A SerpinB2 és a SerpinB10 szerepe a DNS hibajavításban és a tumoros folyamatokban

Our cells are constantly exposed to various DNA damaging agents originated from both endogenous and exogenous sources. Ultraviolet radiation (UV) is one of the most deleterious exogenous DNA damaging agents (targeting mostly the skin cells), which is the major causative factor of several destructive physiological and biological effects, such as immunosuppression, inflammation, DNA damages or initiation of apoptotic processes. In mammalians, approximately 104-106 DNA damages are formed in each cell per day. These DNA damages have to be repaired quickly and precisely, since the improper or delayed repair of the errors can lead to cancerous malformations. Cells have developed several repair mechanisms to eliminate these DNA damages. Nucleotide excision repair (NER) is an exclusive pathway for the recognition and elimination of a wide-range of structurally diverse DNA damages, such as UV-induced cyclobutene-pyrimidine dimers (CPDs), 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs). Since our skin is the first defence layer against exogenous agents, it is the most UV-exposed organ. Two types of malignant skin tumours have been known: melanocytic and non-melanocytic skin cancers (NMSC). NMSC tumour has the highest occurrence rate among the malignant human cancer types in the world. The two most important and common NMSC tumour types are the basal cell carcinoma (BCC) and the squamous cell carcinoma (SCC). SCC tumours are highly invasive tumour types with metastatic predisposition. Although in case of early diagnosis the survival rate increases. The progression of BCC tumours is very slow, and they are just rarely fatal. In case of late diagnosis, they can invade the surrounding tissues and bones, but the occurrence of distant metastasis is infrequent. The BCC is one of the most frequent tumour types in the world. UV radiation strongly initiates the progression of BCC tumours. The serine protease inhibitor superfamily, called Serpins, is one of the largest and functionally the most diverse protein family. According to recent findings, Serpins consists of 1,500 proteins possessing similar domain structure. The role of Serpins has already been described in several processes, such as tumorigenesis, blood clotting, hormone transport, inflammation or immune function. Based on the sequence similarities, the Serpins superfamily is divided into 16 different clades (A-P). SerpinB2 (SPB2) protein was first isolated from human placenta by Uemura et al. in 1970. Initially, the protein was named PAI-2 (Plasminogen activator protein), because it was originally identified as an extracellular inhibitor of the plasminogen activator uPA (urokinase plasminogen activator) and tPA (tissue plasminogen activator) enzymes during pregnancy. Since then, the upregulation of SPB2 mRNA and protein levels has been evaluated in several cell types and it has been also reported that SPB2 is involved in numerous cellular processes, such as signal transduction, inhibition of apoptosis, macrophage survival, monocyte and keratinocyte differentiation, regulation of inflammatory and immune processes. According to the literature, the SPB2 is involved in the progression of various tumour types. Its plasminogen inhibitor function has been described in gastrointestinal, breast and lung cancerous patients. These results suggest that SPB2 could play a role in the regulation of homeostasis upon different kind of injuries, errors, and stress factors. SerpinB10 (SPB10) also known as Bomapin, is a redox-sensitive hematopoietic and myeloid leukaemia specific nuclear protein belonging to the SerpinB family. The protein plays role in the activation of the proliferation and also in the apoptosis induction of myeloid leukaemia cells depending on the presence of specific growth factors. Furthermore, SPB10 mRNA expression is increased in lung cancer patients, while it is decreased in case of breast cancer patients. According to already published data, SerpinB clade is a functionally diverse family. It is assumed that many functions of the SPB clade remain unknown, and it is likely that proteins with a well-characterized function may be involved in other, yet uncharacterized processes

Emerging Roles of Post-Translational Modifications in Nucleotide Excision Repair

Nucleotide excision repair (NER) is a versatile DNA repair pathway which can be activated in response to a broad spectrum of UV-induced DNA damage, such as bulky adducts, including cyclobutane-pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4PPs). Based on the genomic position of the lesion, two sub-pathways can be defined: (I) global genomic NER (GG-NER), involved in the ablation of damage throughout the whole genome regardless of the transcription activity of the damaged DNA locus, and (II) transcription-coupled NER (TC-NER), activated at DNA regions where RNAPII-mediated transcription takes place. These processes are tightly regulated by coordinated mechanisms, including post-translational modifications (PTMs). The fine-tuning modulation of the balance between the proteins, responsible for PTMs, is essential to maintain genome integrity and to prevent tumorigenesis. In this review, apart from the other substantial PTMs (SUMOylation, PARylation) related to NER, we principally focus on reversible ubiquitylation, which involves E3 ubiquitin ligase and deubiquitylase (DUB) enzymes responsible for the spatiotemporally precise regulation of NER

Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA

The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII

DNA double-strand breaks are one of the most deleterious lesions for the cells, therefore understanding the macromolecular interactions of the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. We previously demonstrated that following transcription block, p53 plays a pivotal role in transcription elongation by interacting with S2P RNAPII. In the current study, we reveal that p53 is involved in the fine-tune regulation of S2P RNAPII ubiquitylation. Furthermore, we emphasize the potential role of p53 in delaying the premature ubiquitylation and the subsequent chromatin removal of S2P RNAPII as a response to transcription block

Application of Lacunarity for Quantification of Single Molecule Localization Microscopy Images

The quantitative analysis of datasets achieved by single molecule localization microscopy is vital for studying the structure of subcellular organizations. Cluster analysis has emerged as a multi-faceted tool in the structural analysis of localization datasets. However, the results it produces greatly depend on the set parameters, and the process can be computationally intensive. Here we present a new approach for structural analysis using lacunarity. Unlike cluster analysis, lacunarity can be calculated quickly while providing definitive information about the structure of the localizations. Using simulated data, we demonstrate how lacunarity results can be interpreted. We use these interpretations to compare our lacunarity analysis with our previous cluster analysis-based results in the field of DNA repair, showing the new algorithm’s efficiency

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a “zero set” of potential reference gene candidates

Drosophila as a new tool to study the chromatin structural changes activated by DNA damages

In eukaryotic cells, any processes which involve DNA have to take place in the context of chromatin structure, which affects the probability of the dam-aging agents to cause DNA breaks and the recruit-ment of the repair proteins. The improper repair or persistence of breaks leads to genome instability, which could result in tumor formation. Our goal is to understand what makes cells able to recognize the appearance of DNA break and how the chromatin structure could change around the break. The an-swers to these questions will provide information on whether specific chromatin structures predispose sites for DNA break and whether memory of previ-ous break is retained in the chromatin structure. We started to setup human cell culture-based and Drosophila experimental systems by which we could study how unique histone post-translation-al modifications (PTMs) could affect the DNA repair. We take advantage of the model system where we delete the endogenous histone cluster and we substitute it with mutant histones which permits or mimics unique histone PTMs. We have already started to mutate histone genes and screen 50 different histone PTMs. We will use these flies to check the DNA repair kinetics in those animals which consist of only the mutated histones. Using the Drosophila and the human cell culture based system we have already identified new H3 and H4 histone PTM candidates that play role in chromo-somal rearrangement which could influence the DNA repair processes. The system developed in our laboratory would help in understanding the mecha-nisms, which give rise to frequent chromosomal break points often detected in tumors. Progress in integrating the chromatin dimension in DNA repair will help to understand how DNA damage may impact on genome stability. These results would also help identifying new key targets in DNA dam-age repair and the final goal of the project is to find potential biomarkers which could be used in anti-cancer therapies. Supported by OTKA-PD [112118] and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences