TP53AIP1 induce autophagy via the AKT/mTOR signaling pathway in the breast cancer cells.

Breast cancer ranks the first in the incidence of female cancer and is the most common cancer threatening the life and health of women worldwide.Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) is a pro-apoptotic gene downstream of p53. However, the role of TP53AIP1 in BC needs to be investigated. In vitro and in vivo experiments were conducted to assess the biological functions and associated mechanisms. Several bioinformatics analyses were made, CCK8 assay, wound healing, transwell assays, colony formation assay, EDU, flow cytometry, Immunofluorescence, qRT-PCR and Western-blotting were performed. In our study, we discovered that BC samples had low levels of TP53AIP1 expression, which correlated with a lower survival rate in BC patients. When TP53AIP1 was up-regulated, it caused a decrease in cell proliferation, migration, and invasion. It also induced epithelial-to-mesenchymal transition (EMT) and protective autophagy. Furthermore, the over-expression of TP53AIP1 suppressed tumor growth when tested in vivo. We also noticed that TP53AIP1 up-regulation resulted in decreased levels of phosphorylation in AKT and mTOR, suggesting a mechanistic role. In addition, we performed functional rescue experiments where the activation of AKT was able to counteract the impact of TP53AIP1 on the survival and autophagy in breast cancer cell lines. This suggests that TP53AIP1 acts as an oncogene by controlling the AKT/mTOR pathway. These findings reveal TP53AIP1 as a gene that suppresses tumor growth and triggers autophagy through the AKT/mTOR pathway in breast cancer cells. As a result, TP53AIP1 presents itself as a potential target for novel therapeutic approaches in treating breast cancer.

The Tumor Suppressor Par-4 Regulates Adipogenesis by Transcriptional Repression of PPARγ.

Prostate apoptosis response-4 (Par-4, also known as PAWR) is a ubiquitously expressed tumor suppressor protein that induces apoptosis selectively in cancer cells, while leaving normal cells unaffected. Our previous studies indicated that genetic loss of Par-4 promoted hepatic steatosis, adiposity, and insulin-resistance in chow-fed mice. Moreover, low plasma levels of Par-4 are associated with obesity in human subjects. The mechanisms underlying obesity in rodents and humans are multi-faceted, and those associated with adipogenesis can be functionally resolved in cell cultures. We therefore used pluripotent mouse embryonic fibroblasts (MEFs) or preadipocyte cell lines responsive to adipocyte differentiation cues to determine the potential role of Par-4 in adipocytes. We report that pluripotent MEFs from Par-4-/- mice underwent rapid differentiation to mature adipocytes with an increase in lipid droplet accumulation relative to MEFs from Par-4+/+ mice. Knockdown of Par-4 in 3T3-L1 pre-adipocyte cultures by RNA-interference induced rapid differentiation to mature adipocytes. Interestingly, basal expression of PPARγ, a master regulator of de novo lipid synthesis and adipogenesis, was induced during adipogenesis in the cell lines, and PPARγ induction and adipogenesis caused by Par-4 loss was reversed by replenishment of Par-4. Mechanistically, Par-4 downregulates PPARγ expression by directly binding to its upstream promoter, as judged by chromatin immunoprecipitation and luciferase-reporter studies. Thus, Par-4 transcriptionally suppresses the PPARγ promoter to regulate adipogenesis.

Clinical signature and associated immune metabolism of NLRP1 in pan-cancer.

Inflammations have been linked to tumours, suggesting a potential association between NLRP1 and cancer. Nevertheless, a systematic assessment of NLRP1's role across various cancer types currently absent. A comprehensive bioinformatic analysis was conducted to determine whether NLRP1 exhibits prognostic relevance linked to immune metabolism across various cancers. The study leveraged data from the TCGA and GTEx databases to explore the clinical significance, metabolic features, and immunological characteristics of NLRP1, employing various tools such as R, GEPIA, STRING and TISIDB. NLRP1 exhibited differential expression patterns across various cancers, with elevated expression correlating with a more favourable prognosis in lung adenocarcinoma (LUAD) and pancreatic adenocarcinoma (PAAD). Downregulation of NLRP1 reduced tumour metabolic activity in LUAD. Moreover, the mutational signature of NLRP1 was linked to a favourable prognosis. Interestingly, high NLRP1 expression inversely correlated with tumour stemness while positively correlating with tumour immune infiltration in various cancers including LUAD and PAAD. Through extensive big data analysis, we delved into the role of NLRP1 across various tumour types, constructing a comprehensive role map of its involvement in pan-cancer scenarios. Our findings highlight the potential of NLRP1 as a promising therapeutic target specifically in LUAD and PAAD.

NLRP1-dependent activation of Gasdermin D in neutrophils controls cutaneous leishmaniasis.

Intracellular pathogens that replicate in host myeloid cells have devised ways to inhibit the cell's killing machinery. Pyroptosis is one of the host strategies used to reduce the pathogen replicating niche and thereby control its expansion. The intracellular Leishmania parasites can survive and use neutrophils as a silent entry niche, favoring subsequent parasite dissemination into the host. Here, we show that Leishmania mexicana induces NLRP1- and caspase-1-dependent Gasdermin D (GSDMD)-mediated pyroptosis in neutrophils, a process critical to control the parasite-induced pathology. In the absence of GSDMD, we observe an increased number of infected dermal neutrophils two days post-infection. Using adoptive neutrophil transfer in neutropenic mice, we show that pyroptosis contributes to the regulation of the neutrophil niche early after infection. The critical role of neutrophil pyroptosis and its positive influence on the regulation of the disease outcome was further demonstrated following infection of mice with neutrophil-specific deletion of GSDMD. Thus, our study establishes neutrophil pyroptosis as a critical regulator of leishmaniasis pathology.

Integrated stress response (ISR) activation and apoptosis through HRI kinase by PG3 and other p53 pathway-restoring cancer therapeutics.

Restoration of the p53 pathway has been a long-term goal in the field of cancer research to treat tumors with mutated p53 and aggressive clinical behavior. p53 pathway restoration in p53-deficient cancers can be achieved by small molecules via p53-dependent or p53-independent processes. Hereafter p53-independent restoration of p53-pathway-signaling in p53-deficient/mutated tumors is referred to as 'restoration of the p53 pathway'. We compare activation of p53 target genes by novel compounds PG3 and PG3-Oc, that activate p53-target genes in a p53-independent manner, and four mutant p53-activating compounds while Nutlin-3a is used as negative control. PG3 and PG3-Oc upregulate p21, PUMA, and DR5 in five cancer cell lines with various p53 mutational statuses through ATF4 (Activating Transcriptional Factor 4) and integrated stress response (ISR) independent of p53. Mutant p53-targeting compounds induce expression of the 3 major downstream p53 target genes and ATF4 in a highly variable and cell-type-dependent manner. PG3 treatment activates ATF4 through ISR via kinase HRI (Heme-Regulated Inhibitor). ATF4 mediates upregulation of PUMA, p21, and NAG-1/GDF15 (Nonsteroidal anti-inflammatory drug-activated gene 1). We note that PUMA mediates apoptosis through activation of caspase-8 in HT29 cells and potentially caspase-10 in SW480 cells. We provide a novel mechanism engaged by PG3 to induce cell death via the HRI/ATF4/PUMA axis. Our results provide unique insights into the mechanism of action of PG3 as a novel cancer therapeutic targeting p53 pathway-like tumor suppression.

Activation of the NLRP1B inflammasome by caspase-8.

Cleavage of the innate immune receptor NLRP1B by various microbial proteases causes the proteasomal degradation of its N-terminal fragment and the subsequent release of a C-terminal fragment that forms an inflammasome. We reported previously that metabolic stress caused by intracellular bacteria triggers NLRP1B activation, but the mechanism by which this occurs was not elucidated. Here we demonstrate that TLR4 signaling in metabolically stressed macrophages promotes the formation of a TRIF/RIPK1/caspase-8 complex. Caspase-8 activity, induced downstream of this TLR4 pathway or through a distinct TNF receptor pathway, causes cleavage and activation of NLRP1B, which facilitates the maturation of both pro-caspase-1 and pro-caspase-8. Thus, our findings indicate that caspase-8 and NLRP1B generate a positive feedback loop that amplifies cell death processes and promotes a pro-inflammatory response through caspase-1. The ability of NLRP1B to detect caspase-8 activity suggests that this pattern recognition receptor may play a role in the defense against a variety of pathogens that induce apoptosis.

O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.

Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network. Specifically, we show that O-GlcNAc modified HSP27 binds more strongly to the co-chaperone protein BAG3, which then promotes refolding of a model substrate by HSP70. We use proteomics to identify other potential HSP27 interactions that are changed by O-GlcNAc, including one that we confirm with another sHSP, αB-crystallin. These findings add additional evidence for O-GlcNAc as a switch for regulating protein-protein interactions and for modifications of chaperones as one mechanism by which O-GlcNAc protects against protein aggregation.

PDCD4 promotes inflammation/fibrosis by activating the PPAR­Î³/NF­κB pathway in mouse atrial myocytes.

Fibrosis is the basis of structural remodeling in atrial fibrillation (AF), during which inflammation is crucial. Programmed cell death factor 4 (PDCD4) is a newly identified inflammatory gene, with unknown mechanisms of action in AF. The present study aimed to elucidate the effects of PDCD4 on the inflammation and structural remodeling of atrial myocytes. For this purpose, a PDCD4 overexpression plasmid (oePDCD4) and PDCD4 small interfering (si)RNA (siPDCD4) were used to modulate PDCD4 expression in mouse atrial myocytes (HL­1 cells). The expression of PDCD4 was detected using reverse transcription­quantitative PCR and western blot analysis. The optimal drug concentrations of peroxisome proliferator­activated receptor γ (PPARγ) agonist (pioglitazone hydrochloride), NF­κB inhibitor (CBL0137), PPARγ inhibitor (GW9962) and NF­κB agonist (betulinic acid) were screened using a Cell Counting Kit­8 assay. The levels of inflammatory factors were detected using enzyme­linked immunosorbent assays, the expression levels of fibrosis­related proteins and NF­κB subunits were detected using western blot analysis, and the expression of phosphorylated (p­)p65/p65 was detected using immunofluorescence staining. The results revealed that PDCD4 overexpression increased the levels of fibrotic factors (collagen I, collagen III, fibronectin, α­smooth muscle actin and matrix metalloproteinase 2), pro­inflammatory cytokines (IFN­Î³, IL­6, IL­17A and TNF­α) and p­p65, whereas it reduced the levels of anti­inflammatory cytokines (IL­4) in HL­1 cells. Additionally, treatment with the PPARγ agonist and NF­κB inhibitor reversed the levels of fibrotic­, pro­inflammatory and anti­inflammatory factors in oePDCD4­HL­1 cells. By contrast, PDCD4 silencing exerted the opposite effects on fibrotic factors, pro­inflammatory cytokines, anti­inflammatory cytokines and p­p65. In addition, treatment with the PPARγ inhibitor and NF­κB agonist reversed the levels of fibrotic­, pro­inflammatory and anti­inflammatory factors in siPDCD4­HL­1 cells. In conclusion, the present study demonstrated that PDCD4 may induce inflammation and fibrosis by activating the PPARγ/NF­κB signaling pathway, thereby promoting the structural remodeling of atrial myocytes in AF.

Modulation of m6A RNA modification by DAP3 in cancer cells.

N6-methyladenosine (m6A) RNA methylation is a prevalent RNA modification that significantly impacts RNA metabolism and cancer development. Maintaining the global m6A levels in cancer cells relies on RNA accessibility to methyltransferases and the availability of the methyl donor S-adenosylmethionine (SAM). Here, we reveal that death associated protein 3 (DAP3) plays a crucial role in preserving m6A levels through two distinct mechanisms. First, although DAP3 is not a component of the m6A writer complex, it directly binds to m6A target regions, thereby facilitating METTL3 binding. Second, DAP3 promotes MAT2A's last intron splicing, increasing MAT2A protein, cellular SAM, and m6A levels. Silencing DAP3 hinders tumorigenesis, which can be rescued by MAT2A overexpression. This evidence suggests DAP3's role in tumorigenesis, partly through m6A regulation. Our findings unveil DAP3's complex role as an RNA-binding protein and tumor promoter, impacting RNA processing, splicing, and m6A modification in cancer transcriptomes.

PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells.

Overcoming temozolomide (TMZ)-resistance is a major challenge in glioblastoma therapy. Therefore, identifying the key molecular player in chemo-resistance becomes urgent. We previously reported the downregulation of PDCD10 in primary glioblastoma patients and its tumor suppressor-like function in glioblastoma cells. Here, we demonstrate that the loss of PDCD10 causes a significant TMZ-resistance during treatment and promotes a rapid regrowth of tumor cells after treatment. PDCD10 knockdown upregulated MGMT, a key enzyme mediating chemo-resistance in glioblastoma, accompanied by increased expression of DNA mismatch repair genes, and enabled tumor cells to evade TMZ-induced cell-cycle arrest. These findings were confirmed in independent models of PDCD10 overexpressing cells. Furthermore, PDCD10 downregulation led to the dedifferentiation of glioblastoma cells, as evidenced by increased clonogenic growth, the upregulation of glioblastoma stem cell (GSC) markers, and enhanced neurosphere formation capacity. GSCs derived from PDCD10 knockdown cells displayed stronger TMZ-resistance and regrowth potency, compared to their parental counterparts, indicating that PDCD10-induced stemness may independently contribute to tumor malignancy. These data provide evidence for a dual role of PDCD10 in tumor suppression by controlling both chemo-resistance and dedifferentiation, and highlight PDCD10 as a potential prognostic marker and target for combination therapy with TMZ in glioblastoma.

NLRP1 inhibits lung adenocarcinoma growth through mediating mitochondrial dysregulation in an inflammasome-independent manner.

NLRP1, the first identified inflammasome-forming sensor, is thought to be involved in cancer, yet its definite function in lung adenocarcinoma (LUAD) remains unclear. Herein, we explored the expression and function of NLRP1 in LUAD. Decreased NLRP1 expression was identified in LUAD, which was associated with a poor prognosis. Overexpression of NLRP1 inhibited tumor growth in vitro and in vivo. Mechanically, this effect was observed regardless of inflammasome activation. Further studies revealed that overexpression of NLRP1 downregulated the phosphorylation of DRP1 and promoted mitochondrial fusion, which was mediated by inhibition of NF-κB activity. NF-κB agonist could neutralize the effect of NLRP1 on mitochondrial dynamics. In addition, LUAD sensitivity to cisplatin was enhanced by decreased mitochondrial fission resulting from up-regulated NLRP1. In conclusion, our findings demonstrated an unexpected role of NLRP1 in LUAD by modulating mitochondrial activities, which provides strong evidence for its potential in LUAD treatment.

SMAC mimetic drives microglia phenotype and glioblastoma immune microenvironment.

Tumor-associated macrophages/microglia (TAMs) are highly plastic and heterogeneous immune cells that can be immune-supportive or tumor-supportive depending of the microenvironment. TAMs are the most abundant immune cells in glioblastoma (GB), and play a key role in immunosuppression. Therefore, TAMs reprogramming toward immune-supportive cells is a promising strategy to overcome immunosuppression. By leveraging scRNAseq human GB databases, we identified that Inhibitor of Apoptosis Proteins (IAP) were expressed by TAMs. To investigate their role in TAMs-related immunosuppression, we antagonized IAP using the central nervous system permeant SMAC mimetic GDC-0152 (SMg). On explants and cultured immune cells isolated from human GB samples, SMg modified TAMs activity. We showed that SMg treatment promoted microglia pro-apoptotic and anti-tumoral function via caspase-3 pro-inflammatory cleavage and the inhibition of tumoroids growth. Then we designed a relevant immunogenic mouse GB model to decipher the spatio-temporal densities, distribution, phenotypes and function of TAMs with or without SMg treatment. We used 3D imaging techniques, a transgenic mouse with fluorescent TAM subsets and mass cytometry. We confirmed that SMg promoted microglia activation, antigen-presenting function and tumor infiltration. In addition, we observed a remodeling of blood vessels, a decrease in anti-inflammatory macrophages and an increased level of monocytes and their mo-DC progeny. This remodeling of the TAM landscape is associated with an increase in CD8 T cell density and activation. Altogether, these results demonstrated that SMg drives the immunosuppressive basal microglia toward an active phenotype with pro-apoptotic and anti-tumoral function and modifies the GB immune landscape. This identifies IAP as targets of choice for a potential mechanism-based therapeutic strategy and SMg as a promising molecule for this application.

Induction of the Lipid Droplet Formation Genes in Steatohepatitis Mice by Embryo/Postnatal Nutrient Environment Is Associated with Histone Acetylation around the Genes.

Recently, we have demonstrated that mice, cultured embryos in α-minimum essential medium (αMEM) and subsequent fed a high-fat, high-sugar diet, developed steatohepatitis. In this study, we investigated using these samples whether the expression of lipid droplet formation genes in the liver is higher in MEM mice, whether these expressions are regulated by histone acetylation, writers/readers of histone acetylation, and the transcriptional factors of endoplasmic reticulum stress. Mice were produced by two-cell embryos in αMEM or standard potassium simplex-optimized medium (control) in vitro for 48 h, and implanted into an oviduct for spontaneous delivery. MEM and control-mice were fed a high-fat, high-sugar diet for 18 wk, and then liver samples were collected and analyzed by histology, qRT-PCR, and chromatin immunoprecipitation assay. Gene expression of Cidea, Cidec, and Plin4 were higher in MEM mice and histone H3K9 acetylation, BRD4, and CBP were higher in MEM mice than in control mice around those genes. However, the binding of endoplasmic reticulum stress-related transcription factors (ATF4, CHOP and C/EBPα) around those genes in the liver, was not clearly differed between MEM mice and control mice. The increased expression of Cidea, Cidec and Plin4 in the liver, accompanied by the development of steatohepatitis in mice induced is positively associated with increased histone H3K9 acetylation and CBP and BRD4 binding around these genes.

[Anti-inflammatory mixture alleviates acute lung injury induced by sepsis in rats by modulating Beclin-1-mediated autophagy].

OBJECTIVE: To investigate the protective effects of an anti-inflammatory mixture on acute lung injury (ALI) induced by sepsis in rats, as well as its possible mechanisms. METHODS: A total of 40 Sprague-Dawley (SD) rats were randomly divided into the sham group, septic ALI model group (model group), 3-methyladenine (3-MA) control group, and anti-inflammatory mixture pretreatment group, with 10 rats in each group. Cecal ligation and perforation (CLP) was performed to reproduce a septic ALI model. The rats in the sham group only underwent opening and closing the abdomen without perforation and ligation. Both groups were given saline gavage and intraperitoneal injection for 3 consecutive days before surgery. The 3-MA control group was given intraperitoneal injection of saline and autophagy inhibitor 3-MA 15 mg/kg for 3 consecutive days before modeling. The anti-inflammatory mixture pretreatment group was given 8.8 mL/kg of anti-inflammatory mixture by gavage [the composition of anti-inflammatory mixture: rhubarb 15 g (after the next), coptis chinensis 15 g, baical skullcap root 12 g, magnoliae cortex 12 g, dahurian patrinia herb 30 g] and saline intraperitoneal injection for 3 consecutive days before modeling. The rats in each group were anesthetized 24 hours after surgery and died due to abdominal aortic blood collection. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory cytokines interleukins (IL-1ß and IL-6). Lung tissue was taken and then the bronchoalveolar lavage fluid (BALF) was collected, and the levels of IL-1ß and IL-6 were detected by ELISA. Lung wet/dry weight (W/D) ratio was measured. After hematoxylin-eosin (HE) staining, the histopathological changes of the lungs were observed under light microscopy. Western blotting was used to detect the expression of autophagy markers microtubule-associated protein 1 light chain 3- II/I (LC3- II/I) and Beclin-1 protein in lung tissue. Autophagosomes in lung tissue were observed with transmission electron microscopy. RESULTS: Compared with the sham group, the rats in the model group exhibited severe destruction of lung tissue structure, with significant infiltration of inflammatory cells, the lung W/D ratio and the levels of IL-1ß and IL-6 in serum and BALF were significantly increased, the expressions of LC3- II/I and Beclin-1 protein were down-regulated, the autophagosomes were more. The rats in the 3-MA control group exhibited more severe lung tissue injury as compared with the model group, the lung W/D ratio and the levels of inflammatory cytokines in serum and BALF were further increased, the expressions of LC3- II/I and Beclin-1 protein still showed a decrease tendency as compared with the sham group, and the autophagosomes were less than that in the model group. Compared with the model group, the anti-inflammatory mixture pretreatment group showed milder lung tissue injury with a minimal amount of inflammatory cell infiltration, the lung W/D ratio was significantly reduced (7.07±1.02 vs. 11.33±1.85, P < 0.05), the levels of IL-1ß and IL-6 in both serum and BALF were significantly decreased [IL-1ß (ng/L): 26.04±3.86 vs. 40.83±5.46 in serum, 17.75±2.02 vs. 26.86±4.32 in BALF; IL-6 (ng/L): 91.28±10.15 vs. 129.44±13.05 in serum, 76.06±7.51 vs. 120.91±7.47 in BALF, all P < 0.05], and the ratio of LC3- II/I and Beclin-1 protein expression were significantly increased [LC3- II/I ratio: 1.23±0.02 vs. 0.60±0.02, Beclin-1 protein (Beclin-1/GAPDH): 2.37±0.33 vs. 0.62±0.05, both P < 0.05]. Furthermore, an increase in the number of autophagosomes was observed. CONCLUSIONS: The anti-inflammatory mixture improves lung injury in rats with sepsis induced by CLP and reduce inflammation levels, potentially through upregulation of Beclin-1-mediated autophagy.

Glucose fluctuations aggravate cardiomyocyte apoptosis by enhancing the interaction between Txnip and Akt.

BACKGROUND: Glucose fluctuations may be involved in the pathophysiological process of cardiomyocyte apoptosis, but the exact mechanism remains elusive. This study focused on exploring the mechanisms related to glucose fluctuation-induced cardiomyocyte apoptosis. METHODS: Diabetic rats established via an injection of streptozotocin were randomized to five groups: the controlled diabetic (CD) group, the uncontrolled diabetic (UD) group, the glucose fluctuated diabetic (GFD) group, the GFD group rats with the injection of 0.9% sodium chloride (NaCl) (GFD + NaCl) and the GFD group rats with the injection of N-acetyl-L-cysteine (NAC) (GFD + NAC). Twelve weeks later, cardiac function and apoptosis related protein expressions were tested. Proteomic analysis was performed to further analyze the differential protein expression pattern of CD and GFD. RESULTS: The left ventricular ejection fraction levels and fractional shortening levels were decreased in the GFD group, compared with those in the CD and UD groups. Positive cells tested by DAB-TUNEL were increased in the GFD group, compared with those in the CD group. The expression of Bcl-2 was decreased, but the expressions of Bax, cleaved caspase-3 and cleaved caspase-9 were increased in response to glucose fluctuations. Compared with CD, there were 527 upregulated and 152 downregulated proteins in GFD group. Txnip was one of the differentially expressed proteins related to oxidative stress response. The Txnip expression was increased in the GFD group, while the Akt phosphorylation level was decreased. The interaction between Txnip and Akt was enhanced when blood glucose fluctuated. Moreover, the application of NAC partially reversed glucose fluctuations-induced cardiomyocyte apoptosis. CONCLUSIONS: Glucose fluctuations lead to cardiomyocyte apoptosis by up-regulating Txnip expression and enhancing Txnip-Akt interaction.

RUNX1-PDIA5 Axis Promotes Malignant Progression of Glioblastoma by Regulating CCAR1 Protein Expression.

PDIA5 is responsible for modification of disulfide bonds of proteins. However, its impact on the malignant progression of glioblastoma multiforme (GBM) remains unknown. We analyzed the expression and prognostic significance of PDIA5 in cohorts of GBM and clinical samples. The PDIA5 protein was significantly overexpressed in GBM tissues, and higher expression of PDIA5 was statistically associated with a worse prognosis in patients with GBM. Transcriptional data from PDIA5 knockdown GBM cells revealed that downstream regulatory genes of PDIA5 were enriched in malignant regulatory pathways and PDIA5 enhanced the proliferative and invasive abilities of GBM cells. By constructing a PDIA5 CXXC motif mutant plasmid, we found CCAR1 was the vital downstream factor of PDIA5 in regulating GBM malignancy in vitro and in vivo. Additionally, RUNX1 bound to the promoter region of PDIA5 and regulated gene transcription, leading to activation of the PDIA5/CCAR1 regulatory axis in GBM. The RUNX1/PDIA5/CCAR1 axis significantly influenced the malignant behavior of GBM cells. In conclusion, this study comprehensively elucidates the crucial role of PDIA5 in the malignant progression of GBM. Downregulating PDIA5 can mitigate the malignant biological behavior of GBM both in vitro and in vivo, potentially improving the efficacy of treatment for clinical patients with GBM.

The Role of NLRP1 Inflammasome and Interleukin 1ß in Experimental Neuropathic Pain Model in Rat and the Effect of Tramadol Treatment.

AIM: To evaluate the effects of tramadol on inflammation by measuring NLRP1 and IL-1 beta (IL-1ß) levels in an experimental neuropathic pain model. MATERIAL AND METHODS: Sprague-Dawley rats were divided into three groups: control, chronic constriction injury (CCI), and CCI + tramadol. Neuropathic pain was assessed using mechanical allodynia, thermal hyperalgesia, and cold allodynia. IL-1ß and NLRP1 levels were evaluated using ELISA on sciatic nerve (SN), dorsal root ganglion (DRG), and serum either on day 3 or days 8 postsurgery. RESULTS: On day 3, paw withdrawal latency (PWL) was lower in the CCI and CCI + tramadol groups than the control group in both mechanical and cold allodynia tests. On day 8, the PWL in the CCI group was also lower than in the control group. In contrast, tramadol increased the PWL on day 8 compared to day 3 in the CCI group. During cold allodynia, PWL decreased in the CCI group, however, tramadol reversed this effect on days 3 and 8. Tramadol, therefore, ameliorated pain hypersensitivity in mechanical/cold allodynia tests. Serum IL-1ß levels were higher in the CCI + tramadol and CCI groups than the control group, although serum IL-1ß levels in the CCI and CCI + tramadol groups were comparable. Tramadol decreased the IL-1ß and NLRP1 in DRG compared with the CCI group. A similar trend was observed in the SN samples. CONCLUSION: Our experiments revealed an increase in IL-1ß and NLRP-1 levels in a neuropathic pain model and found that tramadol had an anti-inflammatory effect on the IL-1ß and NLRP1 inflammasomes.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in the 40S small ribosomal subunit.

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

Variants in NLRP2 and ZFP36L2, non-core components of the human subcortical maternal complex, cause female infertility with embryonic development arrest.

The subcortical maternal complex (SCMC), which is vital in oocyte maturation and embryogenesis, consists of core proteins (NLRP5, TLE6, OOEP), non-core proteins (PADI6, KHDC3L, NLRP2, NLRP7), and other unknown proteins that are encoded by maternal effect genes. Some variants of SCMC genes have been linked to female infertility characterized by embryonic development arrest. However, so far, the candidate non-core SCMC components associated with embryonic development need further exploration and the pathogenic variants that have been identified are still limited. In this study, we discovered two novel variants [p.(Ala131Val) and p.(Met326Val)] of NLRP2 in patients with primary infertility displaying embryonic development arrest from large families. In vitro studies using 293T cells and mouse oocytes, respectively, showed that these variants significantly decreased protein expression and caused the phenotype of embryonic development arrest. Additionally, we combined the 'DevOmics' database with the whole exome sequence data of our cohort and screened out a new candidate non-core SCMC gene ZFP36L2. Its variants [p.(Ala241Pro) and p.(Pro291dup)] were found to be responsible for embryonic development arrest. Co-immunoprecipitation experiments in 293T cells, used to demonstrate the interaction between proteins, verified that ZFP36L2 is one of the human SCMC components, and microinjection of ZFP36L2 complementary RNA variants into mouse oocytes affected embryonic development. Furthermore, the ZFP36L2 variants were associated with disrupted stability of its target mRNAs, which resulted in aberrant H3K4me3 and H3K9me3 levels. These disruptions decreased oocyte quality and further developmental potential. Overall, this is the first report of ZFP36L2 as a non-core component of the human SCMC and we found four novel pathogenic variants in the NLRP2 and ZFP36L2 genes in 4 of 161 patients that caused human embryonic development arrest. These findings contribute to the genetic diagnosis of female infertility and provide new insights into the physiological function of SCMC in female reproduction.