Biochimica et biophysica acta. Molecular cell research最新文献

Background

Three-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to replicate physiological tissue organization and functionality. This study investigates the impact of radiation on skeletal muscle response using an innovative in vitro human 3D skeletal muscle organoids (hSMOs) model derived from hPSCs.

Methods

The hSMOs model was established through a differentiation protocol faithfully recapitulating embryonic myogenesis and maturation via paraxial mesodermal differentiation of hPSCs. Key skeletal muscle characteristics were confirmed using immunofluorescent staining and RT-qPCR. Subsequently, the hSMOs were exposed to a clinically relevant dose of 2 Gy of radiation, and their response was analyzed using immunofluorescent staining and RNA-seq.

Results

The hSMO model faithfully recapitulated embryonic myogenesis and maturation, maintaining key skeletal muscle characteristics. Following exposure to 2 Gy of radiation, histopathological analysis revealed deficits in hSMOs expansion, differentiation, and repair response across various cell types at early (30 min) and intermediate (18 h) time points post-radiation. Immunofluorescent staining targeting γH2AX and 53BP1 demonstrated elevated levels of foci per cell, particularly in PAX7⁺ cells, during early and intermediate time points, with a distinct kinetic pattern showing a decrease at 72 h. RNA-seq data provided comprehensive insights into the DNA damage response within the hSMOs.

Conclusions

Our findings highlight deficits in expansion, differentiation, and repair response in hSMOs following radiation exposure, enhancing our understanding of radiation effects on skeletal muscle and contributing to strategies for mitigating radiation-induced damage in this context.

The transcriptional regulator Krüppel-like factor 5 (KLF5) is highly expressed in squamous epithelial cells of the esophagus. Increased KLF5 activity induces tumorigenesis and promotes metastasis in several cancers, although this function appears to be context-dependent. Here, we demonstrate that acute KLF5 inhibition, both genetically and with the potent KLF5 inhibitor ML264, causes non-transformed human primary esophageal squamous epithelial cells to enter the epithelial to mesenchymal transition (EMT). Moreover, chronic KLF5 inhibition with ML264 leads to the development of cells with a mesenchymal phenotype characterized by the expression of mesenchymal markers and functionally by reduced cell growth and increased migration and cellular invasion. This EMT resulting from chronic KLF5 inhibition is not driven by β-Catenin or TGF-β signaling. Pharmacologically, ML264 inhibits KLF5 by promoting proteasomal-mediated degradation. Taken together, we demonstrate that reduced KLF5 activity reprograms epithelial cells towards a mesenchymal phenotype and enhances their migratory and invasive potential. These findings have potential implications not only for esophageal cancers but also for normal processes such as esophageal tissue repair following injury.

Iron‑sulfur (FeS) clusters are inorganic protein cofactors that perform essential functions in many physiological processes. Spectroscopic techniques have historically been used to elucidate details of FeS cluster type, their assembly and transfer, and changes in redox and ligand binding properties. Structural probes of protein topology, complex formation, and conformational dynamics are also necessary to fully understand these FeS protein systems. Recent developments in mass spectrometry (MS) instrumentation and methods provide new tools to investigate FeS cluster and structural properties. With the unique advantage of sampling all species in a mixture, MS-based methods can be utilized as a powerful complementary approach to probe native dynamic heterogeneity, interrogate protein folding and unfolding equilibria, and provide extensive insight into protein binding partners within an entire proteome. Here, we highlight key advances in FeS protein studies made possible by MS methodology and contribute an outlook for its role in the field.

Over the last decade, structural aspects involving iron‑sulfur (Fe/S) protein biogenesis have played an increasingly important role in understanding the high mechanistic complexity of mitochondrial and cytosolic machineries maturing Fe/S proteins. In this respect, solution NMR has had a significant impact because of its ability to monitor transient protein-protein interactions, which are abundant in the networks of pathways leading to Fe/S cluster biosynthesis and transfer, as well as thanks to the developments of paramagnetic NMR in both terms of new methodologies and accurate data interpretation. Here, we review the use of solution NMR in characterizing the structural aspects of human Fe/S proteins and their interactions in the framework of Fe/S protein biogenesis. We will first present a summary of the recent advances that have been achieved by paramagnetic NMR and then we will focus our attention on the role of solution NMR in the field of human Fe/S protein biogenesis.

Vimentin has been considered a canonical marker of epithelial-mesenchymal transition (EMT) and is associated with tumor escape characterized by aberrant PD-L1 expression. However, whether there is a relationship between vimentin and PD-L1 in esophageal squamous cell carcinoma (ESCC) remains poorly understood. The immunological involvement of vimentin in ESCC was first analyzed by multiplex immunofluorescence staining in ESCC tissue microarray followed by a xenografted mouse model. In vivo, C57BL/6 mice were subcutaneously transplanted with AKR cells after stable silencing of vimentin. In vivo results showed that in addition to PD-L1 and PD-L2 expression, vimentin expression was inversely correlated with CD8+ T-cell infiltration. Mechanistically, vimentin can directly interact with PD-L1 and promote nuclear translocation of PD-L1 in AKR cells. In addition, SEMA6C, STC-2 and TRAILR2 were identified as cytokines modulated by vimentin. Blockade of STC-2 and TRAILR2 in co-culture with their own primary antibodies was shown to recruit more CD8+ T cells than controls. Together, these data strongly suggest targeting Vimenin to overcome the immune cycle in ESCC.

Microtubule stabilization is critical for axonal growth and regeneration, and many microtubule-associated proteins are involved in this process. In this study, we found that the knockdown of echinoderm microtubule-associated protein-like 1 (EML1) hindered axonal growth in cultured cortical and dorsal root ganglion neurons. We further revealed that EML1 facilitated the acetylation of microtubules and that the impairment of axonal growth due to EML1 inhibition could be restored by treatment with deacetylase inhibitors, suggesting that EML1 affected tubulin acetylation. Moreover, we verified an interaction between EML1 and the alpha-tubulin acetyltransferase 1, which is responsible for the acetylation of alpha-tubulin. We thus proposed that EML1 might regulate microtubule acetylation and stabilization via alpha-tubulin acetyltransferase 1 and then promote axon growth. Finally, we verified that the knockdown of EML1 in vivo also inhibited sciatic nerve regeneration. Our findings revealed a novel effect of EML1 on microtubule acetylation during axonal regeneration.

We previously reported that integrin alpha 7 (ITGA7) was downregulated in colorectal cancer (CRC) tissues and CRC cell lines and that the lower expression of ITGA7 in CRC tissues was correlated with distant metastasis, suggesting that ITGA7 may function as a suppressor in CRC. The present research was conducted to further investigate the role and mechanisms of ITGA7 in CRC progression. First, bisulfite modification and genomic sequencing (BSP) results showed that the methylation rate of ITGA7 promoter was higher in 10 CRC tissues than in the matched normal tissues. Additionally, 5-Aza-CdR treatment increased ITGA7 expression in CRC cells. Gain-of-function assays revealed the inhibitory role of ITGA7 in CRC cell proliferation and migration. Mechanistically, RNA sequencing, RT-qPCR, and cytoplasm and nuclear separation and rescue assays indicated that knockdown of ITGA7 activated the transcription of MMP9, SETD7, and ADAM15 by enhancing the nuclear translocation of NF-κB. Moreover, CoIP and Western blot suggested a mechanistic model in which ITGA7 binds to CKAP4 to block the interaction of CKAP4 and PI3K p85α and thereby suppress the PI3K/AKT/NF-κB pathway. Accordingly, the current study suggests that ITGA7 functions as a suppressor in CRC progression and that its expression is controlled by promoter methylation.

Chondrocytes rely heavily on glycolysis to maintain the metabolic homeostasis and cartilage matrix turnover. Glycolysis in chondrocytes is remodeled by diverse biochemical and biomechanical factors due to the sporty joint microenvironment. Transforming growth factor-β2 (TGF-β2), one of the most abundant TGF-β superfamily members in chondrocytes, has increasingly attracted attention in cartilage physiology and pathology. Although previous studies have emphasized the importance of TGF-β superfamily members on cell metabolism, whether and how TGF-β2 modulates glycolysis in chondrocytes remains elusive. In the current study, we investigated the effects of TGF-β2 on glycolysis in chondrocytes and explored the underlying biomechanisms. The results showed that TGF-β2 could enhance glycolysis in chondrocytes by increasing glucose consumption, up-regulating liver-type ATP-dependent 6-phosphofructokinase (Pfkl) expression, and boosting lactate production. The TGF-β2 signal entered chondrocytes via TGF-β receptor type I (TβRI), and activated p-Smad3 signaling to regulate the glycolytic pathway. Subsequent experiments employing specific inhibitors of TβRI and p-Smad3 further substantiated the role of TGF-β2 in enhancement of glycolysis via TβRI/p-Smad3 axis in chondrocytes. The results provide new understanding of the metabolic homeostasis in chondrocytes induced by TGF-β superfamily and might shed light on the prevention and treatment of related osteoarticular diseases.

The WhiB-Like (Wbl) proteins are a large family of iron-sulfur (Fe-S) cluster-containing transcription factors exclusively found in the phylum Actinobacteria, including the notable genera like Mycobacteria, Streptomycetes and Corynebacteria. These proteins play pivotal roles in diverse biological processes, such as cell development, redox stress response and antibiotic resistance. Members of the Wbl family exhibit remarkable diversity in their sequences, structures and functions, attracting great attention since their first discovery. This review highlights the most recent breakthroughs in understanding the structural and mechanistic aspects of Wbl-dependent transcriptional regulation.

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLK^K63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLK^K63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLK^K63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLK^K63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.