The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.     

MicroRNA‐148a‐3p suppresses epithelial‐to‐mesenchymal transition and stemness properties via Wnt1‐mediated Wnt/β‐catenin pathway in pancreatic cancer

Although miR‐148a‐3p has been reported to function as a tumour suppressor in various cancers, the molecular mechanism of miR‐148a‐3p in regulating epithelial‐to‐mesenchymal transition (EMT) and stemness properties of pancreatic cancer (PC) cells remains to be elucidated. In the present study, we demonstrated that miR‐148a‐3p expression was remarkably down‐regulated in PC tissues and cell lines. Moreover, low expression of miR‐148a‐3p was associated with poorer overall survival (OS) in patients with PC. In vitro, gain‐of‐function and loss‐of‐function experiments showed that miR‐148a‐3p suppressed EMT and stemness properties as well as the proliferation, migration and invasion of PC cells. A dual‐luciferase reporter assay demonstrated that Wnt1 was a direct target of miR‐148a‐3p, and its expression was inversely associated with miR‐148a‐3p in PC tissues. Furthermore, miR‐148a‐3p suppressed the Wnt/β‐catenin pathway via down‐regulation of Wnt1. The effects of ectopic miR‐148a‐3p were rescued by Wnt1 overexpression. These biological functions of miR‐148a‐3p in PC were also confirmed in a nude mouse xenograft model. Taken together, these findings suggest that miR‐148a‐3p suppresses PC cell proliferation, invasion, EMT and stemness properties via inhibiting Wnt1‐mediated Wnt/β‐catenin pathway and could be a potential prognostic biomarker as well as a therapeutic target in PC.     

TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin‐RING ubiquitin ligase CUL‐2^LRR‐1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC‐48 “unfoldase”. Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome‐associated TIMELESS‐TIPIN complex is required for CUL‐2^LRR‐1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS‐TIPIN, CUL‐2^LRR‐1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM‐7 subunit of CMG. Subsequently, the UBXN‐3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC‐48_UFD‐1_NPL‐4. We show that UBXN‐3 is important in vivo for replisome disassembly in the absence of TIMELESS‐TIPIN. Correspondingly, co‐depletion of UBXN‐3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN‐3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.     

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

Low‐density lipoprotein (LDL)‐cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin‐inducible rapid degradation of oxysterol‐binding protein‐related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL‐derived cholesterol from late endosomes to focal adhesion kinase (FAK)‐/integrin‐positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P₂‐containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P₂ generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P₂ in NPC1‐containing late endosomes in a FAK‐dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL‐cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P₂ exchange between late and recycling endosomes.     

Mitochondrial aconitase 1 regulates age‐related memory impairment via autophagy/mitophagy‐mediated neural plasticity in middle‐aged flies

Age‐related memory impairment (AMI) occurs in many species, including humans. The underlying mechanisms are not fully understood. In wild‐type Drosophila (w¹¹¹⁸ ), AMI appears in the form of a decrease in learning (3‐min memory) from middle age (30 days after eclosion [DAE]). We performed in vivo, DNA microarray, and behavioral screen studies to identify genes controlling both lifespan and AMI and selected mitochondrial Acon1 (mAcon1). mAcon1 expression in the head of w¹¹¹⁸ decreased with age. Neuronal overexpression of mAcon1 extended its lifespan and improved AMI. Neuronal or mushroom body expression of mAcon1 regulated the learning of young (10 DAE) and middle‐aged flies. Interestingly, acetyl‐CoA and citrate levels increased in the heads of middle‐aged and neuronal mAcon1 knockdown flies. Acetyl‐CoA, as a cellular energy sensor, is related to autophagy. Autophagy activity and efficacy determined by the positive and negative changes in the expression levels of Atg8a‐II and p62 were proportional to the expression level of mAcon1. Levels of the presynaptic active zone scaffold protein Bruchpilot were inversely proportional to neuronal mAcon1 levels in the whole brain. Furthermore, mAcon1 overexpression in Kenyon cells induced mitophagy labeled with mt‐Keima and improved learning ability. Both processes were blocked by pink1 knockdown. Taken together, our results imply that the regulation of learning and AMI by mAcon1 occurs via autophagy/mitophagy‐mediated neural plasticity.     

Fat‐1 expression alleviates atherosclerosis in transgenic rabbits

Atherosclerosis is the main cause of cardiovascular diseases. The Fat‐1 gene can express the n‐3 fatty acid desaturase, which converts n‐6 polyunsaturated fatty acids (PUFA) to n‐3 PUFAs. The role of n‐3 PUFAs in atherosclerosis is widely debated. This study explored the effect of n‐3 PUFAs on atherosclerosis in rabbits. In this study, atherosclerosis was induced in Fat‐1 transgenic rabbits and their littermate (WT) rabbits by feeding a high‐cholesterol diet containing 0.3% cholesterol and 3% soybean oil for 16 weeks. Plasma lipid, fatty acid and pathological analyses of atherosclerotic lesions were conducted. Fatty acid composition in the liver and muscle showed that n‐3 PUFAs increased and n‐6 PUFAs decreased in the Fat‐1 group. Plasma high‐density lipoprotein cholesterol (HDL‐C) levels were significantly increased in the Fat‐1 group, and the atherosclerotic lesion area of the aortic arch in Fat‐1 transgenic rabbits was significantly reduced. Histological analysis showed that smooth muscle cells (SMCs) in atherosclerotic lesions decreased significantly. In conclusion, n‐3 PUFAs improve atherosclerosis in Fat‐1 transgenic rabbits, and this process may depend on the increase in plasma HDL‐C and the decrease in the amount of SMCs in atherosclerotic plaques.     

Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

Prohibitin depletion extends lifespan of a TORC2/SGK‐1 mutant through autophagy and the mitochondrial UPR

Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar effect on lifespan. While PHB depletion shortens lifespan of wild‐type animals, it enhances longevity of a plethora of metabolically compromised mutants, including target of rapamycin complex 2 (TORC2) mutants sgk‐1 and rict‐1. Here, we show that sgk‐1 mutants have impaired mitochondrial homeostasis, lipogenesis and yolk formation, plausibly due to alterations in membrane lipid and sterol homeostasis. Remarkably, all these features are suppressed by PHB depletion. Our analysis shows the requirement of SRBP1/SBP‐1 for the lifespan extension of sgk‐1 mutants and the further extension conferred by PHB depletion. Moreover, although the mitochondrial unfolded protein response (UPR^mt) and autophagy are induced in sgk‐1 mutants and upon PHB depletion, they are dispensable for lifespan. However, the enhanced longevity caused by PHB depletion in sgk‐1 mutants requires both, the UPR^mt and autophagy, but not mitophagy. We hypothesize that UPR^mt induction upon PHB depletion extends lifespan of sgk‐1 mutants through autophagy and probably modulation of lipid metabolism.     

The PAX5‐JAK2 translocation acts as dual‐hit mutation that promotes aggressive B‐cell leukemia via nuclear STAT5 activation

While PAX5 is an important tumor suppressor gene in B‐cell acute lymphoblastic leukemia (B‐ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5‐JAK2 encodes a protein consisting of the PAX5 DNA‐binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5‐JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5 ^Jak2/+ mice rapidly developed an aggressive B‐ALL in the absence of another cooperating exogenous gene mutation. The DNA‐binding function and kinase activity of Pax5‐Jak2 as well as IL‐7 signaling contributed to leukemia development. Interestingly, all Pax5 ^Jak2/+ tumors lost the remaining wild‐type Pax5 allele, allowing efficient DNA‐binding of Pax5‐Jak2. While we could not find evidence for a nuclear role of Pax5‐Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5 ^Jak2/+ B‐ALL tumors, implying that nuclear Pax5‐Jak2 phosphorylates STAT5. Together, these data reveal Pax5‐Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.     

Yeast cells accumulate excess endogenous palmitate in phosphatidylcholine by acyl chain remodeling involving the phospholipase B Plb1p

In the yeast Saccharomyces cerevisiae, the molecular species profile of the major membrane glycerophospholipid phosphatidylcholine (PC) is determined by the molecular species-selectivity of the biosynthesis routes and by acyl chain remodeling. Overexpression of the glycerol-3-phosphate acyltransferase Sct1p was recently shown to induce a strong increase in the cellular content of palmitate (C16:0). Using stable isotope labeling and mass spectrometry, the present study shows that wild type yeast overexpressing Sct1p incorporates excess C16:0 into PC via the methylation of PE, the CDP-choline route, and post-synthetic acyl chain remodeling. Overexpression of Sct1p increased the extent of remodeling of PE-derived PC, providing a novel tool to perform mechanistic studies on PC acyl chain exchange. The exchange of acyl chains occurred at both the sn-1 and sn-2 positions of the glycerol backbone of PC, and required the phospholipase B Plb1p for optimal efficiency. Sct1p-catalyzed acyl chain exchange, the acyl-CoA binding protein Acb1p, the Plb1p homologue Plb2p, and the glycerophospholipid:triacylglycerol transacylase Lro1p were not required for PC remodeling. The results indicate that PC serves as a buffer for excess cellular C16:0.     

Cancer‐associated mutations in VAV1 trigger variegated signaling outputs and T‐cell lymphomagenesis

Mutations in VAV1, a gene that encodes a multifunctional protein important for lymphocytes, are found at different frequencies in peripheral T‐cell lymphoma (PTCL), non‐small cell lung cancer, and other tumors. However, their pathobiological significance remains unsettled. After cataloguing 51 cancer‐associated VAV1 mutations, we show here that they can be classified in five subtypes according to functional impact on the three main VAV1 signaling branches, GEF‐dependent activation of RAC1, GEF‐independent adaptor‐like, and tumor suppressor functions. These mutations target new and previously established regulatory layers of the protein, leading to quantitative and qualitative changes in VAV1 signaling output. We also demonstrate that the most frequent VAV1 mutant subtype drives PTCL formation in mice. This process requires the concurrent engagement of two downstream signaling branches that promote the chronic activation and transformation of follicular helper T cells. Collectively, these data reveal the genetic constraints associated with the lymphomagenic potential of VAV1 mutant subsets, similarities with other PTCL driver genes, and potential therapeutic vulnerabilities.     

Circular RNA hsa_circ_0008003 facilitates tumorigenesis and development of non‐small cell lung carcinoma via modulating miR‐488/ZNF281 axis

As one of the most aggressive malignancies, non‐small cell lung carcinoma (NSCLC) has high risks of death. It has been demonstrated that circRNAs accelerate NSCLC progression, but the underlying molecular mechanisms of circRNAs in NSCLC were still obscure. In the first place, the circRNA microarray of NSCLC was investigated in this study, and hsa_circ_0008003 (circ‐0008003) was chosen as the research object. Then, it was unveiled that the expression of circ‐0008003 examined via qRT‐PCR was elevated in tumour tissues relative to the non‐tumour tissues, which was associated with TNM stage and lymphatic metastasis in NSCLC. Additionally, the prognosis of NSCLC patients with high circ‐0008003 level was poor. Besides, circ‐0008003 silencing dampened the invasion and proliferation of NSCLC cells. Next, according to the mechanistic studies, circ‐0008003 functioned as a ceRNA of ZNF281 in NSCLC by acting as the endogenous sponge for miR‐488, which was proved to be a tumour suppressor in NSCLC. Additionally, ZNF281 overexpression and miR‐488 suppression recovered the influences of repressed circ‐0008003 on NSCLC cellular processes. It was validated in this research that circ‐0008003 triggered tumour formation in NSCLC, which was adjusted via miR‐488/ZNF281 axis, casting a novel light on the resultful target for treating NSCLC and predicting the prognosis.     

Syncytiotrophoblast‐derived extracellular vesicles carry apolipoprotein‐E and affect lipid synthesis of liver cells in vitro

In normal pregnancy, hepatic metabolism adaptation occurs with an increase in lipid biosynthesis. Placental shedding of syncytiotrophoblast‐derived extracellular vesicles (STBEVs) into the maternal circulation constitutes a major signalling mechanism between foetus and mother. We investigated whether STBEVs from normal pregnant women might target liver cells in vitro and induce changes in lipid synthesis. This study was performed at the Nuffield Department of Women''s & Reproductive Health, Oxford, UK. STBEVs were obtained by dual‐lobe placental perfusion from 11 normal pregnancies at term. Medium/large and small STBEVs were collected by ultracentrifugation at 10,000g and 150,000g, respectively. STBEVs were analysed by Western blot analysis and flow cytometry for co‐expression of apolipoprotein‐E (apoE) and placental alkaline phosphatase (PLAP). The uptake of STBEVs by liver cells and the effect on lipid metabolism was evaluated using a hepatocarcinoma cell line (HepG2 cells). Data were analysed by one‐way ANOVA and Student''s t test. We demonstrated that: (a) STBEVs carry apoE; (b) HepG2 cells take up STBEVs through an apoE‐LDL receptor interaction; (c) STBEV incorporation into HepG2 cells resulted in (i) increased cholesterol release (ELISA); (ii) increased expression of the genes SQLE and FDPS (microarray) involved in cholesterol biosynthesis; (iii) downregulation of the CLOCK gene (microarray and PCR), involved in the circadian negative control of lipid synthesis in liver cells. In conclusion, the placenta may orchestrate the metabolic adaptation of the maternal liver through release of apoE‐positive STBEVs, by increasing lipid synthesis in a circadian‐independent fashion, meeting the nutritional needs of the growing foetus.     

LOXL1‐AS1 communicating with TIAR modulates vasculogenic mimicry in glioma via regulation of the miR‐374b‐5p/MMP14 axis

At present, growing evidence indicates that long non‐coding RNAs (lncRNAs) participate in the progression of glioma. The function of LOXL1‐AS1 in vasculogenic mimicry (VM) in glioma remains unclear. First, the expressions of TIAR, the lncRNA LOXL1‐AS1, miR‐374b‐5p and MMP14 were examined by qRT‐PCR and Western blot in both, glioma tissues and glioma cell lines. Proliferation, migration, invasion and tube formation assays were conducted to evaluate the roles of TIAR, LOXL1‐AS1, miR‐374b‐5p and MMP14 in malignant cellular behaviours in glioma cells. A nude mouse xenograft model and dual staining for CD34 and PAS were used to assess whether VM was affected by TIAR, LOXL1‐AS1 or miR‐374b‐5p in vivo. In this study, low levels of TIAR and high levels of LOXL1‐AS1 were found in glioma cells and tissues. TIAR downregulated the expression of LOXL1‐AS1 by destabilizing it. LOXL1‐AS1 acted like a miRNA sponge towards miR‐374b‐5p so that downregulation of the former greatly inhibited cell proliferation, migration, invasion and VM. Additionally, miR‐374b‐5p overexpression repressed malignant biological behaviours and VM in glioma by modifying MMP14. In summary, we demonstrated that TIAR combined with LOXL1‐AS1 modulates VM in glioma via the miR‐374b‐5p/MMP14 axis, revealing novel targets for glioma therapy.     

Telmisartan anti‐cancer activities mechanism through targeting N‐cadherin by mimicking ADH‐1 function

This study aimed to investigate if Telmisartan as a novel N‐cadherin antagonist, can overcome cell migration of cancer cells. We investigated the mechanism and influence of Docetaxel and Telmisartan (as an analogous to ADH‐1, which is a well‐known N‐cadherin antagonist) on cancer cells. The effect of ADH‐1 and Telmisartan on cell attachment in PC3, DU145, MDA‐MB‐468 cell lines using recombinant human N‐cadherin was studied. Cell viability assay was performed to examine the anti‐proliferative effects of Telmisartan, ADH‐1 and Docetaxel. Migration was examined via wound healing assay, and apoptosis was determined by flow cytometry. The expression of AKT‐1 as a downstream gene of N‐cadherin signalling pathway was assayed by real‐time PCR. Treatment of PC3, MDA‐MB‐468 and DU145 cells with Telmisartan (0.1 µM) and ADH‐1 (40 µM) resulted in 50%, 58% and approximately 20% reduction in cell attachment to N‐cadherin coated plate respectively. It shows reduction of cell attachment in PC3 and MDA‐MB‐468 cell lines appeared to be more sensitive than that of DU145 cells to the Telmisartan and ADH‐1 treatments. Telmisartan (0.1 µM) and Docetaxel (0.01 nM) significantly reduced cell migration in PC3 and MDA‐MB‐468 cell lines compared with the control group. Using Real‐time PCR, we found that Telmisartan, Docetaxel and ADH‐1 had significant influence on the AKT‐1 mRNA level. The results of the current study for the first time suggest that, Telmisartan, exerts anti‐proliferation and anti‐migration effects by targeting antagonistically N‐cadherin. Also, these data suggest that Telmisartan as a less expensive alternative to ADH‐1 could potentiate Docetaxel anticancer effects.