Background: ACTN4 is an actin-binding protein involved in many cellular processes, including cancer development. High ACTN4 expression is often associated with a poor prognosis. However, it has been identified as a positive marker for platinum-based adjuvant chemotherapy for non-small cell lung cancer (NSCLC). The goal of our study was to investigate the involvement of ACTN4 in the NSCLC cells’ response to the genotoxic drugs. Results: We generated H1299 cells with the ACTN4 gene knock-out (ACTN4 KO), using the CRISPR/Cas9 system. The resistance of the cells to the cisplatin and etoposide was analyzed with the MTT assay. We were also able to estimate the efficiency of DNA repair through the DNA comet assay and gamma-H2AX staining. Possible ACTN4 effects on the non-homologous end joining (NHEJ) and homologous recombination (HR) were investigated using pathway-specific reporter plasmids and through the immunostaining of the key proteins. We found that the H1299 cells with the ACTN4 gene knock-out did not show cisplatin-resistance, but did display a higher resistance to the topoisomerase II inhibitors etoposide and doxorubicin, suggesting that ACTN4 might be somehow involved in the repair of DNA strand breaks. Indeed, the H1299 ACTN4 KO cells repaired etoposide- and doxorubicin-induced DNA breaks more effectively than the control cells. Moreover, the ACTN4 gene knock-out enhanced NHEJ and suppressed HR efficiency. Supporting the data, the depletion of ACTN4 resulted in the faster assembly of the 53BP1 foci with a lower number of the phospho-BRCA1 foci after the etoposide treatment. Conclusions: Thus, we are the first to demonstrate that ACTN4 may influence the resistance of cancer cells to the topoisomerase II inhibitors, and affect the efficiency of the DNA double strand breaks repair. We hypothesize that ACTN4 interferes with the assembly of the NHEJ and HR complexes, and hence regulates balance between these DNA repair pathways.

Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin–proteasome system—the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

Neurons become more vulnerable to stress factors with age, which leads to increased oxidative DNA damage, decreased activity of mitochondria and lysosomes, increased levels of p16, decreased LaminB1 proteins, and the depletion of the dendritic tree. These changes are exacerbated in vulnerable neuronal populations during the development of neurodegenerative diseases. Glu-Asp-Arg (EDR) and Lys-Glu-Asp (KED), and Ala-Glu-Asp-Gly (AEDG) peptides have previously demonstrated neuroprotective effects in various models of Alzheimer’s disease. In this study, we investigated the influence of EDR, KED, and AEDG peptides on the aging of fibroblast-derived induced neurons. We used a new in vitro cellular model of human neuronal aging based on the transdifferentiation of aged dermal fibroblasts from elderly donors into induced cortical neurons. All peptides promote the arborization of the dendritic tree, increasing both the number of primary processes and the total length of dendrites. Tripeptides have no effect on the activity of mitochondria and lysosomes and the level of p16 protein in induced neurons. EDR peptide reduces oxidative DNA damage in induced neurons derived from elderly donor fibroblasts. Short peptides partially protect induced neurons from age-related changes and stimulate dendritogenesis in neurons. They can be recommended for use as neuroprotective agents.

It has been previously shown that some short peptides are involved in various cellular processes, such as transcription modulation and regulation of differentiation mechanisms. In particular, the effect of peptides on the neuronal differentiation of human periodontal ligament stem cells has been demonstrated. The goal of this study was to assess the effect of KED, EDR, and AEDG short peptides in stimulating the transdifferentiation of fetal MSCs into induced neuronal cells and prevention of their senescence. We applied a novel in vitro technique for neuronal cell generation, which combines the use of microRNAs, transcription factors, and small molecules to transdifferentiate fetal mesenchymal stem cells into induced cortical neurons. It was shown that the application of AEDG and KED short peptides at the end of the transdifferentiation process decreases the expression of the cell cycle marker p21 by 15% and beta-galactosidase activity by 1.51–2.4 times. However, short peptides did not affect the expression levels of TUj-1 and LaminB1, whose expression also changes during neuronal differentiation. The experiments indicate the potential of AEDG and KED short peptides as modulators of neurogenesis and geroprotectors and suggest that they can be used as stimulators of neuronal differentiation.