A Comparative Molecular Dynamics Study of Selected Point Mutations in the Shwachman–Bodian–Diamond Syndrome Protein SBDS

The Shwachman–Diamond Syndrome (SDS) is an autosomal recessive disease whose majority of patients display mutations in a ribosome assembly protein named Shwachman–Bodian–Diamond Syndrome protein (SBDS). A specific therapy for treating this rare disease is missing, due to the lack of knowledge of the molecular mechanisms responsible for its pathogenesis. Starting from the observation that SBDS single-point mutations, localized in different domains of the proteins, are responsible for an SDS phenotype, we carried out the first comparative Molecular Dynamics simulations on three SBDS mutants, namely R19Q, R126T and I212T. The obtained 450-ns long trajectories were compared with those returned by both the open and closed forms of wild type SBDS and strongly indicated that two distinct conformations (open and closed) are both necessary for the proper SBDS function, in full agreement with recent experimental observations. Our study supports the hypothesis that the SBDS function is governed by an allosteric mechanism involving domains I and III and provides new insights into SDS pathogenesis, thus offering a possible starting point for a specific therapeutic option.     

Extracellular Vesicles: Messengers of p53 in Tumor–Stroma Communication and Cancer Metastasis

Tumor progression to a metastatic and ultimately lethal stage relies on a tumor-supporting microenvironment that is generated by reciprocal communication between tumor and stromal host cells. The tumor–stroma crosstalk is instructed by the genetic alterations of the tumor cells—the most frequent being mutations in the gene Tumor protein p53 (TP53) that are clinically correlated with metastasis, drug resistance and poor patient survival. The crucial mediators of tumor–stroma communication are tumor-derived extracellular vesicles (EVs), in particular exosomes, which operate both locally within the primary tumor and in distant organs, at pre-metastatic niches as the future sites of metastasis. Here, we review how wild-type and mutant p53 proteins control the secretion, size, and especially the RNA and protein cargo of tumor-derived EVs. We highlight how EVs extend the cell-autonomous tumor suppressive activity of wild-type p53 into the tumor microenvironment (TME), and how mutant p53 proteins switch EVs into oncogenic messengers that reprogram tumor–host communication within the entire organism so as to promote metastatic tumor cell dissemination.     

Cytoplasmic p53β Isoforms Are Associated with Worse Disease-Free Survival in Breast Cancer

TP53 mutations are associated with tumour progression, resistance to therapy and poor prognosis. However, in breast cancer, TP53′s overall mutation frequency is lower than expected (~25%), suggesting that other mechanisms may be responsible for the disruption of this critical tumour suppressor. p53 isoforms are known to enhance or disrupt p53 pathway activity in cell- and context-specific manners. Our previous study revealed that p53 isoform mRNA expression correlates with clinicopathological features and survival in breast cancer and may account for the dysregulation of the p53 pathway in the absence of TP53 mutations. Hence, in this study, the protein expression of p53 isoforms, transactivation domain p53 (TAp53), p53β, Δ40p53, Δ133p53 and Δ160p53 was analysed using immunohistochemistry in a cohort of invasive ductal carcinomas (n = 108). p53 isoforms presented distinct cellular localisation, with some isoforms being expressed in tumour cells and others in infiltrating immune cells. Moreover, high levels of p53β, most likely to be N-terminally truncated β variants, were significantly associated with worse disease-free survival, especially in tumours with wild-type TP53. To the best of our knowledge, this is the first study that analysed the endogenous protein levels of p53 isoforms in a breast cancer cohort. Our findings suggest that p53β may be a useful prognostic marker.     

Preconditioning-Activated AKT Controls Neuronal Tolerance to Ischemia through the MDM2–p53 Pathway

One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2–p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser⁴⁷³, which in turn phosphorylated MDM2 at Ser¹⁶⁶. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2–p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury.     

Gain of Function (GOF) Mutant p53 in Cancer—Current Therapeutic Approaches

Continuous development of personalized treatments is undoubtedly beneficial for oncogenic patients’ comfort and survival rate. Mutant TP53 is associated with a worse prognosis due to the occurrence of metastases, increased chemoresistance, and tumor growth. Currently, numerous compounds capable of p53 reactivation or the destabilization of mutant p53 are being investigated. Several of them, APR-246, COTI-2, SAHA, and PEITC, were approved for clinical trials. This review focuses on these novel therapeutic opportunities, their mechanisms of action, and their significance for potential medical application.     

Impact of Reactive Oxygen and Nitrogen Species Produced by Plasma on Mdm2–p53 Complex

The study of protein–protein interactions is of great interest. Several early studies focused on the murine double minute 2 (Mdm2)–tumor suppressor protein p53 interactions. However, the effect of plasma treatment on Mdm2 and p53 is still absent from the literature. This study investigated the structural changes in Mdm2, p53, and the Mdm2–p53 complex before and after possible plasma oxidation through molecular dynamic (MD) simulations. MD calculation revealed that the oxidized Mdm2 bounded or unbounded showed high flexibility that might increase the availability of tumor suppressor protein p53 in plasma-treated cells. This study provides insight into Mdm2 and p53 for a better understanding of plasma oncology.     

Inhibition of TIGAR Increases Exogenous p53 and Cisplatin Combination Sensitivity in Lung Cancer Cells by Regulating Glycolytic Flux

The metabolism and apoptosis of tumor cells are important factors that increase their sensitivity to chemotherapeutic drugs. p53 and cisplatin not only induce tumor cell apoptosis, but also regulate the tumor cell metabolism. The TP53-induced glycolysis and apoptosis regulator (TIGAR) can inhibit glycolysis and promote more glucose metabolism in the pentose phosphate pathway. We speculate that the regulation of the TIGAR by the combination therapy of p53 and cisplatin plays an important role in increasing the sensitivity of tumor cells to cisplatin. In this study, we found that the combined treatment of p53 and cisplatin was able to inhibit the mitochondrial function, promote mitochondrial pathway-induced apoptosis, and increase the sensitivity. Furthermore, the expression of the TIGAR was inhibited after a combined p53 and cisplatin treatment, the features of the TIGAR that regulate the pentose phosphate pathway were inhibited, the glucose flux shifted towards glycolysis, and the localization of the complex of the TIGAR and Hexokinase 2 (HK2) on the mitochondria was also reduced. Therefore, the combined treatment of p53 and cisplatin may modulate a glycolytic flux through the TIGAR, altering the cellular metabolic patterns while increasing apoptosis. Taken together, our findings reveal that the TIGAR may serve as a potential therapeutic target to increase the sensitivity of lung cancer A549 cells to cisplatin.     

miR-221-5p and miR-186-5p Are the Critical Bladder Cancer Derived Exosomal miRNAs in Natural Killer Cell Dysfunction

Bladder cancer (BC) is the tenth most commonly diagnosed cancer worldwide, and its carcinogenesis mechanism has not been fully elucidated. BC is able to induce natural killer (NK) cell dysfunction and escape immune surveillance. The present study found that exosomes derived from the urinary bladder cancer cell line (T24 cell) contribute in generating NK cell dysfunction by impairing viability, and inhibiting the cytotoxicity of the NK cell on target cells. Meanwhile, T24 cell-derived exosomes inhibited the expression of the important functional receptors NKG2D, NKp30, and CD226 on NK cells as well as the secretion of perforin and granzyme-B. The critical miRNAs with high expression in T24 cell-derived exosomes were identified using high-throughput sequencing. Furthermore, following dual-luciferase reporter assay and transfection experiments, miR-221-5p and miR-186-5p were confirmed as interfering with the stability of the mRNAs of DAP10, CD96, and the perforin gene in NK cells and may be potential targets used in the therapy for BC.     

Evaluation of the Role of p53 Tumour Suppressor Posttranslational Modifications and TTC5 Cofactor in Lung Cancer

Mutations in the p53 tumor suppressor are found in over 50% of cancers. p53 function is controlled through posttranslational modifications and cofactor interactions. In this study, we investigated the posttranslationally modified p53, including p53 acetylated at lysine 382 (K382), p53 phosphorylated at serine 46 (S46), and the p53 cofactor TTC5/STRAP (Tetratricopeptide repeat domain 5/ Stress-responsive activator of p300-TTC5) proteins in lung cancer. Immunohistochemical (IHC) analysis of lung cancer tissues from 250 patients was carried out and the results were correlated with clinicopathological features. Significant associations between total or modified p53 with a higher grade of the tumour and shorter overall survival (OS) probability were detected, suggesting that mutant and/or modified p53 acts as an oncoprotein in these patients. Acetylated at K382 p53 was predominantly nuclear in some samples and cytoplasmic in others. The localization of the K382 acetylated p53 was significantly associated with the gender and grade of the disease. The TTC5 protein levels were significantly associated with the grade, tumor size, and node involvement in a complex manner. SIRT1 expression was evaluated in 50 lung cancer patients and significant positive correlation was found with p53 S46 intensity, whereas negative TTC5 staining was associated with SIRT1 expression. Furthermore, p53 protein levels showed positive association with poor OS, whereas TTC5 protein levels showed positive association with better OS outcome. Overall, our results indicate that an analysis of p53 modified versions together with TTC5 expression, upon testing on a larger sample size of patients, could serve as useful prognostic factors or drug targets for lung cancer treatment.     

Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate P_i and Ca²⁺ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PP_i), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.     

Cytosolic 5′-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.     

The Therapeutic Potential of the Restoration of the p53 Protein Family Members in the EGFR-Mutated Lung Cancer

Despite the recent development of precision medicine and targeted therapies, lung cancer remains the top cause of cancer-related mortality worldwide. The patients diagnosed with metastatic disease have a five-year survival rate lower than 6%. In metastatic disease, EGFR is the most common driver of mutation, with the most common co-driver hitting TP53. EGFR-positive patients are offered the frontline treatment with tyrosine kinase inhibitors, yet the development of resistance and the lack of alternative therapies make this group of patients only fit for clinical trial participation. Since mutant p53 is the most common co-driver in the metastatic setting, therapies reactivating the p53 pathway might serve as a promising alternative therapeutic approach in patients who have developed a resistance to tyrosine kinase inhibitors. This review focuses on the molecular background of EGFR-mutated lung cancer and discusses novel therapeutic options converging on the reactivation of p53 tumor suppressor pathways.     

anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide Treatment Increases Levels of the Proteins p53 and p21WAF1 in the Human Mammary Carcinoma Cell Line MCF-7

The tumor suppressor p53 is involved in the recognition of DNA damage induced by radiation and chemicals. The effect of polycyclic hydrocarbons on p53 was investigated by treating MCF-7 cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), an ultimate carcinogenic metabolite of benzo[a]pyrene. Western blotting of lysates with antibody mAb1801 showed that B[a]PDE doses of 0.1 to 0.5 μM caused detectable increases in p53 protein. In cells treated with 0.3 μM B[a]PDE, p53 protein levels increased by 2 hours after treatment, reached a maximum between 8 and 24 hours and returned to control value by 120 hours after treatment. Levels of p21^WAF1 protein increased by 8 hours after treatment, reached a maximum by 48 hours and returned to control value by 168 hours after treatment. B[a]PDE-DNA adducts were quantitated by [γ-³³P]ATP postlabeling and separation on reverse-phase HPLC. Adduct levels dropped rapidly between 2 and 24 hrs after treatment (48 to 15 pmol/mg DNA), and subsequently decreased more slowly (12 pmol/mg DNA at 48 hrs to 3 at 168 hrs). By 96 hours after treatment, p53 protein levels were comparable to those from untreated controls, although a low level of B[a]PDE-DNA adducts remained. Cells treated with B[a]PDE also incorporated significantly less [³H]thymidine into DNA than did untreated cells for at least 24 hours after treatment. These results provide preliminary evidence that B[a]PDE-induced DNA damage is recognized by a p53-dependent signal transduction pathway and suggest that cell cycle arrest occurs during the repair of the majority of the B[a]PDE-DNA adducts.     

Proteomics Analysis of Andrographolide-Induced Apoptosis via the Regulation of Tumor Suppressor p53 Proteolysis in Cervical Cancer-Derived Human Papillomavirus 16-Positive Cell Lines

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.     

Anchoring Intrinsically Disordered Proteins to Multiple Targets: Lessons from N-Terminus of the p53 Protein

Anchor residues, which are deeply buried upon binding, play an important role in protein-protein interactions by providing recognition specificity and facilitating the binding kinetics. Up to now, studies on anchor residues have been focused mainly on ordered proteins. In this study, we investigated anchor residues in intrinsically disordered proteins (IDPs) which are flexible in the free state. We identified the anchor residues of the N-terminus of the p53 protein (Glu17-Asn29, abbreviated as p53N) which are involved in binding with two different targets (MDM2 and Taz2), and analyzed their side chain conformations in the unbound states. The anchor residues in the unbound p53N were found to frequently sample conformations similar to those observed in the bound complexes (i.e., Phe19, Trp23, and Leu26 in the p53N-MDM2 complex, and Leu22 in the p53N-Taz2 complex). We argue that the bound-like conformations of the anchor residues in the unbound state are important for controlling the specific interactions between IDPs and their targets. Further, we propose a mechanism to account for the binding promiscuity of IDPs in terms of anchor residues and molecular recognition features (MoRFs).     

Effect of pH on the Interaction of Gold Nanoparticles with DNA and Application in the Detection of Human p53 Gene Mutation

Gold nanoparticles (GNPs) are widely used to detect DNA. We studied the effect of pH on the assembly/disassembly of single-stranded DNA functionalized GNPs. Based on the different binding affinities of DNA to GNPs, we present a simple and fast way that uses HCl to drive the assembly of GNPs for detection of DNA sequences with single nucleotide differences. The assembly is reversible and can be switched by changing the solution pH. No covalent modification of DNA or GNP surface is needed. Oligonucleotide derived from human p53 gene with one-base substitution can be distinguished by a color change of the GNPs solution or a significant difference of the maximum absorption wavelength (λ_max), compared with wildtype sequences. This method enables detection of 10 picomole quantities of target DNA.     

A Highly Potent and Cellularly Active β‐Peptidic Inhibitor of the p53/hDM2 Interaction

New and improved : The incorporation of a 6‐chlorotryptophan (6‐Cl‐Trp) into a β‐peptide (M)‐3₁₄ helix leads to a high‐affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.