Molecular and Cellular Biology最新文献

Topoisomerase I (Top1) alleviates DNA supercoiling during replication and transcription, but its catalytic cycle can be hijacked by chemotherapeutic agents such as camptothecin (CPT), stabilizing Top1-DNA covalent complexes (Top1cc) that threaten genome integrity. Efficient resolution of these trapped intermediates is crucial to prevent replication stress, DNA breaks, and cell death. Poly (ADP-ribose) polymerase 1 (PARP1) is a key sensor of Top1cc, facilitating repair by recruiting tyrosyl-DNA phosphodiesterase 1 (TDP1) and modifying chromatin to promote lesion accessibility. Beyond this canonical pathway, emerging evidence highlights PARP1-independent mechanisms such as endo nucleolytic cleavage, proteolytic degradation of Top1 and replication-associated processing. Intriguingly, PARP1 appears to act as a molecular switch between TDP1 and the endonuclease pathway for the repair of Top1cc. This review highlights mechanisms of PARP1-dependent and -independent Top1cc repair pathways, their interplay and redundancy, and how their targeting can enhance Top1-based cancer therapies and overcome resistance.

N6-methyladenosine (m6A), the most prevalent modification in mRNAs, influences mRNA stability, splicing, and translation. Dysregulation of m6A patterns has been linked to various diseases, including cancer, highlighting its significance in cellular homeostasis. However, accurate detection and precise quantification of m6A sites within individual transcripts remains challenging. In this study, we employed nanopore sequencing to achieve transcriptome-wide, base-resolution map of the m6A methylome in human breast cancer cells. By investigating m6A distribution across breast cancer cell lines and implementing a CRISPR/Cas9-based knockout of the major m6A eraser ALKBH5, we provide insights into the differential methylation levels and motif-specific characteristics of m6A transcriptomic sites. We elucidated the m6A epitranscriptome in five well-established breast cancer cell lines derived from distinct molecular subtypes of the disease and confirmed a DRACH-dependent activity of ALKBH5. Comparative methylation analysis with the non-cancerous MCF-10A cell line revealed that MCF-7 and BT-474 breast cancer cells are primarily hypomethylated, while BT-20, MDA-MB-231 and SK-BR-3 cells show widespread hypermethylation. These cell line-based patterns highlight the potential regulatory role of m6A in breast cancer heterogeneity. Overall, our findings enhance the understanding of m6A dynamics in breast cancer.

During mammalian development, production sites of the erythroid growth factor erythropoietin (EPO) shift from the neural tissues to the liver in embryos and to the kidneys in adults. Embryonic neural EPO-producing (NEP) cells, a subpopulation of neuroepithelial and neural crest cells, express the Epo gene between embryonic day (E) 8.5 and E11.5 to promote primitive erythropoiesis in mice. While Epo gene expression in the liver and kidneys is induced under hypoxic conditions through hypoxia-inducible transcription factors (HIFs), the Epo gene regulatory mechanisms in NEP cells remain to be elucidated. Here, we confirmed the presence of cells co-expressing EPO and HIFs in mouse neural tubes, where the hypoxic microenvironment activates HIFs. Chemical activation and inhibition of HIFs demonstrated the hypoxic regulation of EPO expression in human fetal neural progenitors and mouse embryonic neural tissues. In addition, we found that histone deacetylase inhibitors can reactivate EPO production in cell lines derived from NEP cells and human neuroblastoma, as well as in mouse primary neural crest cells, while rejuvenating these cells. Furthermore, the ability of the rejuvenated cells to produce EPO was maintained in hypoxia. Thus, EPO production is controlled by epigenetic mechanisms and hypoxia signaling in the immature state of hypoxic NEP cells.

RNA 5-methylcytosine (m5C) modification has emerged as an important regulatory mechanism in the progression of human cancers, including hepatobiliary tumors. The m5C "reader" Aly/REF export factor (ALYREF) was recently found to be identified as a prognostic biomarker in liver cancer. However, its exact role in intrahepatic cholangiocarcinoma (ICC) progression is unclear. In this study, ALYREF was found to be upregulated in ICC tissues and cells. The gain- and loss-of-function experiments indicated that ALYREF promoted cell proliferation and invasion and suppressed cell apoptosis. Moreover, we found that isocitrate dehydrogenase 1 (IDH1), a metastatic marker of liver cancer, was also upregulated in ICC tissues, displayed a relatively strong positive correlation with the level of ALYREF, and was positively regulated by ALYREF. As an m5C "reader", ALYREF interacted with m5C-IDH1 mRNA and increased its stability. ALYREF knockdown partially eliminated the promotion of IDH1 on ICC cell proliferation and invasion. ALYREF positively regulated NRF2-driven glutathione synthesis in ICC cells, which was reversed by IDH1 silencing. Finally, in a xenograft tumor mouse model, knockdown of ALYREF or treatment with ivosidenib (an IDH1 inhibitor) significantly suppressed tumor growth in vivo. In conclusion, ALYREF promotes ICC progression by increasing IDH1 levels in an m5C-dependent manner.

Lactate, historically considered a metabolic byproduct, has emerged as a key regulator of muscle physiology and metabolism. This study explores its potential as an exercise mimetic to counteract disuse muscle atrophy (DMA) in aging skeletal muscle using a hindlimb suspension model in senescence-accelerated prone 8 (SAMP8) mice. The mice were divided into four groups: Control, lactate-treated control, hindlimb suspension, and hindlimb suspension with lactate intervention. Lactate administration preserved gastrocnemius muscle mass, restored muscle strength, and attenuated oxidative fiber atrophy. Electrophoretic and histological analyses showed increased MyHC I expression, indicating protection of oxidative fibers. Functional assessments revealed improved muscle endurance and contractile force, while metabolomic profiling identified changes in energy metabolism, amino acid metabolism, and protein synthesis pathways. Specifically, lactate improved impaired branched-chain amino acid metabolism, suggesting enhanced protein synthesis. In addition, lactate boosted Cori cycle activity, upregulated hepatic lactate transporters, and increased lactate dehydrogenase B activity, facilitating efficient lactate metabolism and gluconeogenesis. These results provide new insights into the role of lactate as a metabolic regulator and highlight its potential as a therapeutic intervention to combat exercise-induced muscle wasting and preserve muscle function in aging and immobilized individuals.

Protein misfolding and conformational instability drive protein conformational disorders, causing either accelerated degradation and loss-of-function, as in inherited metabolic disorders like lysosomal storage disorders, or toxic aggregation and gain-of-function, as in neurodegenerative diseases like Alzheimer's disease or amyotrophic lateral sclerosis. Classical homocystinuria (HCU), an inborn error of sulfur amino acid metabolism, results from cystathionine beta-synthase (CBS) deficiency. CBS regulates methionine conversion into metabolites critical for redox balance (cysteine, glutathione) and signaling (H₂S). Pathogenic missense mutations in the CBS gene often impair folding, cofactor binding, stability or oligomerization rather than targeting the key catalytic residues of the CBS enzyme. Advances in understanding of CBS folding and assembly as well as CBS interactions with cellular proteostasis network offer potential for therapies using pharmacological chaperones (PCs), i.e., compounds facilitating proper folding, assembly or cellular trafficking. This review discusses progress in identifying PCs for HCU, including chemical chaperones, cofactors, and proteasome inhibitors. We outline future directions, focusing on high-throughput screening and structure-based drug design to develop CBS-specific PCs. These could stabilize mutant CBS, enhance its stability and restore activity, providing new treatments for HCU and possibly other conditions related to dysregulated CBS, such as cancer or Down's syndrome.

Defining the mechanisms that promote development and progression of myeloproliferative neoplasms (MPNs) is important for understanding the mechanisms of malignant hematopoiesis and critical development of new treatment approaches. We provide evidence for a key and essential role of the kinase ULK1 in MPN pathophysiology. Our studies demonstrate that genetic or pharmacological targeting of ULK1 delays substantially disease development in Jak2^V617F-mutant MPN models in vivo and establish that ULK1 activity is required for transcription of genes that control hematopoietic stem cell differentiation. Pharmacological targeting of ULK1 exhibits potent therapeutic effects, resulting in reduction of early stage erythroid progenitors in spleen and bone marrow, decreased levels of hemoglobin, and reduced spleen size in MPN mouse models in vivo. Taken together, these findings provide the first evidence for a novel protumorigenic role for ULK1 downstream of the hyperactive JAK2 signaling in MPNs and raise the potential of ULK1 as a new therapeutic target for the treatment of MPNs.

Iron is an essential micronutrient for eukaryotic organisms. In response to iron deficiency, the yeast Saccharomyces cerevisiae optimizes iron utilization by downregulating nonessential iron-dependent processes, such as mitochondrial respiration. This regulatory mechanism is mediated by a mRNA-binding protein designated Cth2. In response to iron scarcity, Cth2 binds through its tandem zinc-finger (TFZ) domain to multiple mRNAs encoding proteins that are necessary for iron-dependent pathways. This binding limits the expression of these mRNAs by promoting their degradation and inhibiting their translation. In this study, we have examined a set of wild yeast strains that share a G195R mutation within the Cth2 TZF domain. By genetically editing both laboratory and wild yeast strains, we demonstrate that the Cth2-G195R protein is defective in binding and degradation of its target transcripts, and it accumulates in the nucleus of the cell, leading to a significant growth defect in iron-deficient conditions. Some of these wild yeast strains also display enhanced tolerance to high iron conditions, indicating that they have adapted to environments with elevated iron levels and have consequently diminished their capacity to grow in iron-limiting conditions. These findings highlight the crucial function of Cth2 in enabling yeast cells to adapt to iron-deficient environments.

While the cysteine proteases legumain and cathepsins have traditionally been known as "lysosomal" proteases, there is increasing evidence to suggest that they also contribute to a wide range of extralysosomal processes, including in the nucleus. This review aims to provide a comprehensive overview of the current knowledge regarding the translocation of these proteases to the nucleus and their functions on arrival. We discuss possible mechanisms for transporting these proteases to the nucleus, including the presence of a nuclear localization signal sequence or hitchhiking on other proteins that possess this sequence. This transport requires the proteases to first reach the cytosol, which may occur via direct cytosolic translation of truncated proteases or downstream of lysosomal membrane permeabilization. We also discuss the evidence for functions of these proteases upon arrival to the nucleus, including cell cycle progression, cell differentiation, cell death, immune regulation, and epigenetic regulation. As protease substrate profiling methods continue to improve, it is anticipated that many new nuclear substrates and interacting partners will be identified to reveal additional functions for nuclear proteases.

Erythropoiesis, i.e., process of red blood cell (RBC) production, is highly dependent on iron, with 60-70% of the total body iron incorporated into hemoglobin. Iron homeostasis is tightly regulated, given that both iron overload and deficiency can impair RBC development and function. Iron-loading anemias, such as sideroblastic anemia and thalassemia, are associated with ineffective erythropoiesis and systemic iron overload. Recent studies also highlight the role of ferroptosis, i.e., iron-dependent cell death, in erythroid failure under conditions of iron overload. Transcriptional repressor BTB and CNC homology 1 (BACH1), which is regulated by intracellular heme, is a potential key mediator of ferroptosis. In iron deficiency, limited iron availability impairs heme and globin biosynthesis, mitochondrial function, and erythropoietin responsiveness, while also inducing widespread changes in gene expression through DNA methylation, all of which contribute to dysregulated erythropoiesis. Under iron deficiency, BACH1 plays a critical role in maintaining the balance between heme and globin by suppressing globin gene expression, thereby preventing the aggregation of toxic non-heme globin. This review summarizes the current understanding of the mechanisms by which iron imbalance contributes to erythropoietic failure and highlights BACH1 as a potential integrative regulator in the pathophysiology of anemia in both iron-overload and iron-deficient states.