XBP1 variant 1 promotes mitosis of cancer cells involving upregulation of the polyglutamylase TTLL6.

XBP1 variant 1 (Xv1) is the most abundant XBP1 variant and is highly enriched across cancer types but nearly none in normal tissues. Its expression is associated with poor patients' survival and is specifically required for survival of malignant cells, but the underlying mechanism is not known. Here we report that Xv1 upregulates the polyglutamylase tubulin tyrosine ligase-like 6 (TTLL6) and promotes mitosis of cancer cells. Like the canonical XBP1, Xv1 mRNA undergoes unconventional splicing by IRE1α under endoplasmic reticulum stress, but it is also constitutively spliced by IRE1ß. The spliced Xv1 mRNA encodes the active form of Xv1 protein (Xv1s). RNA sequencing in HeLa cells revealed that Xv1s overexpression regulates expression of genes that are not involved in the canonical unfolded protein response, including TTLL6 as a highly upregulated gene. Gel shift assay and chromatin immunoprecipitation revealed that Xv1s bind to the TTLL6 promoter region. Knockdown of TTLL6 caused death of cancer cells but not benign and normal cells, similar to the effects of knocking down Xv1. Moreover, overexpression of TTLL6 partially rescued BT474 cells from apoptosis induced by either TTLL6 or Xv1 knockdown, supporting TTLL6 as an essential downstream effector of Xv1 in regulating cancer cell survival. TTLL6 is localized in the mitotic spindle of cancer cells. Xv1 or TTLL6 knockdown resulted in decreased spindle polyglutamylation and interpolar spindle, as well as congression failure, mitotic arrest and cell death. These findings suggest that Xv1 is essential for cancer cell mitosis, which is mediated, at least in part, by increasing TTLL6 expression.

A novel XBP1 variant is highly enriched in cancer tissues and is specifically required for cancer cell survival.

XBP1 is a basic leucine zipper (bZIP) transcription factor and a key mediator of the endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR). XBP1-mediated transcription facilitates cell adaptation to ER stress and also promotes tumor progression, while suppressing anti-tumor immunity. Here we report a novel XBP1 variant, namely XBP1 variant 1 (XBP1v1, Xv1 for short), that is specifically required for survival of cancer cells. Xv1 contains a cryptic first exon that is conserved only in humans and great apes. Comparing to XBP1, Xv1 encodes a protein with a different N-terminal sequence containing 25 amino acids. Analysis of RNAseq database reveals that Xv1 is broadly expressed across cancer types but almost none in normal tissues. Elevated Xv1 expression is associated with poor survival of patients with several types of cancer. Knockdown of Xv1 induces death of multiple cancer cell lines but has little effect on non-cancerous cells in vitro. Moreover, knockdown of Xv1 also inhibits growth of a xenograft breast tumor in mice. Together, our results indicate that Xv1 is essential for survival of cancer cells.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Inhibits Liver Cancer Through Small Ubiquitin-Related Modifier (SUMO)ylation-Related Suppression of NF-κB/Snail Signaling Pathway and Epithelial-Mesenchymal Transition.

BACKGROUND AND AIMS: Endoplasmic reticulum (ER) stress is associated with liver inflammation and hepatocellular carcinoma (HCC). However, how ER stress links inflammation and HCC remains obscure. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER stress-inducible secretion protein that inhibits inflammation by interacting with the key subunit of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) p65. We hypothesized that MANF may play a key role in linking ER stress and inflammation in HCC. APPROACH AND RESULTS: Here, we found that MANF mRNA and protein levels were lower in HCC tissues versus adjacent noncancer tissues. Patients with high levels of MANF had better relapse-free survival and overall survival rates than those with low levels. MANF levels were also associated with the status of liver cirrhosis, advanced tumor-node-metastasis (TNM) stage, and tumor size. In vitro experiments revealed that MANF suppressed the migration and invasion of hepatoma cells. Hepatocyte-specific deletion of MANF accelerated N-nitrosodiethylamine (DEN)-induced HCC by up-regulating Snail1+2 levels and promoting epithelial-mesenchymal transition (EMT). MANF appeared in the nuclei and was colocalized with p65 in HCC tissues and in tumor necrosis factor alpha (TNF-α)-treated hepatoma cells. The interaction of p65 and MANF was also confirmed by coimmunoprecipitation experiments. Consistently, knockdown of MANF up-regulated NF-κB downstream target genes TNF-α, interleukin (IL)-6 and IL-1α expression in vitro and in vivo. Finally, small ubiquitin-related modifier 1 (SUMO1) promoted MANF nuclear translocation and enhanced the interaction of MANF and p65. Mutation of p65 motifs for SUMOylation abolished the interaction of p65 and MANF. CONCLUSIONS: MANF plays an important role in linking ER stress and liver inflammation by inhibiting the NF-κB/Snail signal pathway in EMT and HCC progression. Therefore, MANF may be a cancer suppressor and a potential therapeutic target for HCC.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects against Aß toxicity via attenuating Aß-induced endoplasmic reticulum stress.

BACKGROUND: Extracellular accumulation of amyloid ß-peptide (Aß) is one of pathological hallmarks of Alzheimer's disease (AD) and contributes to the neuronal loss. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-inducible neurotrophic factor. Many groups, including ours, have proved that MANF rescues neuronal loss in several neurological disorders, such as Parkinson's disease and cerebral ischemia. However, whether MANF exerts its protective effect against Aß neurotoxicity in AD remains unknown. METHODS: In the present study, the characteristic expressions of MANF in Aß1-42-treated neuronal cells as well as in the brains of APP/PS1 transgenic mice were analyzed by immunofluorescence staining, qPCR, and Western blot. The effects of MANF overexpression, MANF knockdown, or recombination human MANF protein (rhMANF) on neuron viability, apoptosis, and the expression of ER stress-related proteins following Aß1-42 exposure were also investigated. RESULTS: The results showed the increased expressions of MANF, as well as ER stress markers immunoglobulin-binding protein (BiP) and C/EBP homologous protein (CHOP), in the brains of the APP/PS1 transgenic mice and Aß1-42-treated neuronal cells. MANF overexpression or rhMANF treatment partially protected against Aß1-42-induced neuronal cell death, associated with marked decrease of cleaved caspase-3, whereas MANF knockdown with siRNA aggravated Aß1-42 cytotoxicity including caspase-3 activation. Further study demonstrated that the expressions of BiP, ATF6, phosphorylated-IRE1, XBP1s, phosphorylated-eIF2α, ATF4, and CHOP were significantly downregulated by MANF overexpression or rhMANF treatment in neuronal cells following Aß1-42 exposure, whereas knockdown of MANF has the opposite effect. CONCLUSIONS: These findings demonstrate that MANF may exert neuroprotective effects against Aß-induced neurotoxicity through attenuating ER stress, suggesting that an applicability of MANF as a therapeutic candidate for AD.

Effects of bone marrow mesenchymal stem cells transfected with Ang-1 gene on hyperoxia-induced optic nerve injury in neonatal mice.

Optic nerve injury triggered retinal ganglion cell (RGC) death and optic nerve atrophy lead to visual loss. Bone marrow mesenchymal stem cells (BMSCs) are stromal cells, capable of proliferating and differentiating into different types of tissues. This aims of this study is to investigate the role of BMSCs transfected with angiopoietin-1 (Ang-1) in optic nerve injury induced by hyperoxia in a neonatal mice model. Ang-1 overexpression vector was constructed and used to transfect BMSCs. Reverse transcription-quantitative polymerase chain reaction was performed to detect Ang-1 expression in BMSCs. The hyperoxia-induced optic nerve injury model was established. The optic nerves at 6-7 mm posterior to the eyeball were extracted, and were treated with luxol fast blue staining, immunohistochemistry, immunofluorescence, and transmission electron microscopy to examine the effects of Ang-1-modified BMSCs on optic nerve injury induced by hyperoxia. The mice in the Ang-1 + BMSCs and BMSCs groups showed remarkably improved myelin sheaths of nerve fibers compared to the hyperoxia saline group. The positive expression and integrated optic density of Ang-1 in the Ang-1 + BMSCs group were significantly higher compared to the air control, hyperoxia saline and BMSCs groups. The number and diameter of myelinated nerve fibers, the diameter of axons and the thickness of myelin sheath in the air control and Ang-1 + BMSCs groups were higher compared to the hyperoxia saline group. Our study provides evidence supporting that Ang-1-modified BMSCs may have preventive and therapeutic effects on hyperoxia-induced optic nerve injury in neonatal mice.

DPF2 regulates OCT4 protein level and nuclear distribution.

The amount of transcription factor OCT4 is strictly regulated. A tight regulation of OCT4 levels is crucial for mammalian embryonic development and oncogenesis. However, the mechanisms underlying regulation of OCT4 protein expression and nuclear distribution are largely unknown. Here, we report that DPF2, a plant homeodomain (PHD) finger protein, is upregulated during H9 cell differentiation induced by retinoic acid. Endogenous interaction between DPF2 and OCT4 in P19 cells was revealed by an immunoprecipitation assay. GST-pull down assay proved that OCT4 protein in H9 cells and recombinant OCT4 can precipitate with DPF2 in vitro. In vitro ubiquitination assay demonstrated DPF2 might serve as an E3 ligase. Knock down of dpf2 using siRNA increased OCT4 protein level and stability in P19 cells. DPF2 siRNAs also up-regulates OCT4 but not NANOG in H9 cells. However, RA fails to downregulates OCT4 protein level in cells infected by lenitviruses containing DPF2 siRNA. Moreover, overexpression of both DPF2 and OCT4 in 293 cells proved the DPF2-OCT4 interaction. DPF2 but not PHD2 mutant DPF2 enhanced ubiquitination and degradation of OCT4 in 293 cells co-expressed DPF2 and OCT4. Both wild type DPF2 and PHD2 mutant DPF2 redistributes nuclear OCT4 without affecting DPF2-OCT4 interaction. Further analysis indicated that DPF2 decreases monomeric and mono-ubiquitinated OCT4, assembles poly-ubiquitin chains on OCT4 mainly through Ub-K48 linkage. These findings contribute to an understanding of how OCT4 protein level and nuclear distribution is regulated by its associated protein.

Identification of ERAD components essential for dislocation of the null Hong Kong variant of α-1-antitrypsin (NHK).

Misfolded proteins or orphan subunits of protein complexes are removed from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD). ERAD requires dislocation, also known as retrotranslocation, of those unwanted proteins from the ER lumen to the cytosol for destruction by the proteasomes. Over one hundred ERAD component proteins have been identified but their role in dislocation remain poorly understood. Here we assessed the requirement of ERAD components for dislocation of NHK in live cells using our recently developed dislocation-induced reconstituted GFP (drGFP) assay. RNAi revealed that 12 out of 21 ERAD components examined are required for efficient dislocation of NHK among which Hrd1, Sel1L, GRP94 and p97/VCP are critically required. In addition, knockdown of 7 of the 21 components enhanced NHK dislocation. This study uncovers a complex functional network of proteins required for NHK dislocation.

Expression of Smyd1b_tv1 by Alternative Splicing in Cardiac Muscle is Critical for Sarcomere Organization in Cardiomyocytes and Heart Function.

Smyd1, a member of the Smyd lysine methyltransferase family, plays an important role in myofibrillogenesis of skeletal and cardiac muscles. Loss of Smyd1b (a Smyd1 ortholog) function in zebrafish results in embryonic death from heart malfunction. smyd1b encodes two isoforms, Smyd1b_tv1 and Smyd1b_tv2, differing by 13 amino acids due to alternative splicing. While smyd1 alternative splicing is evolutionarily conserved, the isoform-specific expression and function of Smyd1b_tv1 and Smyd1b_tv2 remained unknown. Here we analyzed their expression and function in skeletal and cardiac muscles. Our analysis revealed expression of smyd1b_tv1 predominately in cardiac and smyd1b_tv2 in skeletal muscles. Using zebrafish models expressing only one isoform, we demonstrated that Smyd1b_tv1 is essential for cardiomyocyte differentiation and fish viability, whereas Smyd1b_tv2 is dispensable for heart development and fish survival. Cellular and biochemical analyses revealed that Smyd1b_tv1 differs from Smyd1b_tv2 in protein localization and binding with myosin chaperones. While Smyd1b_tv2 diffused in the cytosol of muscle cells, Smyd1b_tv1 was localized to M-lines and essential for sarcomere organization in cardiomyocytes. Co-IP analysis revealed a stronger binding of Smyd1b_tv1 with chaperones and cochaperones compared with Smyd1b_tv2. Collectively, these findings highlight the nonequivalence of Smyd1b isoforms in cardiomyocyte differentiation, emphasizing the critical role of Smyd1b_tv1 in cardiac function.

Translocational attenuation mediated by the PERK-SRP14 axis is a protective mechanism of unfolded protein response.

The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein folding capabilities, and enhancing misfolded protein degradation. After a multi-omics analysis, we find that signal recognition particle 14 (SRP14), an essential component of the SRP, is markedly reduced in cells undergoing ER stress. Further experiments indicate that SRP14 reduction requires PRKR-like ER kinase (PERK)-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation but is independent of ATF4 or ATF3 transcription factors. The decrease of SRP14 correlates with reduced translocation of fusion proteins and endogenous cathepsin D. Enforced expression of an SRP14 variant with elongation arrest capability prevents the reduced translocation of cathepsin D in stressed cells, whereas an SRP14 mutant without the activity does not. Finally, overexpression of SRP14 augments the UPR and aggravates ER-stress-induced cell death. These data suggest that translocational attenuation mediated by the PERK-SRP14 axis is a protective measure for the UPR to mitigate ER stress.

Live cell imaging of protein dislocation from the endoplasmic reticulum.

Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin ß proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.

TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia.

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.

Mutant Atp13a2 proteins involved in parkinsonism are degraded by ER-associated degradation and sensitize cells to ER-stress induced cell death.

Mutations in ATP13A2 (PARK9) have been linked to juvenile parkinsonism with dementia or Kufor-Rakeb syndrome (KRS). The ATP13A2 gene encodes at least three protein isoforms that arise by alternate splicing. A previous study indicated the Atp13a2(Isoform-1) protein is localized to lysosomes, whereas three separate mutations involved in disease cause retention of the protein in the ER. One speculation is that the mutant Atp13a2(Isoform-1) proteins are misfolded and eliminated by the ER-associated degradation pathway (ERAD), which involves the dislocation of proteins from the ER to the cytoplasm for proteasome degradation. We examined whether Atp13a2 proteins are degraded by ERAD and whether the Atp13a2(Isoform-3) protein has similar localization to the Atp13a2(Isoform-1) protein. Through analysis of protein turnover and by disrupting different steps in the ERAD pathway we demonstrate that mutant Atp13a2(Isoform-1) proteins are indeed eliminated by ERAD. Thus, siRNA-mediated knockdown of erasin, a platform for assembly of an ERAD complex, or expression of a dominant negative form of p97/VCP, a protein essential for dislocation of ERAD substrates, or inhibition of the proteasome all slowed degradation of the mutant Atp13a2(Isoform-1) proteins, but not the wild-type Atp13a2(Isoform-1) protein. Immunoprecipitation assays confirmed that the Atp13a2 proteins are ubiquitinated in accord with degradation by ERAD. In contrast to Atp13a2(Isoform-1), we show Atp13a2(Isoform-3) is localized to the ER and rapidly degraded. Lastly, we show Atp13a2 mutants have increased cytotoxicity and predispose cells to ER-stress-induced cell death. These results provide new insight into the properties of wild-type and mutant Atp13a2 proteins involved in KRS.

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis.

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and ß-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and ß-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, ß-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas ß-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the ß-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and ß-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and ß-III-tubulin during the neural differentiation of H9 cells.

Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.

UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.

Importin beta interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant alpha1-antitrypsin.

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin ß, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin ß specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin ß is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin ß cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin ß in ER protein quality control.

Ubiquilin functions in autophagy and is degraded by chaperone-mediated autophagy.

Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.

ER stress response during the differentiation of H9 cells induced by retinoic acid.

Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2α was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2α was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells, but its downregulation was much slower in HEK293 cells. Additionally, two ER-resident E3 ubiquitin ligases, gp78 and Hrd1, were both upregulated in H9 cells following 5 days of exposure to RA. Moreover, the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells, and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29 days, GRP78/Bip, XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA.

Upregulation of mesencephalic astrocyte-derived neurotrophic factor in glial cells is associated with ischemia-induced glial activation.

BACKGROUND: Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line. MANF belongs to a novel evolutionally conserved family of neurotrophic factors along with conserved dopamine neurotrophic factor. In recent years, ever-increasing evidence has shown that both of them play a remarkable protective role against various injuries to neurons in vivo or in vitro. However, the characteristics of MANF expression in the different types of glial cells, especially in astrocytes, remain unclear. METHODS: The model of focal cerebral ischemia was induced by rat middle cerebral artery occlusion. Double-labeled immunofluorescent staining was used to identify the types of neural cells expressing MANF. Primarily cultured glial cells were used to detect the response of glial cells to endoplasmic reticulum stress stimulation. Propidium iodide staining was used to determine dead cells. Reverse transcription PCR and western blotting were used to detect the levels of mRNA and proteins. RESULTS: We found that MANF was predominantly expressed in neurons in both normal and ischemic cortex. Despite its name, MANF was poorly expressed in glial cells, including astrocytes, in normal brain tissue. However, the expression of MANF was upregulated in the glial cells under focal cerebral ischemia, including the astrocytes. This expression was also induced by several endoplasmic reticulum stress inducers and nutrient deprivation in cultured primary glial cells. The most interesting phenomenon observed in this study was the pattern of MANF expression in the microglia. The expression of MANF was closely associated with the morphology and state of microglia, accompanied by the upregulation of BIP/Grp78. CONCLUSIONS: These results indicate that MANF expression was upregulated in the activated glial cells, which may contribute to the mechanism of ischemia-induced neural injury.

Regulation of p27 (Kip1) by Ubiquitin E3 Ligase RNF6.

The cyclin-dependent kinase inhibitor p27 (Kip1) is an important regulator of the G1/S checkpoint. It is degraded by the SCF-SKP2 complex in late G1 thereby allowing cells to progress to the S phase. Here we investigated the role of the E3 ubiquitin ligase RNF6 (Ring Finger Protein 6) in cell cycle progression in prostate cancer cells. Our data demonstrate that RNF6 can promote cell cycle progression by reducing the levels of p27. Knockdown of RNF6 led to an increase in the stability of p27 and to the arrest of cells in the G1 phase. RNF6 interacted with p27 via its KIL domain and this interaction was found to be phosphorylation independent. RNF6 enhanced ubiquitination and subsequent degradation of p27 in the early G0/G1 phase of the cell cycle. Knockdown of RNF6 expression by short hairpin RNA led to inhibition of the CDK2/Cyclin E complex thereby reducing phosphorylation of Retinoblastoma protein (Rb) and to a subsequent decrease in cell cycle progression and proliferation. Our data suggest that RNF6 acts as a negative regulator for p27kip1 leading to its proteasome-dependent degradation in the early G0/G1 phase of the cell cycle.

A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase.

RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.