month-old APP-transgenic female mice with and without knock-out of gene were treated with antibiotics-supplemented or normal drinking water for 2 months. The antibiotic treatment eradicated almost all intestinal bacteria, which led to a reduction in Il-17a-expressing CD4-positive T lymphocytes in the spleen and gut, and to a decrease in bacterial DNA in brain tissue. Depletion of gut bacteria inhibited inflammatory activation in both brain tissue and microglia, lowered cerebral Aβ levels, and promoted transcription of gene in the brain of APP-transgenic mice, all of which effects were abolished by deficiency of Il-17a. As possible mechanisms regulating Aβ pathology, depletion of gut bacteria inhibited β-secretase activity and increased the expression of Abcb1 and Lrp1 in the brain or at the blood-brain barrier, which were also reversed by the absence of Il-17a. Interestingly, a crossbreeding experiment between APP-transgenic mice and knockout mice further showed that deficiency of Il-17a had already increased Abcb1 and Lrp1 expression at the blood-brain barrier. Thus, depletion of gut bacteria attenuates inflammatory activation and amyloid pathology in APP-transgenic mice via Il-17a-involved signaling pathways. Our study contributes to a better understanding of the gut-brain axis in AD pathophysiology and highlights the therapeutic potential of Il-17a inhibition or specific depletion of gut bacteria that stimulate the development of Il-17a-expressing T cells.
Topics: Animals; Alzheimer Disease; Interleukin-17; Gastrointestinal Microbiome; Mice; Mice, Transgenic; Brain; Female; Disease Models, Animal; Mice, Knockout; Amyloid Precursor Protein Secretases; Amyloid beta-Peptides; Anti-Bacterial Agents; Mice, Inbred C57BL; Microglia; CD4-Positive T-Lymphocytes; Blood-Brain Barrier; Humans; Low Density Lipoprotein Receptor-Related Protein-1
PubMed: 38904096
DOI: 10.1080/19490976.2024.2363014 -
International Journal of Biological... 2024Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of...
Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, and . Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress . Endothelium-specific knockout of DKK1 (DKK1/APOE) and endothelium-specific overexpression of DKK1 (DKK1) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation . RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1 and DKK1 mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1/APOE mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.
Topics: Animals; Atherosclerosis; Mice; Intercellular Signaling Peptides and Proteins; Foam Cells; Myocytes, Smooth Muscle; Endothelial Cells; Humans; Ubiquitination; Male; Coculture Techniques; Mice, Knockout; Ubiquitin-Specific Proteases; Mice, Inbred C57BL
PubMed: 38904030
DOI: 10.7150/ijbs.91957 -
International Journal of Biological... 2024The mechanism that maintains ER-to-Golgi vesicles formation and transport is complicated. As one of the adapters, Ninein-like protein (Nlp) participated in assembly and...
The mechanism that maintains ER-to-Golgi vesicles formation and transport is complicated. As one of the adapters, Ninein-like protein (Nlp) participated in assembly and transporting of partial ER-to-Golgi vesicles that contained specific proteins, such as β-Catenin and STING. Nlp acted as a platform to sustain the specificity and continuity of cargoes during COPII and COPI-coated vesicle transition and transportation through binding directly with SEC31A as well as Rab1B. Thus, we proposed an integrated transport model that particular adapter participated in specific cargo selection or transportation through cooperating with different membrane associated proteins to ensure the continuity of cargo trafficking. Deficiency of Nlp led to vesicle budding failure and accumulation of unprocessed proteins in ER, which further caused ER stress as well as Golgi fragmentation, and PERK-eIF2α pathway of UPR was activated to reduce the synthesis of universal proteins. In contrast, upregulation of Nlp resulted in Golgi fragmentation, which enhanced the cargo transport efficiency between ER and Golgi. Moreover, deficient mice were prone to spontaneous B cell lymphoma, since the developments and functions of lymphocytes significantly depended on secretory proteins through ER-to-Golgi vesicle trafficking, including IL-13, IL-17 and IL-21. Thus, perturbations of Nlp altered ER-to-Golgi communication and cellular homeostasis, and might contribute to the pathogenesis of B cell lymphoma.
Topics: Animals; Humans; Mice; COP-Coated Vesicles; Endoplasmic Reticulum; Golgi Apparatus; Protein Transport
PubMed: 38904019
DOI: 10.7150/ijbs.91792 -
International Journal of Biological... 2024Pancreatic cancer is the deadliest malignancy with a poor response to chemotherapy but is potentially indicated for ferroptosis therapy. Here we identified that...
Pancreatic cancer is the deadliest malignancy with a poor response to chemotherapy but is potentially indicated for ferroptosis therapy. Here we identified that cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates NRF2 proteostasis and susceptibility to ferroptosis in pancreatic ductal adenocarcinoma (PDAC). We found that CPEB1 deficiency in cancer cells promotes the translation of p62/SQSTM1 by facilitating mRNA polyadenylation. Consequently, upregulated p62 enhances NRF2 stability by sequestering KEAP1, an E3 ligase for proteasomal degradation of NRF2, leading to the transcriptional activation of anti-ferroptosis genes. In support of the critical role of this signaling cascade in cancer therapy, CPEB1-deficient pancreatic cancer cells display higher resistance to ferroptosis-inducing agents than their CPEB1-normal counterparts and . Furthermore, based on the pathological evaluation of tissue specimens from 90 PDAC patients, we established that CPEB1 is an independent prognosticator whose expression level is closely associated with clinical therapeutic outcomes in PDAC. These findings identify the role of CPEB1 as a key ferroptosis regulator and a potential prognosticator in pancreatic cancer.
Topics: Humans; Ferroptosis; Pancreatic Neoplasms; NF-E2-Related Factor 2; Cell Line, Tumor; Animals; mRNA Cleavage and Polyadenylation Factors; Mice; Proteostasis; Transcription Factors; Carcinoma, Pancreatic Ductal; Kelch-Like ECH-Associated Protein 1; Mice, Nude
PubMed: 38904009
DOI: 10.7150/ijbs.95962 -
Frontiers in Medicine 2024Fabry disease (FD) is an X-linked disorder resulting in a deficiency of α-galactosidase A (GLA) activity. The R112H mutation of GLA is relatively common in Japanese FD...
Fabry disease (FD) is an X-linked disorder resulting in a deficiency of α-galactosidase A (GLA) activity. The R112H mutation of GLA is relatively common in Japanese FD patients, characterized by a late-onset phenotype, almost normal to mild lyso-Gb3 elevation, and mild clinical symptoms, despite low GLA activity. This is due to the structural features of the R112H GLA protein. We herein report the case of a 42-year-old male patient with late-onset FD with a R112H mutation. The patient exhibited only renal involvement with no other organ damage and was successfully treated with galactosidase beta and subsequent migalastat for approximately 10 years. Especially, migalastat was clinically effective in normalizing plasma lyso-Gb3 levels and inhibiting the progression of renal damage associated with FD. Therefore, the use of migalastat in the FD patients with R112H mutation is highly recommended based on this case report.
PubMed: 38903807
DOI: 10.3389/fmed.2024.1383309 -
Frontiers in Microbiology 2024The arginine transporter Can1 is a multifunctional protein of the conventional yeast . Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating...
The arginine transporter Can1 is a multifunctional protein of the conventional yeast . Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the knockout strain is rescued by a functional deficiency of the gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the knockout strain of the pathogenic yeast exhibited slower growth and higher biofilm formation, similar to . More importantly, the gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents.
PubMed: 38903792
DOI: 10.3389/fmicb.2024.1419530 -
Frontiers in Immunology 2024Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells. Toll-like receptor 9 (TLR9) plays a role in autoimmune...
INTRODUCTION
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells. Toll-like receptor 9 (TLR9) plays a role in autoimmune diseases, and B cell-specific TLR9 deficiency delays T1D development. Gut microbiota are implicated in T1D, although the relationship is complex. However, the impact of B cell-specific deficiency of TLR9 on intestinal microbiota and the impact of altered intestinal microbiota on the development of T1D are unclear.
OBJECTIVES
This study investigated how gut microbiota and the intestinal barrier contribute to T1D development in B cell-specific TLR9-deficient NOD mice. Additionally, this study explored the role of microbiota in immune regulation and T1D onset.
METHODS
The study assessed gut permeability, gene expression related to gut barrier integrity, and gut microbiota composition. Antibiotics depleted gut microbiota, and fecal samples were transferred to germ-free mice. The study also examined IL-10 production, Breg cell differentiation, and their impact on T1D development.
RESULTS
B cell-specific TLR9-deficient NOD mice exhibited increased gut permeability and downregulated gut barrier-related gene expression. Antibiotics restored gut permeability, suggesting microbiota influence. Altered microbiota were enriched in Lachnospiraceae, known for mucin degradation. Transferring this microbiota to germ-free mice increased gut permeability and promoted IL-10-expressing Breg cells. Rag mice transplanted with fecal samples from -Cre mice showed delayed diabetes onset, indicating microbiota's impact.
CONCLUSION
B cell-specific TLR9 deficiency alters gut microbiota, increasing gut permeability and promoting IL-10-expressing Breg cells, which delay T1D. This study uncovers a link between TLR9, gut microbiota, and immune regulation in T1D, with implications for microbiota-targeted T1D therapies.
Topics: Animals; Toll-Like Receptor 9; Gastrointestinal Microbiome; Mice, Inbred NOD; Interleukin-10; Mice; Diabetes Mellitus, Type 1; Mice, Knockout; B-Lymphocytes, Regulatory; Female; B-Lymphocytes
PubMed: 38903498
DOI: 10.3389/fimmu.2024.1413177