Bafilomycin A1 Molecular Effect on ATPase Activity of Subcellular Fraction of Human Colorectal Cancer and Rat Liver.

Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.

LAFITE Reveals the Complexity of Transcript Isoforms in Subcellular Fractions.

Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA-sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low-abundance Aware Full-length Isoform clusTEr (LAFITE), to comprehensively analyze the full-length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full-length transcripts, particularly for low-abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3'-UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full-length isoform identification and enables a high-resolution view of the RNA landscape at the isoform level.

Subcellular partitioning of protein kinase activity revealed by functional kinome profiling.

Protein kinases and their substrates form signaling networks partitioned across subcellular compartments to facilitate critical biological processes. While the subcellular roles of many individual kinases have been elucidated, a comprehensive assessment of the synaptic subkinome is lacking. Further, most studies of kinases focus on transcript, protein, and/or phospho-protein expression levels, providing an indirect measure of protein kinase activity. Prior work suggests that gene expression levels are not a good predictor of protein function. Thus, we assessed global serine/threonine protein kinase activity profiles in synaptosomal, nuclear, and cytosolic fractions from rat frontal cortex homogenate using peptide arrays. Comparisons made between fractions demonstrated differences in overall protein kinase activity. Upstream kinase analysis revealed a list of cognate kinases that were enriched in the synaptosomal fraction compared to the nuclear fraction. We identified many kinases in the synaptic fraction previously implicated in this compartment, while also identifying other kinases with little or no evidence for synaptic localization. Our results show the feasibility of assessing subcellular fractions with peptide activity arrays, as well as suggesting compartment specific activity profiles associated with established and novel kinases.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

Bioaccumulation, Detoxification, and Biological Macromolecular Damage of Benzo[a]pyrene in Exposure in Tissues and Subcellular Fractions of Scallop Chlamys farreri.

Because of the persistence and high toxicity of benzo[a]pyrene (B[a]P), the bioaccumulation and detoxification mechanisms of B[a]P have been studied extensively at the tissue level; but the data at the subcellular level in bivalves have not been reported. The present study was conducted to investigate the effects of B[a]P exposure on bioaccumulation, detoxification, and biomacromolecular damage in gills, digestive glands, and their subcellular fractions of the scallop Chlamys farreri. The subcellular fraction contains cytoplasm, mitochondria, microsome, nucleus, cell membrane, and overall organelle. The results demonstrated that B[a]P accumulation showed a clear time-dose effect. Based on the time-dependent accumulation of B[a]P in subcellular fractions, we speculated that the intracellular migration order of B[a]P was cell membrane, organelle, and nucleus in turn. Considering the difference of B[a]P accumulation may be related to B[a]P metabolism, we have further confirmed that the activities of B[a]P metabolizing enzymes in scallop tissues and subcellular fractions were significantly tempted by B[a]P (p < 0.05), including 7-ethoxyresorufin O-deethylase (increased), glutathione-S-transferase (GST; decreased), and superoxide dismutase (increased). First, GST was detected in bivalve cytoplasm and microsome. Second, B[a]P exposure also caused biomacromolecules damage. The results demonstrated that mitochondria and microsome were more vulnerable to lipid peroxidation than cell membrane and nucleus. Taken together, the present study fills some of the gaps in our knowledge of the bioaccumulation and detoxification mechanisms of C. farreri exposed to B[a]P in subcellular fractions and deeply explores the transportation and the main metabolic and damage sites of polycyclic aromatic hydrocarbons (PAHs) in cells, which helped us to comprehensively understand the toxic mechanism of PAHs on bivalves. Environ Toxicol Chem 2022;41:2353-2364. © 2022 SETAC.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF-ERK1/2 signaling pathways.

A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF-ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.

PSL-LCCL: a resource for subcellular protein localization in liver cancer cell line SK_HEP1.

The characterization of subcellular protein localization provides a basis for further understanding cellular behaviors. A delineation of subcellular localization of proteins on cytosolic membrane-bound organelles in human liver cancer cell lines (hLCCLs) has yet to be performed. To obtain its proteome-wide view, we isolated and enriched six cytosolic membrane-bound organelles in one of the hLCCLs (SK_HEP1) and quantified their proteins using mass spectrometry. The vigorous selection of marker proteins and a machine-learning-based algorithm were implemented to localize proteins at cluster and neighborhood levels. We validated the performance of the proposed method by comparing the predicted subcellular protein localization with publicly available resources. The profiles enabled investigating the correlation of protein domains with their subcellular localization and colocalization of protein complex members. A subcellular proteome database for SK_HEP1, including (i) the subcellular protein localization and (ii) the subcellular locations of protein complex members and their interactions, was constructed. Our research provides resources for further research on hLCCLs proteomics. Database URL:  http://www.igenetics.org.cn/project/PSL-LCCL/.

Discovery of an Amino Acid-Modified Near-Infrared Aza-BODIPY Photosensitizer as an Immune Initiator for Potent Photodynamic Therapy in Melanoma.

The discovery of novel photosensitizers with potent phototoxicity and desirable water solubility is an urgent task for photodynamic therapy. Herein, a series of amino acid-modified aza-BODIPY photosensitizers were synthesized and evaluated. These new PSs exhibited enhanced aqueous solubility, increased 1O2 generation efficiency, and an improved photo-dark toxicity ratio. Aspartic acid-modified PS of 1a, which possessed intense NIR absorption and high 1O2 quantum yield, demonstrated the most potent efficacy toward the investigated tumor cell lines without using an emulsifier. Subcellular localization, cell-based ROS production, and cell death pathway of 1a were studied. In vivo fluorescence imaging and ex vivo organ distribution assays manifested that 1a possessed reasonable distribution and clearance. In vivo PDT studies indicated that 1a revealed advantages over Ce6 and our previously optimized PS of BDP-4. It not only afforded an excellent PDT effect with a low drug dose under only single-time photoirradiation but also induced an antitumor immunological response.

Genome-Wide Identification and Characterization of the RCI2 Gene Family in Allotetraploid Brassica napus Compared with Its Diploid Progenitors.

Brassica napus and its diploid progenitors (B. rapa and B. oleracea) are suitable for studying the problems associated with polyploidization. As an important anti-stress protein, RCI2 proteins widely exist in various tissues of plants, and are crucial to plant growth, development, and stress response. In this study, the RCI2 gene family was comprehensively identified and analyzed, and 9, 9, and 24 RCI2 genes were identified in B. rapa, B. oleracea, and B. napus, respectively. Phylogenetic analysis showed that all of the identified RCI2 genes were divided into two groups, and further divided into three subgroups. Ka/Ks analysis showed that most of the identified RCI2 genes underwent a purifying selection after the duplication events. Moreover, gene structure analysis showed that the structure of RCI2 genes is largely conserved during polyploidization. The promoters of the RCI2 genes in B. napus contained more cis-acting elements, which were mainly involved in plant development and growth, plant hormone response, and stress responses. Thus, B. napus might have potential advantages in some biological aspects. In addition, the changes of RCI2 genes during polyploidization were also discussed from the aspects of gene number, gene structure, gene relative location, and gene expression, which can provide reference for future polyploidization analysis.

Overexpression of the Aldehyde Dehydrogenase Gene ZmALDH Confers Aluminum Tolerance in Arabidopsis thaliana.

Aluminum (Al) toxicity is the main factor limiting plant growth and the yield of cereal crops in acidic soils. Al-induced oxidative stress could lead to the excessive accumulation of reactive oxygen species (ROS) and aldehydes in plants. Aldehyde dehydrogenase (ALDH) genes, which play an important role in detoxification of aldehydes when exposed to abiotic stress, have been identified in most species. However, little is known about the function of this gene family in the response to Al stress. Here, we identified an ALDH gene in maize, ZmALDH, involved in protection against Al-induced oxidative stress. Al stress up-regulated ZmALDH expression in both the roots and leaves. The expression of ZmALDH only responded to Al toxicity but not to other stresses including low pH and other metals. The heterologous overexpression of ZmALDH in Arabidopsis increased Al tolerance by promoting the ascorbate-glutathione cycle, increasing the transcript levels of antioxidant enzyme genes as well as the activities of their products, reducing MDA, and increasing free proline synthesis. The overexpression of ZmALDH also reduced Al accumulation in roots. Taken together, these findings suggest that ZmALDH participates in Al-induced oxidative stress and Al accumulation in roots, conferring Al tolerance in transgenic Arabidopsis.

Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX.

Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.

Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation.

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

Mitochondria-Targeted Photoactivatable Real-Time Monitoring of a Controlled Drug Delivery Platform.

The current anticancer therapies are limited by their lack of controlled spatiotemporal release at the target site of action. We report a novel drug delivery platform that provides on-demand, real-time, organelle-specific drug release and monitoring upon photoactivation. The system is comprised of a model anticancer drug doxorubicin, an alkyltriphenylphosphonium moiety to target mitochondria in cancer cells, and a hydroxycinnamate photoactivatable linker that is covalently attached to the drug and mitochondria-targeting moieties such that it can be phototriggered by either UV (one-photon) or NIR (two-photon) light to form a fluorescent coumarin product and facilitate the release of drug payload. The extent of drug release is quantified by the fluorescence intensity of the coumarin formed. Further, the photoactivatable prodrug accumulates in the mitochondria and shows light-triggered temporally controlled cell death. In the future, our platform can be tuned for any biological application of interest, offering immense value in biomedicine.

Label-free multiplexed microtomography of endogenous subcellular dynamics using generalizable deep learning.

Simultaneous imaging of various facets of intact biological systems across multiple spatiotemporal scales is a long-standing goal in biology and medicine, for which progress is hindered by limits of conventional imaging modalities. Here we propose using the refractive index (RI), an intrinsic quantity governing light-matter interaction, as a means for such measurement. We show that major endogenous subcellular structures, which are conventionally accessed via exogenous fluorescence labelling, are encoded in three-dimensional (3D) RI tomograms. We decode this information in a data-driven manner, with a deep learning-based model that infers multiple 3D fluorescence tomograms from RI measurements of the corresponding subcellular targets, thereby achieving multiplexed microtomography. This approach, called RI2FL for refractive index to fluorescence, inherits the advantages of both high-specificity fluorescence imaging and label-free RI imaging. Importantly, full 3D modelling of absolute and unbiased RI improves generalization, such that the approach is applicable to a broad range of new samples without retraining to facilitate immediate applicability. The performance, reliability and scalability of this technology are extensively characterized, and its various applications within single-cell profiling at unprecedented scales (which can generate new experimentally testable hypotheses) are demonstrated.

Relevance of BRAF Subcellular Localization and Its Interaction with KRAS and KIT Mutations in Skin Melanoma.

Although skin melanoma (SKM) represents only one-quarter of newly diagnosed skin malignant tumors, it presents a high mortality rate. Hence, new prognostic and therapeutic tools need to be developed. This study focused on investigating the prognostic value of the subcellular expression of BRAF, KRAS, and KIT in SKM in correlation with their gene-encoding interactions. In silico analysis of the abovementioned gene interactions, along with their mRNA expression, was conducted, and the results were validated at the protein level using immunohistochemical (IHC) stains. For IHC expression, the encoded protein expressions were checked on 96 consecutive SKMs and 30 nevi. The UALCAN database showed no prognostic value for the mRNA expression level of KRAS and BRAF and demonstrated a longer survival for patients with low mRNA expression of KIT in SKMs. IHC examinations of SKMs confirmed the UALCAN data and showed that KIT expression was inversely correlated with ulceration, Breslow index, mitotic rate, and pT stage. KRAS expression was also found to be inversely correlated with ulceration and perineural invasion. When the subcellular expression of BRAF protein was recorded (nuclear vs. cytoplasmatic vs. mixed nucleus + cytoplasm), a direct correlation was emphasized between nuclear positivity and lymphovascular or perineural invasion. The independent prognostic value was demonstrated for mixed expression of the BRAF protein in SKM. BRAF cytoplasmic predominance, in association with KIT's IHC positivity, was more frequently observed in early-stage nonulcerated SKMs, which displayed a low mitotic rate and a late death event. The present study firstly verified the possible prognostic value of BRAF subcellular localization in SKMs. A low mRNA expression or IHC cytoplasmic positivity for KIT and BRAF might be used as a positive prognostic parameter of SKM. SKM's BRAF nuclear positivity needs to be evaluated in further studies as a possible indicator of perineural and lymphovascular invasion.

Hsa-circ_0058106 induces EMT and metastasis in laryngeal cancer via sponging miR-153 and inducing Twist1 nuclear translocation.

PURPOSE: A new circular RNA (hsa-circ_0058106) has been found to be upregulated in laryngeal cancer, but its function remains unknown. Here, we explored its role in the metastasis of laryngeal cancer. METHODS: The level of hsa-circ_0058106 in laryngeal cancer was detected by qRT-PCR. The effect of hsa-circ_0058106 silencing on the metastasis of laryngeal cancer was assessed using scratch wound healing and transwell assays, as well as nude mouse lung metastasis models. Fluorescence in situ hybridization was employed to analyze the cellular localization of hsa-circ_0058106. A luciferase activity assay was used to assess binding between hsa-circ_0058106 and miR-153. Interaction between hsa-circ_0058106 and Twist1 was confirmed using RNA pull-down and RNA immunoprecipitation assays. RESULTS: A high level of hsa-circ_0058106 was found to be associated with lymph node metastasis and advanced clinical stages. Stable knockdown of hsa-circ_0058106 inhibited the migration and invasion of laryngeal cancer cells in vitro and in vivo. Moreover, we found that downregulation of hsa-circ_0058106 suppressed epithelial-mesenchymal transition (EMT), which was underscored by the observation that hsa-circ_0058106 silencing led to decreases in the expression of N-cadherin and Vimentin, and an increase in the expression of E-cadherin. Mechanistically, we found that hsa-circ_0058106 can specifically bind to miR-153 and regulate Snail1 expression by acting as a miR-153 sponge. In addition, we found that knockdown of hsa-circ_0058106 blocked the nuclear translocation of Twist1. CONCLUSIONS: Our results indicate that hsa-circ_0058106 induces EMT and metastasis in laryngeal cancer by sponging miR-153 and inducing Twist1 nuclear translocation. These observations provide new insights into the regulatory effects of circRNAs in laryngeal cancer metastasis.

Characterization of subcellular localization of eukaryotic clamp loader/unloader and its regulatory mechanism.

Proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity clamp for eukaryotic DNA polymerases and a binding platform for many DNA replication and repair proteins. The enzymatic activities of PCNA loading and unloading have been studied extensively in vitro. However, the subcellular locations of PCNA loaders, replication complex C (RFC) and CTF18-RFC-like-complex (RLC), and PCNA unloader ATAD5-RLC remain elusive, and the role of their subunits RFC2-5 is unknown. Here we used protein fractionation to determine the subcellular localization of RFC and RLCs and affinity purification to find molecular requirements for the newly defined location. All RFC/RLC proteins were detected in the nuclease-resistant pellet fraction. RFC1 and ATAD5 were not detected in the non-ionic detergent-soluble and nuclease-susceptible chromatin fractions, independent of cell cycle or exogenous DNA damage. We found that small RFC proteins contribute to maintaining protein levels of the RFC/RLCs. RFC1, ATAD5, and RFC4 co-immunoprecipitated with lamina-associated polypeptide 2 (LAP2) α which regulates intranuclear lamin A/C. LAP2α knockout consistently reduced detection of RFC/RLCs in the pellet fraction, while marginally affecting total protein levels. Our findings strongly suggest that PCNA-mediated DNA transaction occurs through regulatory machinery associated with nuclear structures, such as the nuclear matrix.

Physicochemical and structural characterization, epitope mapping and vaccine potential investigation of a new protein containing Tetratrico Peptide Repeats of Acinetobacter baumannii: An in-silico and in-vivo approach.

Acinetobacter baumannii is an opportunistic multidrug-resistant pathogen that causes a significant mortality rate. The proteins containing Tetratrico Peptide Repeats (TPRs) are involved in the pathogenicity and virulence of bacteria and have different roles such as transfer of bacterial virulence factors to host cells, binding to the host cells and inhibition of phagolysosomal maturation. So, in this study, physicochemical properties of a new protein containing TPRs in A. baumannii which was named PcTPRs1 by this study were characterized and its 3D structure was predicted by in-silico tools. The protein B and T cell epitopes were mapped and its vaccine potential was in-silico and in-vivo investigated. Domain analysis indicated that the protein contains the Flp pilus assembly protein TadD domain which has three TPRs. The helix is dominant in the protein structure, and this protein is an outer membrane antigen which, is extremely conserved among A. baumannii strains; thus, has good properties to be applied as a recombinant vaccine. The best-predicted and refined model was applied in ligand-binding sites and conformational epitopes prediction. Based on epitope mapping results, several epitopes were characterized which could stimulate both immune systems. BLAST results showed the introduced epitopes are completely conserved among A. baumannii strains. The in-vivo analysis indicates that a 101 amino acid fragment of the protein which contains the best selected epitope, can produce a good protectivity against A. baumannii as well as the whole TPR protein and thus could be investigated as an effective subunit and potential vaccines.

p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway.

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53ß, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53ß and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.

A new candidate oncogenic lncRNA derived from pseudogene WFDC21P promotes tumor progression in gastric cancer.

As oncogenes and tumor suppressor genes, long non-coding RNAs (lncRNAs) regulate the biological behavior of gastric cancer (GC) cells such as proliferation, invasion, and metastasis through various signal pathways. At present, although numerous lncRNAs that significantly influence the development and progression of GC have been identified, a considerable number of them have not been found and studied yet. In this study, we identified a new lncRNA derived from pseudogenes WFDC21P, which have not been reported in any previous GC study. LncRNA WFDC21P was significantly upregulated in GC cells and tissues, and clinically associated with the pathological stages of advanced GC. WFDC21P promoted proliferation and metastasis of GC cells both in vitro and in vivo. LncRNA WFDC21P was directly bound to GTPase Ran and it promoted the activity of the Akt/GSK3ß/ß-catenin pathway. Forkhead Box P3 (FOXP3), as a transcription factor of WFDC21P, was directly bound to the promoter region and it positively regulated the transcription of WFDC21P. This finding may provide a novel biomarker and therapeutic target for GC.