Protection-Free Strategy for the Synthesis of Boro-Depsipeptides in Aqueous Media under Microwave-Assisted Conditions.

In this report, 19 boron-containing depsipeptides were synthesized via microwave-assisted Passerini three-component reaction (P-3CR) in an aqueous environment. The linker-free DAHMI fluorescent tagging approach was used on selected boron-containing compounds to study the relationship between their structures and their level of cellular uptake of HEK293 cells. The biological data retrieved from the DAHMI experiments indicated that while the structures of tested compounds may be highly similar, their bio-distribution profile could be vastly distinctive. The reported optimized one-pot synthetic strategy along the linker-free in vitro testing protocol could provide an efficient platform to accelerate the development of boron-containing drugs.

A co-regulatory network of SPIB, AQP8, and GUCA2B related to immune infiltration for early-stage colorectal cancer in silico and in vitro.

In early-stage colorectal cancer (CRC), AQP8, GUCA2B, and SPIB were important suppressor genes and frequently co-expressed. However, the underlying co-regulation effect remains unknown and need to be elucidated. We aimed to investigate the co-regulatory network of AQP8, GUCA2B, and SPIB in CRC using in vitro and in silico methods. Q-PCR, western blot, and immunohistochemistry were used to assess the co-regulatory network of the target genes in the HCT-116 cell line and fresh tumor tissues. Bioinformatical methods were used to validate the findings using the Cancer Genome Atlas COlon ADenocarcinoma and REctum ADenocarcinoma datasets, as well as large scale integrated data sets from Gene Expression Omnibus. In clinical CRC tissues, SPIB, AQP8, and GUCA2B were barely expressed compared to normal mucosa. When compared to 22 well-known genetic biomarkers, they are independent predictors of CRC identification with near 100% accuracy. In the co-regulatory network, they were co-upregulated at the mRNA and protein expression levels. AQP8, GUCA2B and SPIB were linked to immune cell infiltration and GUCA2B and SPIB were negatively associated with tumor purity. The co-regulatory network in miRNA-mRNA analysis was mediated by cancer-related microRNAs miR-182-5p and miR-27a-3. The functional analysis of the co-regulatory network's protein-protein interaction networks reveals three clusters and three major functions: complex interactions of transcription factors in mediating cytokine biology in T cells (SPIB cluster), guanylin, and Intestinal infectious diseases (GUCA2B cluster), and water channel activity balance (AQP8 cluster). The co-regulatory network of SPIB, AQP8, and GUCA2B was confirmed. MiR-27a-3p and miR-182-5p were two possible mediators. The mechanisms of SPIB, AQP8, GUCA2B, miR-182-5p, and miR-27a-3p in CRC merit further investigation.

RNF128 regulates neutrophil infiltration and myeloperoxidase functions to prevent acute lung injury.

Acute lung injury (ALI) is characterised by severe pulmonary inflammation, alveolar-capillary barrier disruption, and pulmonary oedema. Therefore, establishing effective therapeutic targets for ALI prevention is crucial. The present study reports a novel function of RNF128 in regulating LPS-induced ALI. Severe lung damage and increased immune cell infiltration were detected in RNF128-deficient mice. In vitro experiments revealed that RNF128 inhibits neutrophil activation by binding to myeloperoxidase (MPO) and reducing its levels and activity. Moreover, RNF128 regulates alveolar macrophage activation and neutrophil infiltration by interacting with TLR4, targeting it for degradation, and inhibiting NF-κB activation, hence decreasing pro-inflammatory cytokines. Our results demonstrate for the first time that RNF128 is a negative regulator of MPO and TLR4 in neutrophils and alveolar macrophages, respectively. However, AAV9-mediated RNF128 overexpression alleviated lung tissue damage and reduced inflammatory cell infiltration. Thus, RNF128 is a promising therapeutic candidate for pharmacological interventions in ALI.

Zac1, an Sp1-like protein, regulates human p21(WAF1/Cip1) gene expression in HeLa cells.

Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21(WAF1/Cip1) gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity by interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21(WAF1/Cip1) gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.

CRABP1-CaMKII-Agrn regulates the maintenance of neuromuscular junction in spinal motor neuron.

Cellular retinoic acid-binding protein 1 (CRABP1) binds retinoic acid (RA) specifically in the cytoplasm with unclear functions. CRABP1 is highly and specifically expressed in spinal motor neurons (MNs). Clinical and pre-clinical data reveal a potential link between CRABP1 and MN diseases, including the amyotrophic lateral sclerosis (ALS). We established a sequenced MN-muscle co-differentiation system to engineer an in vitro functional 3D NMJ model for molecular studies and demonstrated that CRABP1 in MNs contributes to NMJ formation and maintenance. Consistently, Crabp1 knockout (CKO) mice exhibited an adult-onset ALS-like phenotype with progressively deteriorated NMJs, characterized with behavioral, EchoMRI, electrophysiological, histological, and immunohistochemical studies at 2-20-months old. Mechanistically, CRABP1 suppresses CaMKII activation to regulate neural Agrn expression and downstream muscle LRP4-MuSK signaling, thereby maintaining NMJ. A proof-of-concept was provided by specific re-expression of CRABP1 to rescue Agrn expression and the phenotype. This study identifies CRABP1-CaMKII-Agrn signaling as a physiological pre-synaptic regulator in the NMJ. This study also highlights a potential protective role of CRABP1 in the progression of NMJ deficits in MN diseases.

E3 ubiquitin ligase Grail promotes hepatic steatosis through Sirt1 inhibition.

In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.

Modulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by Zac1 through the antagonistic regulators p53 and histone deacetylase 1 in HeLa Cells.

Zac1 is a novel seven-zinc finger protein which possesses the ability to bind specifically to GC-rich DNA elements. Zac1 not only promotes apoptosis and cell cycle arrest but also acts as a transcriptional cofactor for p53 and a number of nuclear receptors. Our previous study indicated that the enhancement of p53 activity by Zac1 is much more pronounced in HeLa cells compared with other cell lines tested. This phenomenon might be due to the coactivator effect of Zac1 on p53 and the ability of Zac1 to reverse E6 inhibition of p53. In the present study, we showed that Zac1 acted synergistically with either p53 or a histone deacetylase inhibitor, trichostatin A, to enhance p21(WAF1/Cip1) promoter activity. We showed that Zac1 physically interacted with some nuclear receptor corepressors such as histone deacetylase 1 (HDAC1) and mSin3a, and the induction of p21(WAF1/Cip1) gene and protein by Zac1 was suppressed by either overexpressing HDAC1 or its deacetylase-dead mutant. In addition, our data suggest that trichostatin A-induced p21(WAF1/Cip1) protein expression might be mediated through a p53-independent and HDAC deacetylase-independent pathway. Taken together, our data suggest that Zac1 might be involved in regulating the p21(WAF1/Cip1) gene and protein expression through its protein-protein interaction with p53 and HDAC1 in HeLa cells.

Perioperative outcomes of radical lobectomies using robotic-assisted thoracoscopic technique vs. video-assisted thoracoscopic technique: retrospective study of 1,075 consecutive p-stage I non-small cell lung cancer cases.

BACKGROUND: Robotic thoracoscopic surgery was first done in mainland China in 2009 and has gained popularity in the past few years. Here, we present the largest Chinese series of robotic lobectomy for early-stage non-small cell lung cancer (NSCLC) to date. We aimed to compare the perioperative outcomes of our three-arm robotic-assisted lobectomy (RAL3) and video-assisted lobectomy (VAL) for p-stage I NSCLC and report the approach of the robotic anatomic lobar resections of our center. METHODS: We retrospectively collected and analyzed the data of 1075 stage I NSCLC patients who underwent minimally invasive lobectomies (237 RAL3 cases and 838 VAL cases) by the same surgical team from May 2013 to April 2016. Propensity score matching (PSM) was used to minimize the bias between the two groups. Perioperative outcomes were analyzed. RESULTS: Compared to the VALs, the RAL3s had more retrieved lymph nodes (LNs) (9.70 vs. 8.45, P=0.000), less POD1 drain (230.91 vs. 279.79 mL, P=0.001), shorter chest tube duration (3.84 vs. 4.33 d, P=0.003) and shorter postoperative length of stay (4.97 vs. 5.45 d, P=0.004), but a higher cost (¥93,244.84 vs. ¥67,055.82, P=0.000). No significant difference was observed between the RAL3 and VAL groups concerning the average skin-to-skin time (90.84 vs. 92.25 min, P=0.624), conversion rate (1.3% vs. 0.87%, P=1.000) and prolonged postoperative hospital stay (PPHS) rate (3.0% vs. 4.3%, P=0.694). CONCLUSIONS: This study confirms that RAL3 is a safer and more effective technique than VAL for the treatment of early-stage NSCLC.

Importin alpha1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1.

Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1-3 and 4-7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin alpha1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin alpha1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.

Grail attenuates influenza A virus infection and pathogenesis by inhibiting viral nucleoprotein.

Grail is a well-characterized mediator of metabolic disease, tumour progression, and immune response. However, its role in influenza A virus (IAV) infection remains poorly understood. In this study, we demonstrated that Grail knockdown potentiates IAV infection, whereas Grail overexpression blocks IAV replication. The intranasal administration of IAV to Grail KO mice led to a lower survival rate than in similarly infected wild-type mice. Additionally, IAV-infected Grail KO mice had higher viral titres, greater immune cell infiltration, and increased expression of inflammatory cytokines in the lungs. Mechanistically, we showed that Grail interacts with viral nucleoprotein (NP), targeting it for degradation and inhibiting IAV replication. NP expression was increased in Grail knockdown cells and reduced in cells overexpressing Grail. Collectively, our results demonstrate that Grail acts as a negative regulator of IAV infection and replication by degrading viral NP. These data increase our understanding of the host antiviral response to infection with IAV.

Macrophage expression of E3 ubiquitin ligase Grail protects mice from lipopolysaccharide-induced hyperinflammation and organ injury.

Multiple organ dysfunction caused by hyperinflammation remains the major cause of mortality during sepsis. Excessive M1-macrophage activation leads to systemic inflammatory responses. Gene related to anergy in lymphocytes (Grail) is regarded as an important regulator of T cells that functions by diminishing cytokine production. However, its role in regulating macrophage activation and organ injury during sepsis remains unclear. Our aim was to examine the effects of Grail on macrophage reactivity and organ injury in endotoxemic animals. Wild-type and Grail knockout mice were injected with vehicle or Escherichia coli lipopolysaccharide and observed for 24 h. Changes in blood pressure, heart rate, blood glucose, and biochemical variables were then examined. Moreover, levels of neutrophil infiltration, MMP-9, and caspase 3 were analyzed in the lungs of animals. The expression of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages stimulated with LPS were also assessed in the presence or absence of Grail. Results indicated that loss of Grail expression enhances the induction of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages treated with LPS. Furthermore, LPS-induced macrophage hyperactivation was alleviated by ectopic Grail overexpression. In vivo studies showed that Grail deficiency exacerbates organ damage in endotoxemic animals. Levels of neutrophil infiltration, MMP-9, and caspase 3 were significantly increased in the lungs of Grail-deficient endotoxemic mice. Thus, these results suggest that Grail contributes to the attenuation of hyperinflammation caused by activated macrophages and prevents organ damage in endotoxemic mice. We suggest that Grail signaling could be a therapeutic target for endotoxemia.

Regulation of nuclear receptor and coactivator functions by the carboxyl terminus of ubiquitin-conjugating enzyme 9.

Small ubiquitin-related modifier (SUMO) is a protein moiety that is ligated to lysine residues in a variety of target proteins. The SUMO E2 enzyme ubiquitin-conjugating enzyme 9 (Ubc9) is sufficient for substrate recognition and lysine modification of known SUMO targets. Previous studies have demonstrated that mutated Ubc9 that has lost its SUMO-ligating activity retains its enhancement on transactivation mediated by androgen receptor (AR). In contrast to the binding ability to Ubc9, the sumoylation of AR via the association of SUMO-1 and PIAS1 is able to repress AR-dependent transcription. In the present study, we present several lines of evidence to explain the role of over-expressed Ubc9 as a cofactor in the nuclear receptor and coactivator functions, including (i) activity that is independent of its ability to catalyze SUMO-1 conjugation, (ii) an insight into the protein-protein interaction motif in its eight C-terminal residues, (iii) selective coactivator function in nuclear receptor-relevant transactivation activities, and (iv) a non-trichostatin A-sensitive autonomous transcription repression domain in its far C-terminal region. Taken together, our data suggest that the both the protein-protein interaction through the Ubc9 C-terminus and its sumoylation-modifying activity provide the mechanism for regulating nuclear receptor functions.

Human papillomavirus E2 protein associates with nuclear receptors to stimulate nuclear receptor- and E2-dependent transcriptional activations in human cervical carcinoma cells.

Steroid hormones are proposed to act with human papillomaviruses (HPVs) as cofactors in the etiology of cervical cancer. Steroid hormone-activated nuclear receptors (NRs) are thought to bind to specific DNA sequences within transcriptional regulatory regions on the HPV DNA to either increase or suppress transcription of dependent genes. HPV-induced immortalization of epithelial cells usually requires integration of the viral DNA into the host cell genome. The integration event causes disruption of the E2 gene: the E2 protein is a transcription factor that regulates expression of the E6 and E7 oncoproteins by binding to four sites within the viral long control region (LCR). Our previous study suggested that E6 and E7 oncoproteins both directly bind to some NRs and serve as their cofactors. Here, we provide several lines of evidence demonstrating that the E2 protein is an NR coactivator through its physical interaction with NRs. In our study, the NR coactivator function of HPV E2 protein in human cervical carcinoma cells was independent of the type of E2, HPV transformation and the p53 status. Our observations also provide evidence suggesting regulatory mechanisms for the LCR involving interaction between the E2 protein and NRs in HeLa cells.

Receptor-interacting protein 140 as a co-repressor of Heat Shock Factor 1 regulates neuronal stress response.

Heat shock response (HSR) is a highly conserved transcriptional program that protects organisms against various stressful conditions. However, the molecular mechanisms modulating HSR, especially the suppression of HSR, is poorly understood. Here, we found that RIP140, a wide-spectrum cofactor of nuclear hormone receptors, acts as a co-repressor of heat shock factor 1 (HSF1) to suppress HSR in healthy neurons. When neurons are stressed such as by heat shock or sodium arsenite (As), cells engage specific proteosome-mediated degradation to reduce RIP140 level, thereby relieving the suppression and activating HSR. RIP140 degradation requires specific Tyr-phosphorylation by Syk that is activated in stressful conditions. Lowering RIP140 level protects hippocampal neurons from As stress, significantly it increases neuron survival and improves spine density. Reducing hippocampal RIP140 in the mouse rescues chronic As-induced spatial learning deficits. This is the first study elucidating RIP140-mediated suppression of HSF1-activated HSR in neurons and brain. Importantly, degradation of RIP140 in stressed neurons relieves this suppression, allowing neurons to efficiently and timely engage HSR programs and recover. Therefore, stimulating RIP140 degradation to activate anti-stress program provides a potential preventive or therapeutic strategy for neurodegeneration diseases.

The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer.

BACKGROUND: Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. METHODS: Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. RESULTS: Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. CONCLUSIONS: This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.

Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domains.

Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions.

Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes.

Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53α expression and induced the expression of p53ß, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3.

Physical and functional interactions of human papillomavirus E2 protein with nuclear receptor coactivators.

In addition to the human papillomavirus (HPV)-induced immortalization of epithelial cells, which usually requires integration of the viral DNA into the host cell genome, steroid hormone-activated nuclear receptors (NRs) are thought to bind to specific DNA sequences within transcriptional regulatory regions on the long control region to either increase or suppress transcription of dependent genes. In this study, our data suggest that the NR coactivator function of HPV E2 proteins might be mediated through physical and functional interactions with not only NRs but also the NR coactivators GRIP1 (glucocorticoid receptor-interacting protein 1) and Zac1 (zinc-finger protein which regulates apoptosis and cell cycle arrest 1), reciprocally regulating their transactivation activities. GRIP1 and Zac1 both were able to act synergistically with HPV E2 proteins on the E2-, androgen receptor-, and estrogen receptor-dependent transcriptional activation systems. GRIP1 and Zac1 might selectively function with HPV E2 proteins on thyroid receptor- and p53-dependent transcriptional activation, respectively. Hence, the transcriptional function of E2 might be mediated through NRs and NR coactivators to regulate E2-, NR-, and p53-dependent transcriptional activations.

Human spot 14 protein interacts physically and functionally with the thyroid receptor.

Spot 14 (S14) is a small acidic protein with no sequence similarity to other mammalian gene products. Its biochemical function is elusive. Recent studies have shown that, in some cancers, human S14 (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumorigenesis. In this study, we purified untagged hS14 protein and then demonstrated, using various biochemical methods, including analytic ultracentrifugation, that hS14 might form a homodimer. We also found several lines of evidence to suggest physical and functional interactions between hS14 and the thyroid hormone receptor (TR). The ubiquitous expression of hS14 in various cell lines and its cell-type-dependent functions demonstrated in this study suggest that it acts as a positive or negative cofactor of the TR to regulate malic enzyme gene expression. These findings provide a molecular rationale for the role of hS14 in TR-dependent transcriptional activation of the expression of specific genes.