Promyelocytic Leukemia Protein Research Articles

PML restrains p53 activity and cellular senescence in clear cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC), the major subtype of RCC, is frequently diagnosed at late/metastatic stage with 13% 5-year disease-free survival. Functional inactivation of the wild-type p53 protein is implicated in ccRCC therapy resistance, but the detailed mechanisms of p53 malfunction are still poorly characterized. Thus, a better understanding of the mechanisms of disease progression and therapy resistance is required. Here, we report a novel ccRCC dependence on the promyelocytic leukemia (PML) protein. We show that PML is overexpressed in ccRCC and that PML depletion inhibits cell proliferation and relieves pathologic features of anaplastic disease in vivo. Mechanistically, PML loss unleashed p53-dependent cellular senescence thus depicting a novel regulatory axis to limit p53 activity and senescence in ccRCC. Treatment with the FDA-approved PML inhibitor arsenic trioxide induced PML degradation and p53 accumulation and inhibited ccRCC expansion in vitro and in vivo. Therefore, by defining non-oncogene addiction to the PML gene, our work uncovers a novel ccRCC vulnerability and lays the foundation for repurposing an available pharmacological intervention to restore p53 function and chemosensitivity.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

TRIM5 as a promising diagnostic biomarker of hepatocellular carcinoma: integrated analysis and experimental validation.

The TRIM family is associated with the membrane, and its involvement in the progression, growth, and development of various cancer types has been researched extensively. However, the role played by the TRIM5 gene within this family has yet to be explored to a great extent in terms of hepatocellular carcinoma (HCC). The data of patients relating to mRNA expression and the survival rate of individuals diagnosed with HCC were extracted from The Cancer Genome Atlas (TCGA) database. UALCAN was employed to examine the potential link between TRIM5 expression and clinicopathological characteristics. In addition, enrichment analysis of differentially expressed genes (DEGs) was conducted as a means of deciphering the function and mechanism of TRIM5 in HCC. The data in the TCGA and TIMER2.0 databases was utilized to explore the correlation between TRIM5 and immune infiltration in HCC. WGCNA was performed as a means of assessing TRIM5-related co-expressed genes. The "OncoPredict" R package was also used for investigating the association between TRIM5 and drug sensitivity. Finally, qRT-PCR, Western blotting (WB) and immunohistochemistry (IHC) were employed for exploring the differential expression of TRIM5 and its clinical relevance in HCC. According to the results that were obtained from the vitro experiments, mRNA and protein levels of TRIM5 demonstrated a significant upregulation in HCC tissues. It is notable that TRIM5 expression levels were found to have a strong association with the infiltration of diverse immune cells and displayed a positive correlation with several immune checkpoint inhibitors. The TRIM5 expression also displayed promising clinical prognostic value for HCC patients.

Acute Promyelocytic Leukemia: Review of Complications Related to All-Trans Retinoic Acid and Arsenic Trioxide Therapy.

The hallmark of acute promyelocytic leukemia (APL) is the presence of the characteristic fusion transcript of the promyelocytic leukemia gene with the retinoic acid receptor α gene (PML::RARA). The PML::RARA fusion is a molecular target for all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Therapies based on ATRA plus ATO have excellent outcomes in terms of complete remission rates, overall survival, and achievement of deep and durable molecular responses with a very low incidence of relapse. However, although the combination of ATRA and ATO has lower hematologic toxicity than standard chemotherapy, its use is associated with a spectrum of distinctive toxicities, such as differentiation syndrome, liver toxicity, QT interval prolongation, and neurotoxicity. Rigorous monitoring of patients' clinical evolution is indispensable for identifying and addressing each complication. The objective is to maintain an equilibrium between treatment-induced adverse events and therapeutic efficacy. This paper focused on non-hematologic complications associated with the combination of ATRA and ATO. Additionally, we discuss late-onset complications of this therapy. In summary, the majority of treatment-related adverse events are manageable, self-limiting, and reversible. More so, there seems to be a lower incidence rate of secondary neoplasms compared to standard chemotherapy. However, further research is required to assess how the ATRA plus ATO regimen affects the emergence of additional comorbidities.

USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

Ubiquitin-specific peptidase 22 (USP22) is a deubiquitinating enzyme (DUB) that underlies tumorigenicity, proliferation, cell death and differentiation through deubiquitination of histone and non-histone targets. Ubiquitination determines stability, localization and functions of cell fate proteins and controls cell-protective signaling pathways to surveil cell cycle progression. In a variety of carcinomas, lymphomas and leukemias, ubiquitination regulates the tumor-suppressive functions of the promyelocytic leukemia protein (PML), but PML-specific DUBs, DUB-controlled PML ubiquitin sites and the functional consequences of PML (de)ubiquitination remain unclear. Here, we identify USP22 as regulator of PML and the oncogenic acute promyelocytic leukemia (APL) fusion PML-RARα protein stability and identify a destabilizing role of PML residue K394. Additionally, loss of USP22 upregulates interferon (IFN) and IFN-stimulated gene (ISG) expression in APL and induces PML-RARα stabilization and a potentiation of the cell-autonomous sensitivity towards all-trans retinoic acid (ATRA)-mediated differentiation. Our findings imply USP22-dependent surveillance of PML-RARα stability and IFN signaling as important regulator of APL pathogenesis, with implications for viral mimicry, differentiation and cell fate regulation in other leukemia subtypes.

PEDV nucleocapsid antagonizes zinc-finger antiviral protein by disrupting the interaction with its obligate co-factor, TRIM25

The genomes of many pathogens contain high-CpG content, which is less common in most vertebrate host genomes. Such a distinct di-nucleotide composition in a non-self invader constitutes a special feature recognized by its host’s immune system. The zinc-finger antiviral protein (ZAP) is part of the pattern recognition receptors (PRRs) that recognize CpG-rich viral RNA and subsequently initiate RNA degradation as an antiviral defense measure. To counteract such ZAP-mediated restriction, some viruses evolve to either suppress the CpG content in their genome or produce an antagonistic factor to evade ZAP sensing. We have previously shown that a coronavirus, Porcine epidermic diarrhea virus (PEDV), employs its nucleocapsid protein (PEDV-N) to suppress the ZAP-dependent antiviral activity. Here, we propose a mechanism by which PEDV-N suppresses ZAP function by interfering with the interaction between ZAP and its essential cofactor, Tripartite motif-containing protein 25 (TRIM25). PEDV-N was found to interact with ZAP through its N-terminal domain and with TRIM25 through its C-terminal domain. We showed that PEDV-N and ZAP compete for binding to the SPla and the RYanodine Receptor (SPRY) domain of TRIM25, resulting in PEDV-N preventing TRIM25 from interacting with and promoting ZAP. Our result also showed that the presence of PEDV-N in the complex reduces the E3 ligase activity of TRIM25 on ZAP, which is required for the antiviral activity of ZAP. The host-pathogen interaction mechanism presented herein provides an insight into the new function of this abundant and versatile viral protein from a coronavirus which could be a key target for development of antiviral interventions.

Clinical analysis of 82 cases of acute promyelocytic leukemia with PML-RARα short isoform in children and adults.

Acute promyelocytic leukemia (APL) with PML/RARα fusion gene is a distinct variant of acute myeloid leukemia. According to the different break sites of the PML gene, there are three transcripts: Long (bcr1), Variant (bcr2) and Short (bcr3). We retrospectively analyzed 82 APL cases with PML-RARα short isoform. A total of 384 patients with APL were seen, of which 85(22.14%) had PML/RARα short isoform (bcr3) and 82 met the inclusion criteria. The median age was 33.5 years (range, 2-72 years). The incidences of hemorrhage in the intermediate- and high-risk group were higher, but only the incidence between medium and low risk differed statistically (P=0.006), and the incidences of fever, fatigue, splenomegaly, and lymph node enlargement and differentiation syndrome (DS) in those groups were not statistically significant (P>0.05). FLT3 gene mutation rate and the mortality rate of the high-risk group were significantly higher than that of other groups (P=0.040 and P=0.004, P=0.041 and P=0.037, respectively). The mortality rate was lowest (4.26%) in the group treated with ATRA combined with arsenic and anthracycline. The 3-year OS and the 3-year DFS of the low and intermediate-risk group were better (P=0.019 and P=0.017, respectively). ATRA combined with arsenic and anthracycline had significant impact on outcomes in APL with PML-RARα short isoform.

Identification of a novel fusion gene, RARA::ANKRD34C, in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia that is distinguished by the chromosomal translocation t(15;17)(q24;q21), which leads to the fusion of the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA). Recently, we identified a novel fusion gene in APL, RARA::ankyrin repeat domain 34C (ANKRD34C), identified its functions by morphological, cytogenetic, molecular biological and multiplex fluorescence in situ hybridization analyses, and demonstrated the potential therapeutic effect clinically and experimentally of all-trans retinoic acid (ATRA); the findings have important implications for the diagnosis and treatment of atypical APL.

Bioinformatics analysis and validation of the interaction between PML protein and TAB1 protein

To analyze the interaction between PML protein and TAB1 protein using bioinformatic approaches and experimentally verify the results. Using Rosetta software, a 3D model of TAB1 protein was constructed through a comparative modeling approach; the secondary structure of PML protein was retrieved in the PDB database and its crystal structure and 3D structure were resolved. Zdock 3.0.2 software was used to perform protein-protein docking of PML and TAB1, and the best conformation was extracted for molecular structure analysis of the docking model. The interaction between the two proteins was detected using immunoprecipitation in α-MMC-treated M1 inflammatory macrophages. When 6IMQ of PML was used as the docking site, PML protein formed 3 salt bridges, 6 hydrogen bonds and 6 hydrophobic interactions with TAB1 proteins; when 5YUF of PML was used as the docking site, PML protein formed 1 hydrogen bond, 3 electrostatic interactions and 9 hydrophobic interactions with TAB1 proteins, and both of the docking modes formed good molecular docking and interactions. In the M1 inflammatory macrophages treated with α-MMC for 4 h, positive protein bands of PML and TAB1 were detected in the cell lysates in PML-IP group. PML protein can interact strongly with TAB1 protein.

Nuclear Phosphoproteome Reveals Prolyl Isomerase PIN1 as a Modulator of Oncogene-Induced Senescence

Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex and remain to be fully characterized. In this study, we analyzed the changes in the nuclear proteome and phosphoproteome of human lung fibroblast IMR90 cells during the progression of OIS induced by oncogenic RASG12V activation. We found that most of the differentially regulated phosphosites during OIS contained prolyl isomerase PIN1 target motifs, suggesting PIN1 is a key regulator of several promyelocytic leukemia nuclear body proteins, specifically regulating several proteins upon oncogenic Ras activation. We showed that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence, including p21, p16, and p53 with concomitant accumulation of the protein PML and the dysregulation of promyelocytic leukemia nuclear body formation. Collectively, our data demonstrate that PIN1 plays an important role as a tumor suppressor in response to oncogenic ER:RasG12V activation.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Genotypic investigation of drugs resistance and biofilm genes among ciprofloxacin-resistant Pseudomonas aeruginosa isolated from burn patients

Burn patients are the serious targets of hospital-acquired infection occurred by Pseudomonas aeruginosa ( P. aeruginosa) , which is a main cause of burn patients morbidity and mortality, so this study aimed to molecular investigation of genes associated with antibiotic resistance among ciprofloxacin-resistant Pseudomonas aeruginosa. Current study involved 168 burn patients involved 107(63.69%) and 61 (36.3%) were females and male respectively, the results showed 132(78.57%) gram negative bacteria and 27(16.0%) gram positive bacteria while 9(5.3%) no bacterial growth. The results of Vitek-2 compact system recorded 75(44.64%) as P. aeruginosa isolates. Results of ciprofloxacin susceptibility showed 29(38.67%), while 34(45.33%) and 12(16%) of isolates were intermediate and sensitive respectively. Congo red agar assay was applied in current study for qualitative evaluation of pathogenic biofilm were 23/29 (79.31%), and 6/26(20.86%) as high, and moderate biofilm producer among isolates respectively. Result of Polymerase chain reaction (PCR) showed that gyrB was recorded among ciprofloxacin-resistant P. aeruginosa at rate 28/29 (96.55%). At same respect. results of efflux pump genes for oqxA and oqxB genes were detected among this pathogen at rate 26/29 (89.65%) and 29/29(100%) respectively. outer membrane genes, revealed that mexR gene was 27/29 (93.1%), while the spreading of the oprD gene was 26/29(89.65%). Results of molecular detection about colistin resistance genes revealed that the mcr-3 gene was 8/29 (27.58%), but genes of mcr-1 and mcr-2 were no detected in this work, Finally, results showed that 23/29 (79.31%) of ciprofloxacin-resistant P. aeruginosa were harbored pml gene.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.

Suppression of TRIM19 by arterivirus nonstructural protein 1 promotes viral replication

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

Disordered regions mediate the interaction of p53 and MRE11

The genome is frequently targeted by genotoxic agents, resulting in the formation of DNA scars. However, cells employ diverse repair mechanisms to restore DNA integrity. Among these processes, the Mre11-Rad50-Nbs1 complex detects double-strand breaks (DSBs) and recruits DNA damage response proteins such as ataxia-telangiectasia-mutated (ATM) kinase to DNA damage sites. ATM phosphorylates the transactivation domain (TAD) of the p53 tumor suppressor, which in turn regulates DNA repair, growth arrest, apoptosis, and senescence following DNA damage. The disordered glycine-arginine-rich (GAR) domain of double-strand break protein MRE11 (MRE11GAR) and its methylation are important for DSB repair, and localization to Promyelocytic leukemia nuclear bodies (PML-NBs). There is preliminary evidence that p53, PML protein, and MRE11 might co-localize and interact at DSB sites. To uncover the molecular details of these interactions, we aimed to identify the domains mediating the p53-MRE11 interaction and to elucidate the regulation of the p53-MRE11 interaction by post-translational modifications (PTMs) through a combination of biophysical techniques. We discovered that, in vitro, p53 binds directly to MRE11GAR mainly through p53TAD2 and that phosphorylation further enhances this interaction. Furthermore, we found that MRE11GAR methylation still allows for binding to p53. Overall, we demonstrated that p53 and MRE11 interaction is facilitated by disordered regions. We provide for the first time insight into the molecular details of the p53-MRE11 complex formation and elucidate potential regulatory mechanisms that will promote our understanding of the DNA damage response. Our findings suggest that PTMs regulate the p53-MRE11 interaction and subsequently their colocalization to PML-NBs upon DNA damage.

Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells

BackgroundPromyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection.MethodsPML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-β signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays.ResultsOur results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-β signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection.ConclusionsThese findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-β and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

Research on Different Compound Combinations of Realgar-Indigo Naturalis Formula to Reverse APL Arsenic Resistance By Regulating Autophagy through mTOR Pathway

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, and 90% of APL patients carry positive fusion gene of the promyelocytic leukemia protein (PML)- retinoic acid receptor α (RARα), which drives the development of APL. All-trans retinoic acid and arsenic trioxide is able to cure 90% of APL patients by targeting PML-RARα. However, there are still 5%-10% of patients who will relapse due to drug resistance. Compound realgar-indigo naturalis formula (RIF) was developed based on the characteristics of APL pathogenesis. It contains four Chinese herbs: realgar, natural indigo, danshen root and heterophylly falsestarwort root. Clinical studies have confirmed that RIF has a certain efficacy on APL, which shown non-inferiority compared with arsenic trioxide. In addition, RIF combined with all-trans retinoic acid has become the first-line treatment regimen in APL guidelines in China. This study further clarified the efficacy of RIF on arsenic-resistant APL, explored the mechanism, and laid the foundation for further improving the efficacy of APL. O bjective: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. M ethods: The arsenic-resistant cell line HL60-PML A216V-RARα, which could stably express PML A216V-RARα, was established by lentiviral transfection. Among compounds of RIF, the active compound of realgar is tetra-arsenic tetra-sulfide (As 4S 4, A), for natural indigo is indirubin (I), for danshen root is tanshinone IIa (T), and the for heterophylly falsestarwort root is total saponins of Radix Pseudostellariae (S). The four compounds were applied on the HL60-PML A216V-RARα cell line. Cell viability was detected by CCK-8 method. Hoechst staining was used to observe cell morphology, Annexin V/PI double staining was applied to detect cell apoptosis, JC-1 detection was used to detect mitochondrial membrane potential, flow cytometry was used to detect CD33 expression to evaluate cell differentiation, and Western blotting was used to observe the changes in the levels of PML A216V-RARα, apoptosis-related factors, PI3K/AKT/mTOR and autophagy in the control, A, ITS and AITS groups. The PI3K inhibitor LY294002 and the autophagy inhibitor Baf A1 were combined and applied to observe the changes in the levels of PML A216V-RARα, mTOR and p62 after blocking the PI3K or autophagy pathway. Results: The arsenic-resistant cell line HL60-PML A216V-RARα was successfully established. Compared with the control group, A, ITS and AITS groups all inhibited the growth of HL60-PML A216V-RARα cells in a time-dependent manner ( P < 0.0001). The IC50 value of AITS was significantly lower than the other groups ( P < 0.0001), and AITS group exhibited the strongest inhibitory effect, followed by ITS and A. Hoechst staining and JC-1 detection both showed that AITS had the strongest effect in inducing apoptosis. Annxin V/PI detection showed that the apoptosis rate of the control group was 10.04%, 15.19% for A group, 12.46% for ITS group, and 28.05% for AITS group. The differences between the groups were statistically significant ( P < 0.0001). Detection of CD33 expression showed: control group (79.72%), ITS group (79.19%), A group (28.60%), AITS group (24.46%), and there were significant differences between groups ( P < 0.001 or P < 0.0001). AITS could reduce PML A216V-RARα protein, caspase3, Bcl-2 levels more than ITS and A ( P < 0.05 or P < 0.0001), and ITS was better than AITS and A in increasing cleaved PARP-1 levels ( P < 0.0001). AITS decreased the levels of PI3K, mTOR, P-mTOR, AKT, p-AKT and p62 and increased the level of LC3B ( P < 0.001 or P < 0.0001). Electron microscopic detection of autophagy showed that AITS had the highest level of autophagy, AITS combined with LY294002 increased the level of p62 and decreased the level of mTOR ( P < 0.0001) compared to the AITS group; while when AITS was combined with Baf A1, the level of p62 increased ( P < 0.0001) and the level of mTOR also increased ( P < 0.001). Autophagy could induced by serum starvation. As the concentration of fetal bovine serum decreased, the effects of degrading PML A216V-RARα and p62, and LC3B level increasing were stronger. C onclusion: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PML A216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy.

Hypoxic preconditioned MSCs-derived small extracellular vesicles for photoreceptor protection in retinal degeneration

Photoreceptor apoptosis is an important pathogenesis of retinal degeneration and a primary cause of vision loss with limited treatment methods. Mesenchymal stem/stromal cells-derived small extracellular vesicles (MSC-sEVs) have shown therapeutic value in various ocular disorders. Recent studies have revealed that hypoxic preconditioning can improve the effectiveness of MSC-sEVs in tissue regeneration. However, whether hypoxic preconditioned MSC-sEVs (Hyp-sEVs) exert superior effects on photoreceptor protection relative to normoxic conditioned MSC-sEVs (Nor-sEVs) remains unclear. Here, we reported that Hyp-sEVs further improved retinal structure, recovered retinal function, and suppressed photoreceptor apoptosis in N-methyl-N-nitrosourea (MNU)-induced mouse model compared with Nor-sEVs. Hyp-sEVs also exhibited enhanced anti-apoptotic roles in MNU-provoked 661 W cell injury in vitro. We then analyzed the protein profiles of Nor-sEVs and Hyp-sEVs by LC-MS/MS and found that growth-associated protein 43 (GAP43) was enriched in Hyp-sEVs. The knockdown of GAP43 abolished the retinal therapeutic effects of Hyp-sEVs. Mechanistically, hypoxic stimulation-induced hypoxia-inducible factor-1α (HIF-1α) activation was responsible for preventing tripartite motif-containing protein 25 (TRIM25)-mediated GAP43 ubiquitination and degradation, leading to the upregulation of GAP43 in Hyp-sEVs. Together, our findings uncover the efficacy and mechanism of Hyp-sEVs-based photoreceptor protection and highlight the potential of Hyp-sEVs as optimized therapeutics for retinal degeneration.Graphical abstract

The effect of TRIM5 variants on the susceptibility to HIV-1 infection and disease progression in the Polish population.

Tripartite motif containing 5α protein is a factor contributing to intracellular defense mechanisms against human immunodeficiency virus-1 (HIV-1) infection. The studies of TRIM5 variants effects on the risk of HIV-1 infection and the clinical course of disease provided inconclusive results in different ethnic groups. The aim of this study was to investigate the influence of TRIM5 variants on susceptibility to HIV-1 infection and clinical parameters among Polish HIV-1-infected patients. In our study, we investigated 301 HIV-1-infected patients and 186 age-matched seronegative controls. Seven variants of the TRIM5 gene (rs7127617, rs3824949, rs3740996, rs11601507, rs10838525, rs11038628, and rs28381981) were genotyped using both sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. The frequencies of rs7127617 TT genotype and T allele occurrence were lower in HIV-1-infected subjects compared to controls (0.14 vs. 0.26 for T/T genotype and 0.45 vs. 0.54 for T allele), suggesting their possible protective effect (p=0.005 and p=0.007, respectively). Heterozygosity and presence of the T allele at rs3740996 were enriched in controls compared to HIV-1-infected group (0.19 vs. 0.12 for C/T genotype and 0.11 vs. 0.07 for T allele; p=0.03 and p=0.02, respectively). Moreover, rs3824949 CC genotype carriers had a lower viral load than patients bearing rs3824949 GG/CG genotypes (4.0 vs. 4.6 log copies/mL; p=0.049); however, none of the variants affected CD4+ cell count. In conclusion, our data confirm the role of TRIM5 variants in the HIV-1 transmission and the clinical course of HIV-1 infection. The presence of rs7127617 TT genotype and T allele seems to protect against HIV-1 transmission in examined population.

Degradation of AZGP1 suppresses the progression of breast cancer cells via TRIM25.

Alpha-2-glycoprotein 1, zinc-binding (AZGP1) is a secreted protein, which has been shown to be a potential biomarker of cancer progression; however, its roles in breast cancer are still unclear. Currently, we analyzed the online datasets and found that AZGP1 was highly expressed in breast cancer tissues and its expression was negatively correlated with the survival of breast cancer patients. Functional experiments through AZGP1 knockdown revealed that AZGP1 could promote the proliferation, migration, and invasion ability of breast cancer cells. In vivo experiments obtained a consistent result. Mechanistically, it was found that AZGP1 interacted with tripartite motif-containing protein 25 (TRIM25), which subsequently promoted AZGP1 degradation through facilitating the ubiquitination. Furthermore, overexpression of TRIM25 partially reversed the promoting effects of AZGP1 overexpression on breast cancer progression. Therefore, this study indicates that AZGP1 might be a potential therapeutic target for breast cancer treatment.