Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair.

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/- ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.

Polysaccharides from Basella alba Protect Post-Mitotic Neurons against Cell Cycle Re-Entry and Apoptosis Induced by the Amyloid-Beta Peptide by Blocking Sonic Hedgehog Expression.

The amyloid-beta peptide (Aß) is the neurotoxic component in senile plaques of Alzheimer's disease (AD) brains. Previously we have reported that Aß toxicity is mediated by the induction of sonic hedgehog (SHH) to trigger cell cycle re-entry (CCR) and apoptosis in post-mitotic neurons. Basella alba is a vegetable whose polysaccharides carry immunomodulatory and anti-cancer actions, but their protective effects against neurodegeneration have never been reported. Herein, we tested whether polysaccharides derived from Basella alba (PPV-6) may inhibit Aß toxicity and explored its underlying mechanisms. In differentiated rat cortical neurons, Aß25-35 reduced cell viability, damaged neuronal structure, and compromised mitochondrial bioenergetic functions, all of which were recovered by PPV-6. Immunocytochemistry and western blotting revealed that Aß25-35-mediated induction of cell cycle markers including cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) in differentiated neurons was all suppressed by PPV-6, along with mitigation of caspase-3 cleavage. Further studies revealed that PPV-6 inhibited Aß25-35 induction of SHH; indeed, PPV-6 was capable of suppressing neuronal CCR and apoptosis triggered by the exogenous N-terminal fragment of sonic hedgehog (SHH-N). Our findings demonstrated that, in the fully differentiated neurons, PPV-6 exerts protective actions against Aß neurotoxicity via the downregulation of SHH to suppress neuronal CCR and apoptosis.

Targeting proliferating cell nuclear antigen (PCNA) for cancer therapy.

Proliferating cell nuclear antigen (PCNA) is an essential scaffold protein in many cellular processes. It is best known for its role as a DNA sliding clamp and processivity factor during DNA replication, which has been extensively reviewed by others. However, the importance of PCNA extends beyond its DNA-associated functions in DNA replication, chromatin remodelling, DNA repair and DNA damage tolerance (DDT), as new non-canonical roles of PCNA in the cytosol have recently been identified. These include roles in the regulation of immune evasion, apoptosis, metabolism, and cellular signalling. The diverse roles of PCNA are largely mediated by its myriad protein interactions, and its centrality to cellular processes makes PCNA a valid therapeutic anticancer target. PCNA is expressed in all cells and plays an essential role in normal cellular homeostasis; therefore, the main challenge in targeting PCNA is to selectively kill cancer cells while avoiding unacceptable toxicity to healthy cells. This chapter focuses on the stress-related roles of PCNA, and how targeting these PCNA roles can be exploited in cancer therapy.

Structural Basis for the Interaction Between Yeast Chromatin Assembly Factor 1 and Proliferating Cell Nuclear Antigen.

Proliferating cell nuclear antigen (PCNA), the homotrimeric eukaryotic sliding clamp protein, recruits and coordinates the activities of a multitude of proteins that function on DNA at the replication fork. Chromatin assembly factor 1 (CAF-1), one such protein, is a histone chaperone that deposits histone proteins onto DNA immediately following replication. The interaction between CAF-1 and PCNA is essential for proper nucleosome assembly at silenced genomic regions. Most proteins that bind PCNA contain a PCNA-interacting peptide (PIP) motif, a conserved motif containing only eight amino acids. Precisely how PCNA is able to discriminate between binding partners at the replication fork using only these small motifs remains unclear. Yeast CAF-1 contains a PIP motif on its largest subunit, Cac1. We solved the crystal structure of the PIP motif of CAF-1 bound to PCNA using a new strategy to produce stoichiometric quantities of one PIP motif bound to each monomer of PCNA. The PIP motif of CAF-1 binds to the hydrophobic pocket on the front face of PCNA in a similar manner to most known PIP-PCNA interactions. However, several amino acids immediately flanking either side of the PIP motif bind the IDCL or C-terminus of PCNA, as observed for only a couple other known PIP-PCNA interactions. Furthermore, mutational analysis suggests positively charged amino acids in these flanking regions are responsible for the low micromolar affinity of CAF-1 for PCNA, whereas the presence of a negative charge upstream of the PIP prevents a more robust interaction with PCNA. These results provide additional evidence that positive charges within PIP-flanking regions of PCNA-interacting proteins are crucial for specificity and affinity of their recruitment to PCNA at the replication fork.

Immunohistochemical Analysis of Dentigerous Cysts and Odontogenic Keratocysts Associated with Impacted Third Molars-A Systematic Review.

OBJECTIVE: This systematic review investigates the diagnostic, prognostic, and therapeutic implications of immunohistochemical markers in dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) associated with impacted third molars. MATERIALS AND METHODS: A comprehensive search strategy was employed across major databases including MEDLINE/PubMed, EMBASE, and Web of Science, from the inception of the databases to March 2024. Keywords and Medical Subject Heading (MeSH) terms such as "dentigerous cysts", "odontogenic keratocysts", "immunohistochemistry", "Ki-67", and "p53" were used. The PRISMA 2020 guidelines were followed to ensure methodological rigor. Inclusion criteria encompassed studies on humans and animals providing definitive diagnoses or specific signs and symptoms related to DCs and OKCs, with results on protein expression derived from immunohistochemistry, immune antibody, proteomics, or protein expression methods. RESULTS: Of the 159 studies initially identified, 138 met the inclusion criteria. Our analysis highlighted significantly higher expressions of Ki-67 (22.1% ± 4.7 vs. 10.5% ± 3.2, p < 0.001), p53 (15.3% ± 3.6 vs. 5.2% ± 1.9, p < 0.001), and Bcl-2 (18.4% ± 3.2 vs. 8.7% ± 2.4, p < 0.001) in OKCs compared to DCs, indicating a higher proliferative index, increased cellular stress, and enhanced anti-apoptotic mechanisms in OKCs. Additionally, PCNA levels were higher in OKCs (25.6% ± 4.5 vs. 12.3% ± 3.1, p < 0.001). Genetic mutations, particularly in the PTCH1 gene, were frequently observed in OKCs, underscoring their aggressive behavior and potential malignancy. CONCLUSIONS: The findings emphasize the significant role of immunohistochemical markers in distinguishing between DCs and OKCs, with elevated levels of Ki-67, p53, Bcl-2, and PCNA in OKCs suggesting a higher potential for growth and recurrence. Genetic insights, including PTCH1 mutations, further support the need for personalized treatment approaches. These markers enhance diagnostic accuracy and inform targeted therapeutic strategies, potentially transforming patient management in oral and maxillofacial surgery.

Insights into microstructure and expression of markers of proliferation, apoptosis and T cells in the spleen of cattle egret (Bubulcus ibis).

The spleen is the largest secondary lymphoid organ with significant roles in pathogen clearance. It is involved in several avian diseases. The cattle egret is a wild insectivorous bird of agricultural and socioeconomic importance. Data related to microstructural features of cattle egret spleen are lacking. The present study investigated the gross anatomical, histological and immunohistochemical characteristics of the cattle egret spleen. Proliferation (PCNA and PHH3), apoptosis (cleaved caspase 3, C.CASP3) and T-cell (CD3 and CD8) markers were assessed. Grossly, the spleen appeared brownish red, oval-shaped and located at the oesophago-proventricular junction. Histologically, the spleen was surrounded by a thin capsule sending a number of trabeculae which contained branches of the splenic vessels. The white pulp consisted of the periarteriolar lymphoid sheath and periellipsoidal lymphatic sheath (PELS). The red pulp was formed of sinusoids and cords. The penicillar capillaries, which represent the terminal segments of the splenic arterial tree were highly branched, wrapped by prominent ellipsoids and directly connected to the splenic sinusoids, suggesting a closed type of circulation. Immunohistochemically, proliferating cell nuclear antigen (PCNA)-expressing cells were distributed with high counts throughout the splenic parenchyma, being highest within the splenic cords and PELS. Both PHH3- and C.CASP3-expressing cells revealed a similar pattern to that of PCNA, although with fewer counts. Large numbers of T cells were observed throughout the splenic parenchyma, mainly within the cords, as revealed by CD3 and CD8 immunoreaction. The present study provides a clear insight into the precise structure of the spleen in cattle egrets and thus improves our understanding about birds' immunity.

PCNA molecular recognition of different PIP motifs: Role of Tyr211 phosphorylation.

The coordination of enzymes and regulatory proteins for eukaryotic DNA replication and repair is largely achieved by Proliferating Cell Nuclear Antigen (PCNA), a toroidal homotrimeric protein that embraces the DNA duplex. Many proteins bind PCNA through a conserved sequence known as the PCNA interacting protein motif (PIP). PCNA is further regulated by different post-translational modifications. Phosphorylation at residue Y211 facilitates unlocking stalled replication forks to bypass DNA damage repair processes but increasing nucleotide misincorporation. We explore here how phosphorylation at Y211 affects PCNA recognition of the canonical PIP sequences of the regulatory proteins p21 and p15, which bind with nM and µM affinity, respectively. For that purpose, we have prepared PCNA with p-carboxymethyl-L-phenylalanine (pCMF, a mimetic of phosphorylated tyrosine) at position 211. We have also characterized PCNA binding to the non-canonical PIP sequence of the catalytic subunit of DNA polymerase δ (p125), and to the canonical PIP sequence of the enzyme ubiquitin specific peptidase 29 (USP29) which deubiquitinates PCNA. Our results show that Tyr211 phosphorylation has little effect on the molecular recognition of p21 and p15, and that the PIP sequences of p125 and USP29 bind to the same site on PCNA as other PIP sequences, but with very low affinity.

The roles of TonEBP in the DNA damage response: From DNA damage bypass to R-loop resolution.

Tonicity-responsive enhancer binding protein (TonEBP) is a stress-responsive protein that plays a critical role in the regulation of gene expression and cellular adaptation to stressful environments. Recent studies uncovered the novel role of TonEBP in the DNA damage response, which significantly impacts genomic stability. This review provides a comprehensive overview of the novel role of TonEBP in DNA damage repair, including its involvement in the DNA damage bypass pathway and the recognition and resolution of DNA damage-induced R-loop structures.

Corrigendum: Expression of constitutive fusion of ubiquitin to PCNA restores the level of immunoglobulin A/T mutations during somatic hypermutation in the Ramos cell line.

[This corrects the article DOI: 10.3389/fimmu.2022.871766.].

Evaluation of Phototoxicity of Short-Wavelength Laser Light Utilizing PCNA Accumulation.

In recent years, diseases such as age-related macular degeneration and retinal pigment degeneration caused by excessive exposure to short-wavelength visible light have become significant concerns. With the aim of quantitatively evaluating the toxicity of short-wavelength light, proliferating cell nuclear antigen (PCNA) accumulation at the irradiation site was investigated using live cell imaging techniques to irradiate individual living cells with short-wavelength laser light. By examining the dependency of PCNA accumulation on the irradiation site within the cells and their cell cycle, it was observed that PCNA accumulation occurred only when the cell nucleus of cells in the S phase of the cell cycle was irradiated. We investigated the accumulation of PCNA at the laser irradiation site using laser light at wavelengths of 405 nm and 375 nm, with intensities ranging from 0.5 µW to 9.0 µW. The results confirmed an increase in PCNA accumulation with increasing intensity, and a higher accumulation was observed with laser light irradiation at a wavelength of 375 nm compared to 405 nm. By comparing the PCNA accumulation and 24 h cell viability, we demonstrated the feasibility of quantitatively assessing laser light toxicity through the measurement of PCNA accumulation.

Excessive proliferating cell nuclear antigen attenuates endometrial adhesive capacity and decidualization in patients with recurrent implantation failure.

STUDY QUESTION: Does the expression of proliferating cell nuclear antigen (PCNA) in the endometrium regulate endometrial receptivity in patients with recurrent implantation failure (RIF)? SUMMARY ANSWER: A high abundance of PCNA attenuates endometrial adhesive capacity and decidualization in patients with RIF. WHAT IS KNOWN ALREADY: Aberrant expression of PCNA has been discovered in multiple infertility-related disorders. However, the expression pattern and role of PCNA in the establishment of endometrial receptivity and endometrial decidualization in patients with RIF remain unclear. STUDY DESIGN, SIZE, DURATION: We analysed the expression of PCNA in mid-secretory endometrial tissues from 24 patients with RIF and 24 healthy women. Additionally, PCNA expression levels were measured in proliferative and mid-secretory phase endometrial tissue samples from women with regular menstrual cycles and in decidual tissue samples taken from ten women during normal early pregnancy (n = 10 per phase for each group). The function and regulatory mechanisms of PCNA in endometrial adhesive capacity and endometrial decidualization were investigated using BeWo spheroids, Ishikawa cells, and human endometrial stromal cells (HESCs). PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression of PCNA in mid-secretory endometrial tissues of patients with RIF and women with normal endometrium and in endometrial tissue at different stages of the menstrual cycle and in decidualized tissues was analysed by RT-qPCR, western blot, and immunohistochemistry staining (IHC). Furthermore, the number of BeWo spheroids directly attached to the Ishikawa cell monolayers, and the potential molecular mechanisms involved, were compared between cells overexpressing PCNA and a control group. Additionally, the effect and regulatory mechanisms of PCNA on the decidualization of HESCs in vitro were investigated. MAIN RESULTS AND THE ROLE OF CHANCE: Our findings indicated that the abundance of PCNA was dramatically greater in mid-secretory endometrial tissues from patients with RIF than in those from women with healthy endometrium. The expression of PCNA increased in the proliferative phase of the menstrual cycle but decreased gradually in the mid-secretory phase and in decidual tissues. Interestingly, PCNA was expressed in both human endometrial epithelial cells (HEECs) and HESCs. In Ishikawa cells, PCNA overexpression dramatically reduced the endometrial adhesive capacity by inhibiting the expression of adhesion molecules (E-cadherin and integrin ß3) and activating the FAK/paxillin signalling pathway. Furthermore, in HESCs, PCNA overexpression attenuated endometrial decidualization by activating the AKT/ß-catenin signalling pathway and increasing tight junctions between cells by upregulating ZO-1 and occludin expression. In addition, PCNA-ELAVL1 interactions were confirmed by coimmunoprecipitation in decidualized HESCs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The functional analysis of PCNA was limited by the number of human endometrial tissues. A larger sample size is required to further explore the potential roles of PCNA during embryo implantation. Moreover, the present results should be taken with caution, as only a few of the embryos that were transferred in RIF patients population underwent preimplantation genetic testing for embryonic chromosome aneuploidies (PGT-A), despite embryo ploidy testing being significant in the diagnosis of unexplained RIF. WIDER IMPLICATIONS OF THESE FINDINGS: High PCNA expression attenuates endometrial adhesive capacity and decidualization in patients with RIF. These findings provide new insights into the potential mechanisms underlying the occurrence of implantation failure. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (82101698), Shandong Provincial Natural Science Foundation (ZR2021MH012), and the Science and Technology Plan of Yantai (2023YD021 and 2022YD031). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Comparison of Proliferating Cell Nuclear Antigen (PCNA) Expression among Nasal Polyp and Chronic Rhinosinusitis.

BACKGROUND AND OBJECTIVE: Chronic rhinosinusitis is a common inflammatory disorder in sinonasal mucosa that could be developed with or without nasal polyps. Cellular proliferation is suggested as a possible mechanism of nasal polyp development. However, conducted studies in this context are limited. So, the present study's aim is the comparison of Proliferating cell nuclear antigen (PCNA) expression in nasal polyps and chronic rhinosinusitis. MATERIALS AND METHODS: In this cross-sectional study, 70 nasal polyp and 60 chronic rhinosinusitis samples from patients referred to Mostafa Khomeini Hospital, Tehran from 2017 to 2022 were immunohistochemically stained by PCNA marker. The percentage of PCNA nuclear expression was determined in two groups and its association with the type of pathological lesion and the patient's age and sex was analyzed by SPSS statistic software version 24 statistical software (IBM Statistics, USA). RESULTS: The mean expression of PCNA in nasal polyp and chronic rhinosinusitis samples was 16.55% ±â€¯13.66 and 17.58% ±â€¯12.68 respectively (ranging from 0 to 57% in both groups) however, there was no significant statistical difference between the two groups (p = 0.479). No relationship was found between PCNA expression with age and sex in none of the chronic rhinosinusitis and nasal polyp groups. CONCLUSION: Proliferative activity of the nasal epithelial cell is similar in chronic rhinosinusitis with and without nasal polyps and it is considered that the increase of epithelial cell proliferative activity probably has no role in nasal polyp development in patients with chronic rhinosinusitis.

Reno-protective effect of nicorandil and pentoxifylline against potassium dichromate-induced acute renal injury via modulation p38MAPK/Nrf2/HO-1 and Notch1/TLR4/NF-κB signaling pathways.

BACKGROUND: Occupational and environmental exposure to chromium compounds such as potassium dichromate (PDC) (K2Cr2O7) has emerged as a potential aetiologic cause for renal disease through apoptotic, and inflammatory reactions. The known potent antioxidants such as nicorandil (NIC) and/or pentoxifylline (PTX) were studied for their possible nephroprotective effect in PDC-treated rats. METHODS: Forty male Wistar rats were divided into five groups; control, PDC group, NIC+PDC, PTX+PDC group, and combination+PDC group. Nephrotoxicity was evaluated histopathologically and biochemically. Invasive blood pressure, renal function parameters urea, creatinine, uric acid and albumin, glomerular filtration rate markers Cys-C, Kim-1 and NGAL, inflammatory markers IL-1ß, IL-6, TNF-α, TGF-ß, COX-II, p38MAPK, NF-κB and TLR4, oxidative stress SOD, GSH, MDA, MPO, HO-1 and Nrf2 and apoptotic mediators Notch1 and PCNA were evaluated. Besides, renal cortical histopathology was assayed as well. RESULTS: PDC led to a considerable increase in indicators for kidney injury, renal function parameters, invasive blood pressure, oxidative stress, and inflammatory markers. They were markedly reduced by coadministration of PDC with either/or NIC and PTX. The NIC and PTX combination regimen showed a more significant improvement than either medication used alone. Our results demonstrated the nephroprotective effect of NIC, PTX, and their combined regimen on PDC-induced kidney injury through suppression of oxidative stress, apoptosis, and inflammatory response. CONCLUSION: Renal recovery from PDC injury was achieved through enhanced MAPK/Nrf2/HO-1 and suppressed Notch1/TLR4/NF-κB signaling pathways. This study highlights the role of NIC and PTX as effective interventions to ameliorate nephrotoxicity in patients undergoing PDC toxicity.

The bacterial DNA sliding clamp, ß-clamp: structure, interactions, dynamics and drug discovery.

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.

DNAJC24 acts directly with PCNA and promotes malignant progression of LUAD by activating phosphorylation of AKT.

Heat shock proteins (HSPs) are a group of highly conserved proteins found in a wide range of organisms. In recent years, members of the HSP family were overexpressed in various tumors and widely involved in oncogenesis, tumor development, and therapeutic resistance. In our previous study, DNAJC24, a member of the DNAJ/HSP40 family of HSPs, was found to be closely associated with the malignant phenotype of hepatocellular carcinoma. However, its relationship with other malignancies needs to be further explored. Herein, we demonstrated that DNAJC24 exhibited upregulated expression in LUAD tissue samples and predicted poor survival in LUAD patients. The upregulation of DNAJC24 expression promoted proliferation and invasion of LUAD cells in A549 and NCI-H1299 cell lines. Further studies revealed that DNAJC24 could regulate the PI3K/AKT signaling pathway by affecting AKT phosphorylation. In addition, a series of experiments such as Co-IP and mass spectrometry confirmed that DNAJC24 could directly interact with PCNA and promoted the malignant phenotypic transformation of LUAD. In conclusion, our results suggested that DNAJC24 played an important role in the progression of LUAD and may serve as a specific prognostic biomarker for LUAD patients. The DNAJC24/PCNA/AKT axis may be a potential target for future individualized and precise treatment of LUAD patients.

Adaptive use of error-prone DNA polymerases provides flexibility in genome replication during tumorigenesis.

Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics.

EIF3B stabilizes PCNA by counteracting SYVN1-mediated ubiquitination to serve as a promotor in cholangiocarcinoma.

Cholangiocarcinoma, a prevalent hepatic malignancy, exhibits a progressively rising incidence. While Eukaryotic translation initiation factor 3 subunit B (EIF3B) has been implicated in the occurrence and development of various cancers, its specific roles in cholangiocarcinoma remain unexplored. Immunohistochemical (IHC) analysis was employed to detect EIF3B/PCNA expression in cholangiocarcinoma. Cells were manipulated using short hairpin RNA (shRNA)-mediated lentiviruses or overexpression plasmids. Statistical significance was assessed using the Student's t-test and one-way ANOVA, with P < 0.05 considered statistically significant. EIF3B exhibited robust expression in cholangiocarcinoma, demonstrating a significant correlation with the pathological grade of cholangiocarcinoma patients. Furthermore, modulation of EIF3B expression, either depletion or elevation, demonstrated the ability to inhibit or enhance cholangiocarcinoma cell survival and migration in vitro. Mechanistically, we identified Proliferating Cell Nuclear Antigen (PCNA) as a downstream gene of EIF3B, driving cholangiocarcinoma. EIF3B stabilized PCNA by inhibiting PCNA ubiquitination, a process mediated by E3 ligase SYVN1. Similar to EIF3B, PCNA levels were also abundant in cholangiocarcinoma, and knocking down PCNA impeded cholangiocarcinoma development. Intriguingly, silencing PCNA attenuated the promotion induced by EIF3B overexpression. Furthermore, the elevated P21 protein level in shEIF3B RBE cells was partially attenuated after UC2288 (P21 signaling pathway inhibitor) treatment. Our findings underscored the potential of EIF3B as a therapeutic target for cholangiocarcinoma. Unraveling its functions holds promise for the development of more specific and effective targeted therapy strategies.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

Srs2 binding to PCNA and its sumoylation contribute to RPA antagonism during the DNA damage response.

Activation of the DNA damage checkpoint upon genotoxin treatment induces a multitude of cellular changes, such as cell cycle arrest, to cope with genome stress. After prolonged genotoxin treatment, the checkpoint can be downregulated to allow cell cycle and growth resumption. In yeast, downregulation of the DNA damage checkpoint requires the Srs2 DNA helicase, which removes the ssDNA binding complex RPA and the associated Mec1 checkpoint kinase from DNA, thus dampening Mec1 activation. However, it is unclear whether the 'anti-checkpoint' role of Srs2 is temporally and spatially regulated to both allow timely checkpoint termination and to prevent superfluous RPA removal. Here we address this question by examining regulatory elements of Srs2, including its phosphorylation, sumoylation, and protein-interaction sites. Our genetic analyses and checkpoint level assessment suggest that the RPA countering role of Srs2 is promoted by Srs2 binding to PCNA, which is known to recruit Srs2 to subsets of ssDNA regions. RPA antagonism is further fostered by Srs2 sumoylation, which we found depends on the Srs2-PCNA interaction. Srs2 sumoylation is additionally reliant on Mec1 and peaks after Mec1 activity reaches maximal levels. Collectively, our data provide evidence for a two-step model wherein checkpoint downregulation is facilitated by PCNA-mediated Srs2 recruitment to ssDNA-RPA filaments and the subsequent Srs2 sumoylation stimulated upon Mec1 hyperactivation. We propose that this mechanism allows Mec1 hyperactivation to trigger checkpoint recovery.

Modulatory effects of Moringa oleifera leaf extract on sodium nitrate-induced experimental colitis via regulation of P53, Ki-67 and PCNA biomarkers.

BACKGROUND: Ulcerative colitis is a risk factor for colorectal carcinoma. Different mechanisms are related to colitis like apoptosis and hyperproliferation. Moringa oleifera leaves extract (MO) provides a promising option to overcome the risk. PURPOSE: To examine the colonic changes in a rat model of colitis induced by sodium nitrate (SN) and study the effects of MO. STUDY DESIGN: Eight adult male rats were allocated in each of the three group; control (distilled water), SN (100 mg/kg/day, orally via gastric gavage), and SN + MO (100 mg/kg/day, orally via gastric gavage). METHODS: Body weight was measured after the end of the experiment. Colonic homogenates were tested for levels of oxidative stress indicators. Immunohistochemistry for P53, PCNA and Ki-67 was performed. Fresh colon specimens were used for quantitative real-time PCR for assessment of P53, PCNA and Ki-67 gene expression. RESULTS: SN group revealed a significant decreased weight (p = 0.002). MDA and NO levels were higher with SN administration than with MO co-administration (p= 0.04, 0.01 respectively). GSH level was reduced in SN group (p = 0.02) and significantly increased with MO intake (p = 0.04). SN-induced colonic destructive changes were reversed with MO. P53, PCNA and Ki-67 levels of gene expression were reduced in SN + MO group than SN group (P = 0.007, 0.02, 0.001 respectively). CONCLUSION: MO protected the colonic mucosa against SN-induced changes regulating apoptosis, and cell proliferation.