PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Despite the immense success of immune checkpoint blockade (ICB) in cancer treatment, many tumors, including melanoma, exhibit innate or adaptive resistance. Tumor-intrinsic T-cell deficiency and T-cell dysfunction have been identified as essential factors in the emergence of ICB resistance. Here, we found that protein arginine methyltransferase 1 (PRMT1) expression was inversely correlated with the number and activity of CD8+ T cells within melanoma specimen. PRMT1 deficiency or inhibition with DCPT1061 significantly restrained refractory melanoma growth and increased intratumoral CD8+ T cells in vivo. Moreover, PRMT1 deletion in melanoma cells facilitated formation of double-stranded RNA derived from endogenous retroviral elements (ERV) and stimulated an intracellular interferon response. Mechanistically, PRMT1 deficiency repressed the expression of DNA methyltransferase 1 (DNMT1) by attenuating modification of H4R3me2a and H3K27ac at enhancer regions of Dnmt1, and DNMT1 downregulation consequently activated ERV transcription and the interferon signaling. Importantly, PRMT1 inhibition with DCPT1061 synergized with PD-1 blockade to suppress tumor progression and increase the proportion of CD8+ T cells as well as IFNγ+CD8+ T cells in vivo. Together, these results reveal an unrecognized role and mechanism of PRMT1 in regulating antitumor T-cell immunity, suggesting PRMT1 inhibition as a potent strategy to increase the efficacy of ICB. SIGNIFICANCE: Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Transcriptional analysis of the dimorphic fungus Umbilicaria muehlenbergii reveals the molecular mechanism of phenotypic transition.

The lichen-forming fungus Umbilicaria muehlenbergii undergoes a phenotypic transition from a yeast-like to a pseudohyphal form. However, it remains unknown if a common mechanism is involved in the phenotypic switch of U. muehlenbergii at the transcriptional level. Further, investigation of the phenotype switch molecular mechanism in U. muehlenbergii has been hindered by incomplete genomic sequencing data. Here, the phenotypic characteristics of U. muehlenbergii were investigated after cultivation on several carbon sources, revealing that oligotrophic conditions due to nutrient stress (reduced strength PDA (potato dextrose agar) media) exacerbated the pseudohyphal growth of U. muehlenbergii. Further, the addition of sorbitol, ribitol, and mannitol exacerbated the pseudohyphal growth of U. muehlenbergii regardless of PDA medium strength. Transcriptome analysis of U. muehlenbergii grown in normal and nutrient-stress conditions revealed the presence of several biological pathways with altered expression levels during nutrient stress and related to carbohydrate, protein, DNA/RNA and lipid metabolism. Further, the results demonstrated that altered biological pathways can cooperate during pseudohyphal growth, including pathways involved in the production of protectants, acquisition of other carbon sources, or adjustment of energy metabolism. Synergistic changes in the functioning of these pathways likely help U. muehlenbergii cope with dynamic stimuli. These results provide insights into the transcriptional response of U. muehlenbergii during pseudohyphal growth under oligotrophic conditions. Specifically, the transcriptomic analysis indicated that pseudohyphal growth is an adaptive mechanism of U. muehlenbergii that facilitates its use of alternative carbon sources to maintain survival.

A RhoA structure with switch II flipped outward revealed the conformational dynamics of switch II region.

Small GTPase RhoA switches from GTP-bound state to GDP-bound state by hydrolyzing GTP, which is accelerated by GTPases activating proteins (GAPs). However, less study of RhoA structural dynamic changes was conducted during this process, which is essential for understanding the molecular mechanism of GAP dissociation. Here, we solved a RhoA structure in GDP-bound state with switch II flipped outward. Because lacking the intermolecular interactions with guanine nucleotide, we proposed this conformation of RhoA could be an intermediate after GAP dissociation. Further molecular dynamics simulations found the conformational changes of switch regions are indeed existing in RhoA and involved in the regulation of GAP dissociation and GEF recognition. Besides, the guanine nucleotide binding pocket extended to switch II region, indicating a potential "druggable" cavity for RhoA. Taken together, our study provides a deeper understanding of the dynamic properties of RhoA switch regions and highlights the direction for future drug development.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

Application of spectral features for separating homochromatic foreign matter from mixed congee.

Foreign matter (FM) in mixed congee not only reduces the quality of the congee but may also harm consumers. However, the common computer vision methods with poor recognition ability for the homochromatic FM. This study used hyperspectral reflectance images with the pattern recognition model to detect homochromatic FM on the mixed congee surface. First, spectral features corresponding to homochromatic FM and background were extracted from hyperspectral images. Then, based on the optimal spectral preprocessing method, LDA, K-nearest neighbor, backpropagation artificial neural network, and support vector machine (SVM) were used to classify the spectral features. The results revealed that the SVM model input with raw spectra principal components exhibited optimal identification rates of 99.17%. Finally, most of the pixels for homochromatic FM were classified correctly by using the SVM model. To summarized, hyperspectral images combined with pattern recognition are an effective method for recognizing homochromatic FM in mixed congee.

Abnormal expression of miR-135a in patients with depression and its possible involvement in the pathogenesis of the condition.

At present, due to the increasing pressures on society and the stress of everyday living, the number of individuals suffering from depression has increased. Therefore, the treatment of depression has also received increasing attention. MicroRNA (miRNA/miR)-135a is a well-studied miRNA. It has been reported that miR-135a is significantly downregulated in patients with depression and may be a potential marker for the diagnosis of the condition. However, the specific mechanisms of action of miR-135a in patients with depression remain unclear. In the present study, it was found that miR-135a was downregulated in patients with depression, and in a mouse model of depression. The effects of miR-135a on depression-related symptoms in mice were then explored. In the mice with chronic unpredictable mild stress (CUMS) that were treated with miR-135a for 3 weeks, a significantly reduced level of weight gain was observed in comparison with the control group. In addition, treatment with miR-135a mimic significantly increased sucrose preference in the sucrose preference test in the mice, and reduced the immobility time in the forced swimming test and tail suspension test. Treatment with miR-135a mimic also inhibited CUMS-induced hippocampal cell apoptosis. Furthermore, treatment with miR-135a mimic and fluoxetine significantly reduced the CUMS-induced increase in the expression levels of inflammatory factors (IL-1ß, IL-6 and TNF-α) in the hippocampus of the mice. Subsequently, reverse transcription-quantitative polymerase chain reaction and western blot analysis revealed that treatment with miR-135a mimic significantly inhibited the expression of Toll-like receptor 4 in the mouse hippocampus. In conclusion, the findings of the present study indicate that miR-135a may be a novel potential target for the treatment of depression.

miR­146a reduces depressive behavior by inhibiting microglial activation.

Depression is one of the major psychiatric diseases affecting the quality of life for individuals worldwide. Numerous reports have investigated depression, although its etiology remains to be elucidated. microRNA (miR)­146a is suggested to regulate innate immune and inflammatory responses. However, it is unclear whether miR­146a is involved in depression. Depression model mice were established using lipopolysaccharide­induced depression and chronic unpredictable mild stress, separately. miR­146a mimic and short interfering RNA were used to treat depressed mice. Depression­like behaviors and levels of pro­inflammatory cytokines were measured, while ionized calcium binding adapter molecule 1 (Iba­1) expression in hippocampus was quantified by immunohistochemistry. Neuroinflammatory factor levels in hippocampus were measured by western blotting. BV­2 cells were used to confirm that miR­146a suppressed microglia activation. Compared with control mice, the two depressed mouse models showed clearly decreased sucrose preference and significantly increased immobility time in the forced swimming test and tail suspension test (P<0.05). miR­146a overexpression significantly increased sucrose preference and reduced immobility time in depressed mice (P<0.05). However, total distance traveled in the locomotor activity test did not differ among groups. Compared with controls, expression levels of Iba­1, inducible nitric oxide, IL­1ß, TNF­α, interleukin 1 receptor associated kinase 1 (IRAK1), TNF receptor­associated factor 6 (TRAF6) and phosphorylated NF­κB p65 were significantly increased in depressed mice (P<0.05). miR­146a overexpression effectively inhibited expression of these neuroinflammatory proteins, while miR­146a silencing significantly upregulated their expression (P<0.05). Consistent with these in vivo results, miR­146a mimic treatment inhibited TNF­α, IL­1ß, IRAK1 and TRAF6 expression in BV­2 cells. miR­146a improved depressive behaviors in depressed model mice by inhibiting microglial activation and neuroinflammatory factor expression.

Covalent Inhibitors Allosterically Block the Activation of Rho Family Proteins and Suppress Cancer Cell Invasion.

The Rho family GTPases are crucial drivers of tumor growth and metastasis. However, it is difficult to develop GTPases inhibitors due to a lack of well-characterized binding pockets for compounds. Here, through molecular dynamics simulation of the RhoA protein, a groove around cysteine 107 (Cys107) that is relatively well-conserved within the Rho family is discovered. Using a combined strategy, the novel inhibitor DC-Rhoin is discovered, which disrupts interaction of Rho proteins with guanine nucleotide exchange factors (GEFs) and guanine nucleotide dissociation inhibitors (GDIs). Crystallographic studies reveal that the covalent binding of DC-Rhoin to the Cys107 residue stabilizes and captures a novel allosteric pocket. Moreover, the derivative compound DC-Rhoin04 inhibits the migration and invasion of cancer cells, through targeting this allosteric pocket of RhoA. The study reveals a novel allosteric regulatory site within the Rho family, which can be exploited for anti-metastasis drug development, and also provides a novel strategy for inhibitor discovery toward "undruggable" protein targets.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 µM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 µM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

The saturated fatty acid palmitate induces insulin resistance through Smad3-mediated down-regulation of FNDC5 in myotubes.

Elevated plasma free fatty acid (FFA) levels are associated with insulin resistance and can cause lipotoxicity in skeletal muscles. In response to FFAs, skeletal muscle can secrete a variety of cytokines. Irisin, one such muscle-secreted cytokine, can improve glucose tolerance, glucose uptake, and lipid metabolism. It is produced by the transmembrane protein fibronectin type Ⅲ domain containing 5 (FNDC5) by specific proteases. The purpose of this study was to investigate the regulatory mechanisms of the FNDC5 response to palmitate and their relationships with insulin resistance in C2C12 myotubes. RNA sequencing analysis results from C2C12 myotubes treated with palmitate showed that palmitate could activate the TGF-ß signaling pathway. Palmitate directly affected the expression of Smad3, but not its phosphorylation level, in C2C12 myotubes. Furthermore, knockdown and knockout of Smad3 alleviated the inhibitory effect of palmitate on the expression of FNDC5. In contrast, overexpression of Smad3 aggravated the inhibition of FNDC5 expression. There is a Smad3 binding motif in the -660 bp to -649 bp region of the Fndc5 promoter. CRISPR/Cas9 knockout of this region also alleviated the inhibition of FNDC5 expression in response to palmitate. More importantly, inhibition of FNDC5 expression mediated by Smad3 led to a decrease in insulin sensitivity in C2C12 myotubes. Collectively, these findings suggest that palmitate could induce insulin resistance through Smad3-mediated down-regulation of the Fndc5 gene.

C/EBPß mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes.

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.

Glucosamine induces increased musclin gene expression through endoplasmic reticulum stress-induced unfolding protein response signaling pathways in mouse skeletal muscle cells.

Glucosamine (GlcN) is a dietary supplement that is widely used to promote joint health. Reports have demonstrated that oral GlcN adversely affects glucose metabolism. Here, we found that oral administration of GlcN induced insulin resistance (IR) and increased plasma glucose levels in mice. Musclin is a muscle-secreted cytokine that participates in the development and aggravation of diabetes. In this study, we found that increased expression of the musclin plays a pathogenic role in GlcN-induced IR in mice. Additional in vivo and in vitro studies showed that 4-PBA inhibited GlcN-induced endoplasmic reticulum (ER) stress and reduced musclin expression, indicating that ER stress might be closely linked to musclin expression. Moreover, the inhibition of musclin gene expression was also observed when sh-RNAs and small molecular compound inhibitors inhibited ER stress-induced PERK and IRE1-associated unfolding protein response (UPR) signaling pathways, and the CRISPR/Cas9 genome editing technology knockout the ATF6-associated UPR pathway in C2C12 myotubes cells. Silencing of the expression of musclin effectively relieved GlcN-affected phosphorylation of Akt, glucose intake and glycogen synthesis. These results suggest that GlcN increased musclin gene expression though UPR, and musclin represents an important mechanism underlying GlcN-induced IR in mice.

Coccomyxagreatwallensis sp. nov. (Trebouxiophyceae, Chlorophyta), a lichen epiphytic alga from Fildes Peninsula, Antarctica.

A single-celled green alga Coccomyxagreatwallensis Shunan Cao & Qiming Zhou, sp. nov., isolated from a specimen of Antarctic lichen Psoromahypnorum (Vahl) Gray, is described and illustrated based on a comprehensive investigation of morphology, ultrastructure, ecology and phylogeny. The cells of C.greatwallensis are ovoid to long ellipsoidal and measured 3-5 µm × 6-12 µm. The new species has distinct ITS rDNA and SSU rDNA sequences and differs from the phylogenetic closely related species C.antarctica, C.arvernensis and C.viridis in cell size, distribution and habitat.

Coccomyxa antarctica sp. nov. from the Antarctic lichen Usnea aurantiacoatra.

The single celled green alga Coccomyxa antarctica Shunan Cao & Qiming Zhou, sp. nov. was isolated from the Antarctic torrential lichen Usnea aurantiacoatra (Jacq.) Bory. It is described and illustrated based on a comprehensive study of its morphology, ultrastructure, ecology and phylogeny. C. antarctica is a lichenicolous alga which has elongated cells and contains a parietal chloroplast as observed under the microscope. C. antarctica is clearly different from other species by phylogenetic analysis (ITS rDNA and SSU rDNA sequences), also it differs from its phylogenetic closely species C. viridis by its larger cell size.

Exploring the roles of substrate-binding surface of the chaperone site in the chaperone activity of trigger factor.

Trigger factor (TF) is a key component of the prokaryotic chaperone network, which is involved in many basic cellular processes, such as protein folding, protein trafficking, and ribosome assembly. The major chaperone site of TF has a cradle-like structure in which protein substrate may fold without interference from other proteins. Here, we investigated in vivo and in vitro the roles of hydrophobic and charged patches on the edge and interior of cradle during TF-assisted protein folding. Our results showed that most of the surface of the cradle was involved in TF-assisted protein folding, which was larger than found in early studies. Although the inner surface of cradle was mostly hydrophobic, both hydrophobic and electrostatic patches were indispensable for TF to facilitate correct protein folding. However, hydrophobic patches were more important for the antiaggregation activity of TF. Furthermore, it was found that the patches on the surface of cradle were involved in TF-assisted protein folding in a spatial and temporal order. These results suggest that the folding-favorable interface between the cradle and substrate was dynamic during TF-assisted protein folding, which enabled TF to be involved in the folding of substrate in an aggressive manner rather than acting as a classic holdase.-Fan, D., Cao, S., Zhou, Q., Zhang, Y., Yue, L., Han, C., Yang, B., Wang, Y., Ma, Z., Zhu, L., Liu, C. Exploring the roles of substrate-binding surface of chaperone site in the chaperone activity of trigger factor.

Hypoxia in 3T3-L1 adipocytes suppresses adiponectin expression via the PERK and IRE1 unfolded protein response.

Adiponectin, an adipocytokine produced by adipocytes, functions as an anti-inflammatory and anti-apoptotic substance, while also enhancing insulin sensitivity. Patients or model animals with obesity or diabetes typically present attenuated expression of adiponectin. Moreover, obesity and diabetes are often accompanied with hypoxia in adipose tissue, which may result in endoplasmic reticulum (ER) stress as well as low expression of adiponectin. The purpose of this study was to investigate the specific role of the unfolded protein response (UPR) involved in the low expression of adiponectin induced by hypoxia. Subjecting 3T3-L1 adipocytes to hypoxia significantly reduced adiponectin expression and activated the PERK and IRE1 signaling pathways in a time-dependent manner. The ATF6 signaling pathway showed no obvious changes with hypoxia treatment under a similar time course. Moreover, the down-regulated expression of adiponectin induced by hypoxia was relieved once the PERK and IRE1 signaling pathways were suppressed by the inhibitors GSK2656157 and 4µ8C, respectively. Overall, these data demonstrate that hypoxia can suppress adiponectin expression and activate the PERK and IRE1 signaling pathways in differentiated adipocytes, and this two pathways are involved in the suppression of adiponectin expression induced by hypoxia.

Global Analysis of the Impact of Deleting Trigger Factor on the Transcriptome Profile of Escherichia coli.

Trigger factor (TF) is a key component of prokaryotic chaperone network, which is involved various basic cellular processes such as nascent peptide folding, protein trafficking, ribosome assembly. To better understanding the physiological roles of TF, global transcriptome profiles of a variety of TF deletion mutant strains of Escherichia coli were determined. We found that deletion of the tig gene, encoding TF, led to a dramatic alteration of transcriptome profile, not only affecting the gene expression of members of the chaperone network, but also changing the levels of quite a few RNAs related to metabolism and other cellular processes. Further studies showed that this alteration was only partially recovered by knockin of TF domain-deletion mutants into the endogenous tig locus, indicating that structural integrity is crucial for the biological function of TF. Finally, by combining the transcriptome and phenotype results, a physiological mechanism underlying the impact of TF deletion on the transcriptome profile was proposed. J. Cell. Biochem. 118: 141-153, 2017. © 2016 Wiley Periodicals, Inc.

Functional characterization of the Helicobacter pylori chaperone protein HP0795.

Trigger factor (TF) is one of the multiple bacterial chaperone proteins interacting with nascent peptides and facilitating their folding in bacteria. While TF is well-characterized in E. coli, HP0795, a TF-like homologue gene identified earlier in the pathogenic Helicobacter pylori (H. pylori), has not been studied biochemically to date. To characterize its function as a chaperone, we performed 3D-modeling, cross-linking and in vitro enzyme assays to HP0795 in vitro. Our results show that HP0795 possesses peptidyl prolyl cis-trans isomerase activity and exhibits a dimeric structure in solution. In addition, stable expression of HP0795 in a series of well-characterized E. coli chaperone-deficient strains rescued the growth defects in these mutants. Furthermore, we showed that the presence of HP0795 greatly reduced protein aggregation caused by deficiencies of chaperones in these strains. In contrast to other chaperone genes in H. pylori, gene expression of HP0795 displays little induction under acidic pH conditions. Together, our results suggest that HP0795 is a constitutively expressed TF-like protein of the prokaryotic chaperone family that may not play a major role in acid response. Given the pathogenic properties of H. pylori, our insights might provide new avenues for potential future medical intervention for H. pylori-related conditions.