International Journal of Molecular Sciences

Chemoproteomic analysis of the BET inhibitors, JQ1, AZD5153, and I-BET151, identified an extremely large signature of ubiquitin modified proteins associating in vitro with a recombinant BRD4 N-terminal protein fragment. The identified proteins included those with known functions in BRD4 complexes for transcriptional and epigenetic control (acetylated histones, the MED complex, BAF complex, RNA pol II transcription complexes, and chromatin-associated complexes). The BRD4 interactome in response to BET inhibitors is suggested to be orchestrated by compound-specific differential actions of up to 16 E3 ligases, 4 deubiquitinase enzymes, and 51 accessory proteins of the ubiquitin proteasome system (UPS). The UPS response of BET inhibition also involves proteins necessary for Myc enhancer binding and Myc response gene expression. A large cohort of UPS substrates commonly responsive to JQ1 and AZD5153 treatments suggests the existence of distinct mechanisms, one involving compound-activated UPS proteins, and another via their direct actions on BRD4. The findings raise the intriguing possibility that UPS triggers promoting proteostasis changes to the BRD4 interactome may be mechanistically coupled with BRD4 function in a proximity-dependent, chromatin-associated manner. Consequently, BET inhibitors and their downstream effects present highly complex environments which may lead to polypharmacology, the phenotypic outcomes or overall clinical benefits of which are hard to assess. However, many new targets and small molecule combinations suggested in this study may afford a path forward for narrowly and more selectively targeting Myc in the clinic with potentially cleaner profiles compared with BET inhibitors or BRD4 as target.

DNA repair pathways are critical processes that need both spatial and temporal fine regulation. Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions, while excluding non-interacting components. Proteins disordered domains, as well as RNA, favor condensation to modulate this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses. In this context, it is emerging that RNA-processing pathways and PARylation events, through the addition of an ADP-ribose moiety to both proteins and DNA, participate in different aspects of the DNA Damage Response (DDR). Remarkably, defects in these regulatory connections are associated with genomic instability and human pathologies. In addition, it has been recently noticed that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway of non-distorting DNA lesions, bearing additional non-canonical DNA-repair functions associated with processes coping with RNA metabolism. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in several tumorigenic and amyloidogenic events. This work represents a paradigmatical pipeline for evaluating the relevance of LLPS in DDR. Statement of significanceIn this work, we aimed to test the hypothesis of an involvement of phase-separation in regulating the molecular mechanisms of the multifunctional enzyme APE1 starting from the analysis of its recently-characterized protein-protein interactome (PPI). We compared APE1-PPI to phase-separation databases and we performed functional enrichment analysis, uncovering links between APE1 and already known demixing factors, establishing an association with liquidliquid phase separation. This analysis could represent a starting point for implementing downstream experimental validations, using in vitro and in vivo approaches, to assess actual demixing.

UNC45B, a UCS class myosin chaperone that facilitates Hsp90-myosin interactions, plays a crucial role in early myofibrillogenesis, though its specific function sustaining cardiac contractility remains obscure. Here we identified seven non-synonymous (p.L486F, p.C487Y, p.A492T, p.D496Y, p.R721Q, p.A780V and p.C786Y) and two synonymous (p.Y769= and p.A780=) variants in UNC45B through whole-exome sequencing (WES) and subsequent genetic screening of Dilated Cardiomyopathy (DCM) patients by Sanger sequencing. The majority of the variants (77.7%) are localized within the highly conserved UCS domain of UNC45B, a critical region for chaperone-like property, also known for myosin binding site. In-vitro analysis by immunofluorescence revealed cytoplasmic mislocalization of mutant UNC45B and pronounced hypertrophic morphology, most evident in p.R721Q and p.A780V mutants. Nuclear fragmentation, a hallmark of cellular stress and apoptosis, was also prominently detected in these variants. Complementary ultrastructural analysis by TEM further substantiated these observations, revealing nuclear deformation, increased cytoplasmic vacuolation, and marked chromatin disorganization. To assess the downstream molecular consequences, qRT-PCR was performed which revealed robust up-regulation of hypertrophic markers (Nppa, Mef2c, Myh6, Myh7, Actc1, Ttn, Nfatc1, and Nfatc2), with Nppa and Mef2c showing >12-fold induction in p.R721Q, p.A780V and p.C786Y variants. Additionally, an increased apoptosis marker Bax/Bcl2 ratio, particularly in p.R721Q (=27.6) and p.A780V (=18.3), confirmed activation of apoptotic signalling pathways. Additionally, 2D and 3D in-silico modeling revealed notable conformational changes which strongly corroborate in-vitro findings. Collectively, these variants weaken the structure and function of UNC45B, causing disrupted sarcomeric organization, pronounced cellular hypertrophy, and subsequent activation of apoptotic pathways leading to cardiac dysfunction.

BackgroundPlacentae of women with preeclampsia (PE) exhibit reduced levels of kynurenine (Kyn), a biological compound derived from tryptophan metabolism with antioxidant, vasorelaxant, and hypotensive properties. Little is known regarding functional levels of the Kyn metabolizing enzymes (KYNME) in women with preeclampsia. Since high circulating levels of syncytiotrophoblast extracellular vesicles (STB-EVs) have been associated with preeclampsia onset, we aimed to study whether Kyn reduction in preeclampsia may be attributed to increased degradation by KYNME present in STB-EVs. MethodsWe conducted a study that included women with normal (n=9) and early-onset preeclamptic (EOPE) pregnancies (n=9). From them, STB-EVs were isolated by dual-lobe placental perfusions from normal (n=3) and EOPE (n=3). KYNME were identified using placental immunohistochemistry and western blot in placental and STB-EV extractions. Serum Kyn levels were measured using gas chromatography mass spectrometry. ResultsCargo of STB-EVs consist of functional KYNME, which break down Kyn in a dose and time-dependent manner. No significant differences in the content of Kyn metabolizing enzymes were found in STB-EVs between normal and EOPE pregnancies. However, decreased serum levels of Kyn were found in women with EOPE relative to normal pregnancies. ConclusionSTB-EVs carry functional KYNME and may regulate the levels of circulating Kyn during normal pregnancy and preeclampsia. Due to the dose effect of increased STB-EVs in EOPE, the functional KYNME content of said vesicles may contribute to reduced levels of Kyn. This finding opens a new avenue for investigating the potential benefits of KYNME inhibitors in conjunction with kynurenine replacement for managing preeclampsia.

Mitofusin 2 (MFN2), a key outer mitochondrial membrane GTPase, regulates mitochondrial fusion, mitophagy, calcium homeostasis, and cellular bioenergetics. This study investigated the role of MFN2 variants in patients with Dilated Cardiomyopathy (DCM) using whole-exome sequencing (WES) of 5 familial and 10 sporadic DCM cases. A rare de-novo MFN2 variant, c.932A>G (p. N311S), was identified in a DCM patient, which is absent in 100 healthy controls as well as in the 1000 Genomes, IndiGenomes, databases while it shows very low MAF (0.0000081) in gnomAD. Structural modelling predicted the variant to be highly deleterious and revealed marked conformational distortion of the mutant protein (RMSD = 8.95 A). Molecular docking further showed a weakened interaction between MFN2-N311S and PRKN (Parkin), indicating impaired mitophagy and defective mitochondrial quality control. Moreover, functional analysis in stable H9c2 cardiomyoblast cell lines demonstrated significantly reduced MFN2 mutant protein expression, extensive mitochondrial clustering and fragmentation. The mutant protein also indicated significant reduction in mitochondrial membrane potential, ATP production, and oxygen consumption rate (OCR), together with elevated cytosolic Ca2+ and reactive oxygen species (ROS) levels. qRT-PCR analysis further revealed activation of the PI3K/AKT/mTOR signalling pathway and increased expression of hypertrophic markers Myh6, Nppa, Nfatc1, and Nfatc2. The above findings collectively highlight the significant impact of the MFN2 mutation on mitochondrial dynamics and cellular health, suggesting a significant correlation with the pathogenesis of DCM. This finding could further open a door to develop a potential therapeutic target for DCM.

IntroductionRibosomal (rDNA), satellite III (f-SatIII) and telomere (TR) tandem repeats perform a variety of functions in the human cell. Copy number variations (CNVs) of these repeats contributes to the global chromatin architecture and genome expression changes in response to stress or pathology. An elevated rDNA abundance and lowered contents of f-SatIII and TR were found in blood leukocytes of patients with schizophrenia. The main question of the study was: are the CNVs of the three repeat types in blood leukocytes a universal phenomenon linked to a patho4logy associated with chronic oxidative stress? Cystic fibrosis (CF), a monogenic disease was chosen as an object of the study. Materials and MethodsWe determined the rDNA, f-SatIII and TR content in the blood leukocyte genomes of 545 subjects aged 0.2 to 50 years. The subjects were divided into three groups: Control (HC-group, N = 267), CF group (N= 186) and CF(d) group (severe patients, who died after some time upon sampling, aged 17 to 40 years, N = 92). For each patient, the type of the mutation in CFTR gene had been determined earlier. Non-radioactive quantitative hybridization technique was applied to quantify the repeats. Resultsthe rDNA abundance was elevated in the DNA of CF and CF(d) groups (565{+/-}105 copies per genome, N=278) compared to HC group (445{+/-}112 copies, N=267). A patients age 3 to 16 years was associated with "severe" mutations in CFTR (98% of cases), increased f-SatIII repeat counts and decreased telomere repeat (TR) contents in genome DNA compared to the age-matched controls. The genomes of deceased patients from CF(d) group also harbored increased numbers of f-SatIII and decreased numbers of TR. Patients above 16 years with a milder course of the disease and relatively low content of "severe" CFTR mutations contained less f-SatIII and more TR in their genomic DNA. A parameter rDNA{middle dot}(f-SatIII/TR) showed a maximum difference between patients with relatively mild (age 17 to 40) and severe (age 17 to 40) forms of the pathology according to ROC analysis data (AUC = 0.86). ConclusionCystic fibrosis was associated with an increase in rDNA abundance and altered f-SatIII and TR contents in the DNA of cases compared to the controls. The severe course of the disease was characterized with high f-SatIII contents and shortened telomeres. Whereas mild CF cases were associated with low contents of f-SatIII and normal or slightly reduced telomere length. The index rDNA{middle dot} (f-SatIII/TR) might be a predictor of the patients life expectancy.

SummaryFabry disease is a lysosomal disease due to -galactosidase A (a-GalA) deficiency. Since 2001, Enzyme replacement therapy (ERT) has been used as specific treatment of Fabry disease, with variable effects depending on patient gender and affected organs. In Fabry cells, the endososomal/lysosomal system is highly altered. Consequently, the exogenous enzyme may be mistargeted and trapped into intracellular vesicles instead of reaching the lysosomes. In this study, we aimed to investigate the mechanisms underlying the processing of the exogenous enzyme. We used Fabry cells (cultured fibroblasts and podocyte cell line) to study the enzyme internalization and its effects on the catabolism of the main a-Gal A substrate, globotriaosylceramide (Gb3), upon autophagy inhibition. The exogenous enzyme reaches the early endosome in a similar timeframe in Fabry and control cells, while its targeting to lysosomes is delayed in Fabry cells. Gb3 concentration is lowered upon therapeutic enzyme addition or wortmannin treatment with a synergetic effect. These findings illustrate the positive impact of autophagy inhibition on enzyme trafficking and processing, allowing the increase of functional enzyme rate within the lysosome. Given the high cost of ERT, a better understanding of the cellular fate of the exogenous enzyme may lead to improve its targeting to the lysosome.

Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with poor prognosis and limited therapies. To elucidate its post-transcriptional regulation, ceRNA networks were reconstructed from TCGA-ACC and GTEx 2025 data. Tumor networks exhibited compact topology and tumor-specific hub miRNAs (miR-466, miR-940, miR-507), which were downregulated, inversely correlated with oncogenic transcripts, and associated with adverse survival. Candidate targets were identified through dual validation by miRTarBase and miRDB, further confirmed with TargetScan, and filtered for inverse correlation, significant upregulation in tumors, and treatment-response association. The intersection of these stringent layers yielded four robust oncogenic biomarkers; CKS2, ERP44, ERG28, and FAM32A that were consistently upregulated and annotated to key processes: chromatin remodeling and cell cycle control (CKS2), apoptosis regulation (FAM32A), ER proteostasis and redox balance (ERP44), and sterol biosynthesis (ERG28). Network analysis highlighted CKS2 as central PPI hubs. Prognostic modeling demonstrated strong survival divergence, with CKS2 conferring the highest risk (HR = 4.49, p = 1.1e-06), while integration of the four-gene panel achieved near-perfect classification accuracy (AUC = 0.99). Collectively, these findings define CKS2, ERP44, ERG28, and FAM32A as high-confidence biomarkers in ACC, derived through multi-layered overlap filtering, and underscore their diagnostic and prognostic relevance.

Atrial fibrillation (AF) increases energy demand in atrial myocytes, yet the mitochondrial mechanisms underlying this stress remain poorly defined. Using previously published proteomic data from left atrial tissue of AF and sham-operated goats, we performed organelle-specific bioinformatic analyses of the mitochondrial fraction. Over-representation and consensus pathway analyses consistently highlighted enrichment of oxidative phosphorylation (OXPHOS) subunits. Gene set enrichment and network analyses implicated Heat Shock Protein Family A Member 9 (HSPA9) as a potentially central regulatory hub coordinating the dysregulation of Complex I and III subunits, with 69% of regulatory relationships showing pathway concordance. These results indicate a coordinated, system-wide mitochondrial adaptation in AF, integrating energy production, proteostasis, and respiratory chain regulation.

Mutations in LRRK2 are the most common genetic cause of Parkinsons disease. Despite substantial research efforts, the physiological and pathological role of this multidomain protein remains poorly defined. In this study, we used a systematic approach to construct the general protein-protein interactome around LRRK2, which was then differentiated into 15 tissue-specific interactomes taking into consideration the differential expression patterns and the co-expression behaviours of the LRRK2 interactors in different healthy tissues. The LRRK2 interactors exhibited distinct expression features in the brain as compared to the peripheral tissues analysed. Moreover, a high degree of similarity was found for the LRRK2 interactors in putamen, caudate and nucleus accumbens, thus defining a potential LRRK2 functional cluster within the striatum. We also explored the functions highlighted by the "core LRRK2 interactors" within each tissue and illustrated how the LRRK2 interactomes can be used as a tool to trace the relationship between LRRK2 and specific interactors of interest, here exemplified with a study focused on the LRRK2 interactors belonging to the Rab protein family.

Cell-adhesion molecules (CAM) are membrane proteins responsible for cell-cell interactions or cell-extracellular matrix interactions. Among these proteins, claudins (CLDN), occludin (OCLN), and junctional adhesion molecules (JAM) are components of the tight junction (TJ), the single proteic structure tasked with safeguarding the paracellular space. The TJ is responsible for controlled permeability of blood-tissue barriers, regulating the passage of molecule passage by size and charge. Currently there is no translational solution to manipulate the TJ with the exception of Focused Ultra-sound (FUS) and Micro bubbling (MB) techniques, still in clinical trials. Here we describe the expression of TJ proteins in the outer membrane of E. coli and report its consequences. When expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). The adhesion properties of the aggregates are representative of the individual membrane proteins expressed. This method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows the high-throughput interrogation of small-molecules influence on paracellular permeability, enabling for the first time the discovery of its modulators for therapeutic strategies.

Drug Discovery is a lengthy and costly process and has faced a period of declining productivity within the last two decades. As a consequence, integrative data-driven approaches are nowadays on the rise in pharmaceutical research, making use of an inter-connected (network) view on diseases. In addition, evidence-based decisions are alleviated by studying the time evolution of innovation trends in drug discovery.\n\nIn this paper a new approach leveraging data mining and data integration for inspecting target innovation trends protein family-wise is presented. The study highlights protein families which are receiving emerging interest in the drug discovery community (mainly kinases and G protein coupled receptors) and those with areas of interest in target space that have just emerged in the scientific literature (mainly kinases and transporters) highlighting novel opportunities for drug intervention.\n\nIn order to delineate the evolution of target-driven research interest from a biological perspective, trends in biological process annotations from Gene Ontology (GO) and disease annotations from DisGeNet for major target families are captured. The analysis reveals an increasing interest in targets related to immune system processes, and a recurrent trend for targets involved in circulatory system processes. At the level of disease annotations, targets associated to e.g., cancer-related pathologies as well as to intellectual disability and schizophrenia are increasingly investigated nowadays.\n\nCan this knowledge be used to study the \"movement of targets\" in a network view and unravel new links between diseases and biological processes? We tackled this question by creating dynamic network representations considering data from different time periods. The dynamic network for immune system process-associated targets suggest that e.g. breast cancer as well as schizophrenia are linked to the same targets (cannabinoid receptor CB2 and VEGFR2) thus suggesting similar treatment options which could be confirmed by literature search. The methodology has the potential to identify other drug repurposing candidates and enables researchers to capture trends in research attention in target space at an early stage.\n\nThe KNIME workflows and R scripts used in this study are publicly available from https://github.com/BZdrazil/Moving_Targets.\n\nAuthor summaryIn this study we have investigated target innovation in drug discovery over a period of 22 years (1995-2016) by extracting time trends of research interest (as published in the scientific literature and stored in the ChEMBL database) in certain protein classes inspecting different measures (numbers of pharmacological measurements, targets, papers, and drugs). Focusing on the most relevant protein classes in drug discovery (G protein-coupled receptors, kinases, ion channels, nuclear receptors, proteases, and transporters), we further linked single targets to Gene Ontology (GO) biological process annotations and inspected steep increasing or decreasing trends of GO annotations within target families over time. We also tracked trends in disease annotations from DisGeNET by filtering out diseases linked to targets with emerging trends in research interest. Finally, targets, GO terms, and diseases are interconnected in network representations and shifts in research foci are investigated over time. This new methodology which utilizes data mapping and data analysis can be used to explore trends in research attention target family-wise, to uncover previously unknown links between diseases and biological processes and to identify potential candidates for drug repurposing.

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

The CB1 cannabinoid receptor is implicated in a broad range of physiological processes and disease states, however CB1-targeting drugs in clinical use remain based on tetrahydrocannabinol (THC). Ligands that exhibit functional selectivity for different intracellular signalling pathways are currently an area of rapid development, and hold significant potential as therapeutic agents. Improved understanding of the exact molecular mechanisms underpinning biased activation of intracellular effector proteins for CB1 is necessary to enable drug development of biased CB1 ligands into candidates for treatment of human disease. Using molecular dynamics, this study shows that CB1conformations resulting from activation by the orthosteric ligands CP55940, {Delta}9-THC or 5F-MDMB-PICA exhibit differences in the dynamic organisation of key residues and receptor substructures involved in coupling to G proteins and {beta}-arrestins, that leads to selective activation of downstream signalling pathways. The identification of conformationally distinct CB1-agonist complexes that demonstrate different functional profiles provides an important step in unravelling the molecular determinants for biased signalling, and lays a platform for future rational design of novel therapeutic leads.

The limitations of virus-specific antiviral drugs became apparent during the current COVID-19 pandemic. The search for broad range antiviral proteins of a new kind to answer current and future pandemics has become an even more pressing matter. Here, the author further describes the expected anti-SARS-CoV-2 mechanisms of a novel broad range antiviral chimeric protein constructed between ricin A chain and pokeweed antiviral proteins. The latest in protein-ligand docking software were used to determine binding affinity of RTA-PAPs to SARS-CoV-2 frameshift stimulation element and elucidate the preferential post-infection entry mechanisms of RTA-PAPs into virus infected cells over non-infected ones, by doing a comparative analysis between in vitro and in silico results on numerous viruses. The results obtained strongly suggest that the post-infection preferential entry of RTA-PAPs into infected cells is mediated by the presence of viroporins integrated into the host cell membrane. The discovery of this mechanism revealed RTA-PAPs, and proteins like them, to be a new class of broad range antivirals that target with high specificity viroporin producing viruses, and with gain of functions in antiviral activities, post-infection.

Integrating transcriptome, translatome, and proteome data remains challenging because changes in mRNA, ribosome occupancy, and protein abundance do not always occur simultaneously. To address this, we applied tensor-decomposition-based unsupervised feature extraction to a tri-omics dataset generated under branched-chain amino acid starvation. The three layers were organized into a tensor and analyzed to extract singular value vectors representing coordinated variation across omics, conditions, and replicates. This approach distinguished patterns consistent with ribosome stacking, in which the transcriptome and translatome increase while the proteome decreases, from those of translational buffering, in which the proteome remains stable despite variations in upstream layers. Using components associated with these signatures, we selected 1,781 genes related mainly to reduced translational efficiency and 221 related to buffer-ing. Functional interpretation via enrichment analysis, supported by generative artificial intelligence and a manual literature review, revealed six major biological units: genome replication and maintenance, extracellular matrix remodeling, mitochondrial biogenesis and oxidative phosphorylation, proteostasis and secretion, vesicle transport and signal integration, and epigenetic/RNA regulation. These results indicate that tensor decomposition can effectively integrate triomics data and uncover the biologically meaningful gene clusters and mechanisms underlying cell fate transitions. This framework is valuable for interpreting complex multilayer regulation beyond conventional pairwise analyses.

STUDY QUESTIONIs Tcte1 mutation causative for male infertility? SUMMARY ANSWERCollected data underline the complex and devastating effect of the single-gene mutation on testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADYLatest data revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity, and is built from DRC1-DRC7 proteins. DRC5 (TCTE1) - one of N-DRC element, has been already reported as a candidate for abnormal sperm flagella beating, however, only in restricted manner with no clear explanation of respective observations. STUDY DESIGN, SIZE, DURATIONUsing CRISPR/Cas9 genome editing technique, mouse knockout line of Tcte1 gene was created on the basis of C57Bl/6J strain. Then, the mouse reproductive potential, semen characteristics, testicular gene expression level, sperm ATP and testis apoptosis level measurements have been performed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n=248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS, SETTING, METHODSTo check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild type (WT - control group), heterozygous Tcte1+/-, and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) has been done for mice testis tissues. STRING interactions have been checked for protein-protein interactions, based on changed expression level of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the ATP amount in spermatozoa, the luminescence level was measured. Also, immunofluorescent in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n=137 non-obstructive azoospermia or cryptozoospermia, n=111 samples with spectrum of oligoasthenoteratozoospermia, including n=47 with asthenozoospermia) has been extracted to perform genomic sequencing (WGS, WES or Sanger). Protein prediction modeling of human identified variants and the exon 3 structure deleted in mouse knockout has been also performed. MAIN RESULTS AND THE ROLE OF CHANCENo progeny at all was found for homozygous males with revealed oligoasthenoteratozoospermia, while heterozygous animals (fertile) manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing, linked with apoptosis, or spermatogenesis. In Tcte1-/- males the protein revealed only residual amounts in sperm head nucleus, and was not transported to sperm flagella, as other N-DRC components. Decreased ATP level (2.4-fold lower) was found in spermatozoa of homozygous mice, together with disturbed tail:midpiece ratio, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at similar level in all three mouse genotypes. Mutation screening of human infertile males revealed 1 novel and 5 ultrarare heterogeneous variants (predicted as disease causing) in 6.05% of patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus, suggesting the disrupted TCTE1 interaction with its binding partners located within the axoneme. What does this mean for patients?Abnormal semen parameters (sperm count, motility and morphology) are known as one of the first symptoms that may be related to male fertility problems. This study aimed to determine the role of Tcte1 in male infertility using mouse knockout model. Tcte1 protein is one of the structural elements building N-DRC, a complex within an axoneme that is responsible for coordination of the sperm flagella elements activity, strictly related to sperm motility. We have found that mutations in mouse Tcte1 knockout model revealed two phenotypes, dependent on zygosity: infertile oligoasthenoteratozoospermic homozygotes, and fertile oligozoospermic heterozygotic males, suggesting haploinsufficiency mechanism. Pilot study on human samples with TCTE1 variants revealed a wide spectra of semen quality (from non-obstructive azoospermia, via cryptozoospermia, to severe oligoasthenozoospermia). Thus, TCTE1 gene is the next one that should be added to the male infertility list because of its crucial role in spermatogenesis influencing the variety of testicular molecular networks (incl. energy machinery processing) and proper sperm function.

DNA double-strand breaks are the most deleterious lesions for the cells, therefore understanding the macromolecular interactions in 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. Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) are the two main regulatory kinases of homologous recombination and non-homologous end joining, respectively. Although these kinases are involved in the activation of different repair pathways, they have common target proteins, such as P53. We previously demonstrated that following transcription block, P53 plays a pivotal role in transcription elongation process by interacting with S2P RNAPII. In the current study, we reveal that P53, ATM and DNAPK are involved in the fine-tune regulation of the ubiquitin-proteasome system-related degradation of S2P RNAPII. However, they act differently in this process: P53 delays the removal of S2P RNAPII, while ATM and DNAPK participate in the activation of members of E3 ligase complexes involved in the ubiquitylation of S2P RNAPII. We also demonstrate that WW domain-containing protein 2 (WWP2) and Cullin-3 (CUL3) are interaction partners of S2P RNAPII, thus forming a complex with the transcribing RNAPII complex. Simple SummaryTo ensure the proper repair following DNA double-strand breaks, the eviction of the arrested elongating RNA polymerase II (S2P RNAPII) is required. Here, we report an emerging role of P53, Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) in the ubiquitin-proteasome system-dependent removal of S2P RNAPII. We also identified interactions between S2P RNAPII and WW domain-containing protein 2 (WWP2) or Cullin-3 (CUL3) (members of E3 ligase complexes), which are involved in the ubiquitylation of S2P RNAPII following DNA damage. Furthermore, the RNAPII-E3 ligase complex interactions are mediated by P53, ATM and DNAPK, which suggests potential participation of all three proteins in the effective resolution of transcription block at the damage site. Altogether, our results provide a better comprehension of the molecular background of transcription elongation block-related DNA repair processes and highlight an indispensable function of P53, ATM and DNAPK in these mechanisms.

BackgroundFacioscapulohumeral muscular dystrophy type 2 (FSHD2) poses a significant challenge within the domain of neuromuscular disorders, marked by a progressive decline in muscle strength accompanied by tissue wasting. FSHD2 results from chromosomal deletions triggering the activation of a dormant gene known as DUX4. While DUX4 typically regulates early embryonic development, its activation in adult muscle cells leads to premature cell death. Despite this understanding, the exact pathology of FSHD2 remains unclear. To date, no effective treatment for FSHD2 exists. MethodWe acquired single-cell RNA sequencing (RNA-Seq) data (GSE143452) from primary myoblasts for FSHD2 from the United States National Institutes of Health (NIH) portal website. Our analysis encompassed a comprehensive examination of differentially expressed genes, alongside associated compounds sourced from the Chemical Entities of Biological Interest (ChEBI) database. Employing rigorous statistical methods, we pinpointed the most prominently upregulated and downregulated genes. Subsequently, we determined the compounds capable of modulating the expression of these top genes, either enhancing or reducing their activity. ResultsBisphenol S (BPS) can upregulate 52 of 100 top downregulated genes in FSHD2 without downregulating any other genes and Bisphenol F (BPF) can upregulate 45 of 100 downregulated genes with downregulating only one other gene. The enrichment analysis of both sets of 52 genes related to BPS and 45 genes corresponding to BPF highlights their significant involvement in various aspects of muscle biology, particularly as pertaining to the function and dysfunction of cardiac and skeletal muscle. ConclusionsLeveraging single-cell RNA-Seq data and computational analysis, we identified key dysregulated genes in FSHD2 and elucidated their modulation by compounds such as BPS and BPF. While effective treatments for FSHD2 remain elusive, our study provides valuable insights into potential therapeutic targets and pathways for further investigation in the pursuit of effective interventions for this debilitating condition. However, more research is needed to understand whether the roles of BPS and F are constructive or destructive.

Overactive bladder (OAB) is a urological symptom complex defined by urinary urgency. It can have a devastating impact on an individuals quality of life and leads to significant financial cost. Insulin-like growth factor 1 (IGF-1) is a protein hormone involved in a broad range of processes including cell proliferation and differentiation. IGF-1 is also regulated through alternative splicing. While the primary IGF-1Ea transcript is highly expressed in liver, the alternative IGF-1Ec transcript encodes the proteolytically-derived MGF peptide and has been primarily studied in skeletal muscle. MGF has been shown to stimulate satellite cell proliferation following tissue mechanical stretch or injury, but the role of MGF in smooth muscle, such as the detrusor muscle of the bladder, has been little explored. The aim of this study was to explore the expression of MGF in bladder biopsies from patients with OAB and age-matched controls. We show using immunohistochemistry that MGF is widely expressed in bladder tissue. Quantification of MGF expression by western blot showed that average MGF expression is more than doubled in OAB biopsies compared to controls (mean MGF in OAB=0.51{+/-}0.1, n=23; mean MGF in controls=0.22{+/-}0.07, n=9; p=0.05). Furthermore, there is an inverse correlation between MGF protein levels and symptom severity, as determined by the urodynamic parameter maximum cystometric capacity (correlation=0.53, p=0.03 n=16). MGF expression was highest in OAB biopsies with strong expression of the muscle cell marker DES. Combined with our observation that MGF induces cell proliferation in primary bladder cultures, our data suggests that high MGF expression in OAB patients may represent an attempted protective response in the bladder.