Bioscience Reports最新文献

The androgen receptor (AR) is the main driver of nearly all prostate cancer (PCa). It alters gene expression by binding to specific cis-regulatory elements on the DNA. Where the AR binds in the genome determines what genes are expressed. However, the AR cistrome is not static and dramatically changes during PCa initiation and progression to activate distinct transcriptional programs that fuel disease growth and therapeutic resistance. Emerging evidence suggests that these changes in DNA binding are not caused by chromatin accessibility but rather from interactions with AR coregulators. These proteins influence AR at every step of its activity and play a critical role in DNA binding and gene activation. These context-specific coregulator interactions can stabilize AR binding with DNA that has low- to moderate-affinity and also affect locus-specific epigenetic modifications to promote transcription. Given their critical role in this process, alterations to coregulator proteins define the normal and oncogenic cistrome and profoundly affect AR-mediated gene transcription. In this review, we aim to provide a new perspective on the role of AR coregulators in transcriptional activity, how these interactions evolve through different stages of PCa and their potential as therapeutic targets in advanced disease.

Epidermolysis bullosa simplex (EBS) is a rare genetic disorder, resulting from mutations in keratin 5 and keratin 14 (KRT14), and is characterised by skin fragility, herpetiform blistering, and the development of confluent palmoplantar keratoderma and nail dystrophy. Inflammation, pain and itch are the most common complications of severe EBS. However, pathophysiological mechanisms remain poorly characterised at a molecular level. Recently, RNA N6-methyladenosine (m6A) nucleotide modification has been implicated in several cutaneous physiological processes, including epidermal differentiation, inflammation, adaptive immune responses, host-pathogen interactions, wound healing and tissue repair. Nevertheless, the role of m6A in EBS has yet to be defined. In this pilot study, we investigated the gene expression of key regulators of m6A, such as writers Methyltransferase-like 3 and 4 (METTL3 and METTL14), readers YTH domain-containing proteins (YTHDC1, YTHDC2, YTHDC3) and YTH domain-containing family proteins ( YTHDF1 and YTHDF2) and erasers fat mass and obesity-associated (FTO) and alkB homolog 5 (ALKBH5), as well as total RNA m6A levels in the EB keratinocites cell line (KEB-7) derived from a patient with severe EBS, carrying the KRT14 R125P mutation. NEB-1 cells, derived from a healthy donor, were employed as controls. RNAseq and quantitative RT-PCR demonstrated up-regulation of the writer METTL14, while FTO was down-regulated. Moreover, the total RNA m6A colorimetric assay reported higher levels of m6A in severe EBS cells (KEB-7). Additionally, increased expression of the reader of YTHDC1 suggests a dysregulation of downstream pathways. These findings suggest a potential role for m6A in determining complications in severe EBS; however, its role and effects need to be fully elucidated.

Erythrocytes contain a significant amount of membrane cholesterol, which is continuously exchanged with lipoproteins. Recent studies suggest that erythrocyte cholesterol content correlates positively with atherosclerotic cardiovascular disease (ASCVD) severity independent of low-density lipoprotein levels, potentially reflecting residual ASCVD risk. However, conventional methods for measuring erythrocyte cholesterol content require labor-intensive lipid extraction procedures, limiting their clinical applicability. In this study, we developed a novel enzymatic assay that enables direct quantification of erythrocyte total cholesterol content using two denaturants to eliminate hemoglobin interference. This simple method demonstrated high accuracy and precision, as confirmed by intra-assay repeatability, between-day precision, linearity, and spike-recovery tests. Using this assay, we determined the erythrocyte cholesterol content per volume (154.8 ± 2.9 mg/dl in men, 155.9 ± 6.9 mg/dl in women) and per cell (139.0 ± 5.2 fg/cell in men, 140.8 ± 5.3 fg/cell in women) (n = 12, healthy subjects). While erythrocyte cholesterol content per volume correlated with the conventional method, the erythrocyte cholesterol content per cell showed no such correlation. Moreover, neither measure was associated with serum lipid levels, suggesting their potential as independent biomarkers for ASCVD. Additionally, we evaluated erythrocyte cholesterol content across different maturation stages and found that older erythrocytes had significantly lower cholesterol content, consistent with mass spectrometry results. These findings further validated the physiological relevance of the proposed method. In conclusion, we successfully established a simple and clinically applicable enzymatic method for measuring erythrocyte cholesterol content, providing novel insights into erythrocyte cholesterol metabolism and its potential role in ASCVD risk assessment.

Enzymatic halogenation in natural products has been intensely investigated due to its potential utility as a tool to improve pharmacological and pharmaceutical properties of drug leads. Chlortetracycline (CTC), the first tetracycline (TC) antibiotic discovered nearly eight decades ago, contains a chlorine group. This chlorine is installed enzymatically by the flavin adenine dinucleotide (FAD)-dependent halogenase CtcP. CtcP and the FAD reductase CtcQ, which is also encoded in the CTC biosynthetic gene cluster, function as a two-component system. Structural information on CtcP and CtcQ has been lacking. In this study, we determined crystal structures of CtcP from Kitasatospora aureofaciens in a complex with polyethylene glycol and sulfate ions and in a complex with FAD, and a crystal structure of CtcQ in a complex with FAD and NAD. The structures of CtcP revealed a close similarity of this enzyme to the phenolic halogenase PltM, despite a large difference in the sizes of their respective substrates, presumably TC and phloroglucinol. The CtcP structure showed a conserved dimeric organization also found in PltM crystals. We showed that dimerization of CtcP is allosterically influenced by a distant C-terminal helical hairpin. A closed substrate-binding cavity of CtcP suggested that conformational changes were required to allow a substrate, likely not TC, to bind CtcP. We demonstrated that CtcP and CtcQ weakly bound each other. The dimeric structures of CtcP and CtcQ prompted us to propose approximate models of a 2:2/CtcP:CtcQ complex, where FAD(H2) would shuttle between the two enzymes for chlorination and reduction.

The secretome of adipose-derived stromal cells (ASCs) presents a promising avenue for cell-free therapies due to its rich mixture of bioactive molecules. Different culture conditions can modulate the composition of this mixture, but how this affects the functional properties of the secretome remains to be investigated. This study investigated the in vitro effects of normoxia, cytokines, high glucose, hypoxia, and hypoxia + high glucose-derived ASC secretomes on angiogenesis (tube formation assay), collagen deposition (Picrosirius red staining), and immunomodulation (one-way mixed lymphocyte reaction in combination with an antibody-mediated cell-dependent cytotoxicity assay). The data showed that normoxia- and hypoxiaderived secretomes consistently exhibited potent proangiogenic effects in both human and rat models. These secretomes also demonstrated positive influences on collagen deposition and immunomodulation. Interestingly, the human ASC hypoxia + high glucose-derived secretome emerged as a stimulator of collagen deposition and modulator of the immune system. Conversely, cytokines and high glucose-derived secretomes have shown less strong effects in almost all functional parameters. In conclusion, our findings indicate that modulating culturing conditions results in secretomes with different functional properties and emphasizes the multifaceted role of ASC secretomes in regenerative processes.