An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Knockdown of EIF4G1 in NSCLC induces CXCL8 secretion.

Non-small cell lung cancer (NSCLC) is the most common type of lung tumor; however, we lack effective early detection indicators and therapeutic targets. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is vital to initiate protein synthesis, acting as a scaffolding protein for the eukaryotic protein translation initiation factor complex, EIF4F, which regulates protein synthesis together with EIF4A, EIF4E, and other translation initiation factors. However, EIF4G1's function in NSCLC cancer is unclear. Herein, transcriptome sequencing showed that knockdown of EIF4G1 in H1299 NSCLC cells upregulated the expression of various inflammation-related factors. Inflammatory cytokines were also significantly overexpressed in NSCLC tumor tissues, among which CXCL8 (encoding C-X-C motif chemokine ligand 8) showed the most significant changes in both in the transcriptome sequencing data and tumor tissues. We revealed that EIF4G1 regulates the protein level of TNF receptor superfamily member 10a (TNFRSF10A) resulting in activation of the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) pathways, which induces CXCL8 secretion, leading to targeted chemotaxis of immune cells. We verified that H1299 cells with EIF4G1 knockdown showed increased chemotaxis compared with the control group and promoted increased chemotaxis of macrophages. These data suggested that EIF4G1 is an important molecule in the inflammatory response of cancer tissues in NSCLC.

Prognostic and functional roles of EIF4G1 in lung squamous cell carcinoma.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is highly expressed in many cancers and affects their occurrence and development. However, the effect of EIF4G1 on the prognosis, biological function and the relevant mechanism in lung squamous cell carcinoma (LSCC) is unclear. Through clinical cases, Cox's proportional hazard model and Kaplan-Meier plotter survival analysis, we find the expression levels of EIF4G1 are dependent on age and clinical stage, high expression of EIF4G1 could be used to predict the overall survival of LSCC patients. LSCC cell line NCI-H1703, NCI-H226 and SK-MES-1infected with EIF4G1 siRNA are used to detect the function of EIF4G1 with cell proliferation and tumorigenesis in vivo and vitro. The data show that EIF4G1 promotes tumor cell proliferation and the G1/S transition of cell cycle in LSCC, then the biological function of LSCC is effected by the AKT/mTOR pathway. Above all, these results have demonstrated that EIF4G1 promotes LSCC cell proliferation and may represent an indicator of prognosis in LSCC.

Cationic amino acid transporter-1 (CAT-1) promotes fibroblast-like synoviocyte proliferation and cytokine secretion by taking up L-arginine in rheumatoid arthritis.

BACKGROUND: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. METHODS: The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo. RESULTS: L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1ß, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. CONCLUSION: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Blockade of the amino acid transporter SLC6A14 suppresses tumor growth in colorectal Cancer.

BACKGROUND: The amino acid transporter SLC6A14, which transports 18 of the 20 proteinogenic amino acids, is too low to be detected in healthy normal tissues but is significantly increased in some solid cancers. However, little is known about the roles of SLC6A14 in colorectal cancer (CRC). METHODS: The mRNA and protein levels of SLC6A14 were detected using TCGA database, real-time polymerase chain reaction, western blot, and tissue microarrays, respectively. Amino acids concentration was determined by LC-MS/MS. Cell proliferation and apoptosis were determined using MTT assay and flow cytometry. Transwell invasion assay and wound healing assay were employed to analyze cell migration and invasion. The protein levels of Akt-mTOR signaling pathway and MMPs proteins were detected by western blot. RESULTS: Both of the mRNA and protein levels of SLC6A14 were upregulated in CRC tissues, and the protein levels of SLC6A14 were closely related to the tumor cells differentiation: the higher the expression of SLC6A14 was, the poorer the differentiation of the tumor cells was. Further knockdown SLC6A14 with siRNA or treatment with α-MT in CRC cell lines reduced cell proliferation and migration in vitro and inhibited xenograft tumor growth in vivo. Mechanistically, SLC6A14 was demonstrated to regulate the expression and phosphorylation of Akt-mTOR, which mediates the promoting tumor growth function of SLC6A14. Blockade of SLC6A14 with α-MT inhibited the activation of mTOR signaling. CONCLUSION: SLC6A14 was upregulated in CRC and could promote tumor progression by activating the Akt-mTOR signaling pathway, which may serve as an effective molecular target for the treatment of CRC.

The Prognostic Value and Immune Infiltration of USP10 in Pan-Cancer: A Potential Therapeutic Target.

Ubiquitin-specific peptidase 10 (USP10) can sustain cellular functions and regulate cellular processes. It plays an essential role in cancer inhibition or facilitation by reversing ubiquitin-proteasome degradation. Studies have identified USP10 to be involved in tumor progression in various cancers. However, the pan-cancer expression pattern of USP10, its prognostic value, and the association between tumor immune cell infiltration and USP10 expression remain to be discussed and thus comprised the aims of the present study. Based on clinical samples and bioinformatic analyses, high USP10 expression was observed in most cancer tissues except for ovarian cancer. High USP10 expression correlated with pathological stage and node metastasis and predicted poor patient prognosis. In addition, further analyses at the TIMER and GEPIA databases showed that USP10 is involved in the infiltration of multiple immune cells and regulated the infiltration levels of specific immune cell subpopulations, particularly in pancreatic adenocarcinoma (PAAD) and liver hepatocellular carcinoma (LIHC). Importantly, USP10 might influence survival by modulating immune infiltration in patients with PAAD and LIHC. These results identified USP10 as a potential biomarker for pan-cancer prognosis, and in certain cancers, USP10 could identify clinical prognosis linked to tumor immune infiltration.

Elevation of EIF4G1 promotes non-small cell lung cancer progression by activating mTOR signalling.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), as the key component of the transcription initiation factor complex EIF4F, is significantly upregulated in multiple solid tumours, including lung cancer. However, the function and mechanism of EIF4G1 in the regulation of non-small-cell lung cancer (NSCLC) remain unclear. Here, using the clinical samples and the comprehensive survival analysis platforms Kaplan-Meier plotter, we observed aberrant upregulation of EIF4G1 in NSCLC tissues; furthermore, high expression of EIF4G1 showed association with low differentiation of lung cancer cells and poor overall survival in NSCLC patients. Non-small-cell lung cancer cell line A549 and H1703 stably infected with EIF4G1 shRNA were used to determine the function of EIF4G1 in regulating cell proliferation and tumorigenesis in vitro and in vivo. The results demonstrated that EIF4G1 promoted the G1/S transition of the cell cycle and tumour cell proliferation in non-small cell lung cancer. Mechanistically, EIF4G1 was found to regulate the expression and phosphorylation of mTOR (Ser2448), which mediates the tumorigenesis-promoting function of EIF4G1. The inhibition of mTOR attenuated the EIF4G1-induced development and progression of tumours. These findings demonstrated that EIF4G1 is a new potential molecular target for the clinical treatment of non-small cell lung cancer.

Functionality of an absolutely conserved glycine residue in the chimeric relaxin family peptide R3/I5.

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.

Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Our recent study demonstrated that selectivity of the chimeric relaxin family peptide R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of the highly conserved nonchiral B23Gly or B24Gly with some natural l-amino acids. To investigate the mechanism of this modulating effect, in the present study we incorporated unnatural amino acids into the B23 or B24 position of a semi-synthetic R3/I5 that was prepared by a novel sortase-catalysed ligation approach using synthetic relaxin-3 B-chain and recombinant INSL5 A-chain. R3/I5 was a weak agonist for RXFP3 after B23Gly was replaced by D-Ala or D-Ser, but a strong antagonist for this receptor after B23Gly was replaced by corresponding l-amino acids. However, these replacements always resulted in a weak agonist for RXFP4. Thus, configuration of the B23 residue of R3/I5 affected activation of RXFP3 but not RXFP4. For the B24 residue, both size and configuration affected receptor selectivity of R3/I5. l-amino acids with an appropriate size, such as L-Ser and L-Abu, had the greatest effect on increasing the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. Our present results provided new insights into receptor selectivity of R3/I5, and would facilitate design of novel agonists or antagonists for RXFP3 and RXFP4 in future studies.

Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

Targeting EIF4F complex in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) accounts for about 85-90% of lung cancer cases, which represents the leading cause of cancer-related death in the world. The majority of lung cancer patients doesn't respond well to conventional chemo-/radio-therapeutic regimens and have a poor prognosis. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but their outcome/prognosis is far from satisfactory. The translation initiation EIF4F complex has been shown to play important roles in cancer progression, but its functional role and therapeutic effect in lung cancers especially NSCLC remain largely unknown. In this current review, we summarize recent findings regarding the role of EIF4F complex in NSCLC progression and targeted therapy potentials. We also discuss the unanswered questions and future directions in this field.

Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation.

Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1-4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5. Saturation binding assays demonstrated that the NanoLuc-ligated R3/I5 retained high binding affinity to RXFP3 and RXFP4, with the calculated dissociation constants (K d) of 4.34 ± 0.33 nM (n = 3) and 5.66 ± 0.54 nM (n = 3), respectively. Using the NanoLuc-ligated R3/I5 as a tracer in competition binding assays, binding potencies of various ligands towards RXFP3 and RXFP4 were conveniently quantified. This work provides a simple method for rapid preparation of bioluminescent tracers for relaxin family peptides and other protein/peptide hormones for ligand-receptor interaction studies.

Development of a selective agonist for relaxin family peptide receptor 3.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.

A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

Glutamine decreases intestinal mucosal injury in a rat model of intestinal ischemia-reperfusion by downregulating HMGB1 and inflammatory cytokine expression.

Intestinal ischemia-reperfusion (IR) is a common clinical pathophysiological process that is common in severe trauma, major surgery, and in post-resuscitation. Glutamine (Gln) reduces intestinal IR injury, however, its mechanism of action remains to be determined. High mobility group box 1 (HMGB1) protein, nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1) are mediators involved in the pathophysiology of intestinal IR injury. The aim of the present study was to investigate the effects of Gln on the intestinal mucosa of HMGB1 expression following IR to determine whether Gln relieved intestinal IR injury in the intestinal mucosal barrier. Forty-eight Sprague-Dawley rats were included in the present study. A model of intestinal ischemia-reperfusion injury was established by clamping the superior mesenteric artery of the rats to cause ischemia, followed by restoring blood flow. The animals were randomly divided into the control (n=24) and the Gln (n=24) groups for the experiments. The two groups of rats were given enteral nutrition with equal heat, nitrogen (heat 125.4 kJ/kg/day, nitrogen 0.2 g/kg/day). The Gln group of rats was fed with enteral nutrition plus 3% Gln, while the control rats were fed with enteral nutrition plus 3% soybean protein. After 7 days, the HMGB1 and plasma levels of NF-κB, TNF-α, IL-1, Gln, D-lactic acid and diamine oxidase (DAO) were observed. The changes in the morphology of intestinal mucosa were observed using electron microscopy. The plasma levels of TNF-α, IL-1, D-lactic acid and DAO, and the level of HMGB1, NF-κB, TNF-α and IL-1 in intestinal mucosa were significantly higher after IR (p<0.05), while the plasma level of Gln was lower in the two groups. In the control group, the plasma level of IL-1, TNF-α, DAO and D-lactic acid, and that of HMGB1, NF-κB, TNF-α, and IL-1 in intestinal mucosa were significantly higher, while the plasma level of Gln was lower than that prior to modeling on the 3rd and 7th days of the experiment. In the Gln group, the plasma level of IL-1, TNF-α, DAO and D-lactic acid, and that of HMGB1, NF-κB, IL-1, and TNF-α in intestinal mucosa were significantly higher (p<0.05) compared to the control on the 3rd and 7th days of the experiment. By contrast, after the 7th day, the plasma level of IL-1, TNF-α, DAO and D-lactic acid, and the level of HMGB1, NF-κB, IL-1, TNF-α in intestinal mucosa were significantly lower in the Gln group, while the plasma level of Gln was significantly higher than those in control group and after IR on the 7th day of the experiment. Additionally, the structure of villi and recess was damaged, villi was sparse and short, and considerable inflammatory cell influx embellished the lamina propria, lymphangiectasia, and edema after IR. On the 7th day, compared to after IR, the intestinal villi and recess structure of the controls was significantly restored in the Gln group. In conclusion, Gln repaired the intestinal mucosal injury in IR by reducing the expression of HMGB1 and inflammatory cytokines, and reducing the permeability of the intestinal mucosa.

Sleep Duration and Midday Napping with 5-Year Incidence and Reversion of Metabolic Syndrome in Middle-Aged and Older Chinese.

STUDY OBJECTIVES: Prospective evidence on the association of sleep duration and midday napping with metabolic syndrome (MetS) is limited. We aimed to examine the associations of sleep duration and midday napping with risk of incidence and reversion of MetS and its components among a middle-aged and older Chinese population. METHODS: We included 14,399 subjects from the Dongfeng-Tongji (DFTJ) Cohort Study (2008-2013) who were free of coronary heart disease, stroke, and cancer at baseline. Baseline data were obtained by questionnaires and health examinations. Odds ratios (ORs) and 95% confidence interval (CI) were derived from multivariate logistic regression models. RESULTS: After controlling for potential covariates, longer sleep duration (≥ 9 h) was associated with a higher risk of MetS incidence (OR, 1.29; 95% CI, 1.08-1.55) and lower reversion of MetS (OR, 0.80; 95% CI, 0.66-0.96) compared with sleep duration of 7 to < 8 h; whereas shorter sleep duration (< 6 h) was not related to incidence or reversion of MetS. For midday napping, subjects with longer napping (≥ 90 min) was also associated with a higher risk of MetS incidence and a lower risk of MetS reversion compared with those with napping of 1 to < 30 min (OR, 1.48; 95% CI, 1.05-2.10 and OR, 0.70; 95% CI, 0.52-0.94, respectively). Significance for incidence or reversion of certain MetS components remained in shorter and longer sleepers but disappeared across napping categories. CONCLUSIONS: Both longer sleep duration and longer midday napping were potential risk factors for MetS incidence, and concurrently exert adverse effects on MetS reversion.

Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family.

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies.

KAP1 is overexpressed in hepatocellular carcinoma and its clinical significance.

BACKGROUND: The transcriptional regulator in embryonic development, KAP1, has been proved could promote cell proliferation and metastatic progression in a variety of human cancers. However, the role of KAP1 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this study is to investigate the relationship of KAP1 expression with the progression and prognosis of HCC. METHODS: We measured the expression level of KAP1 in both human hepatoma cell lines and HCC tissues obtained from HCC patients by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. Furthermore, the effect of KAP1 expression on hepatoma cell proliferation was investigated through KAP1 knock-down strategy. Besides that, the correlation between KAP1 expression and HCC progression was analyzed. RESULTS: KAP1 overexpression was proved broadly existed in the human hepatoma cell lines. Furthermore, down-regulate the expression of KAP1 by specific siRNA could inhibit cell proliferation which was partly originated from the activation of p53 mediated signal pathway. Moreover, comparisons between the cancer tissues and noncancerous tissues proved the expression level of KAP1 was significant higher in tumor tissues obtained from HCC patients. In addition, KAP1 overexpression was significantly correlated with tumor size and tumor stage and also a predictor for poor prognosis of HCC patients. CONCLUSION: Our results presented here demonstrate that KAP1 plays an important role in HCC and could be regarded as a valuable biomarker for tumor diagnosis and prognosis prediction, as well as a potential target for the treatment of HCC.

Functional role of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in NSCLC.

Eukaryotic translation initiation factor 4 gamma 1(EIF4G1) is related to tumorigenesis and tumor progression. However, its role and the underlying mechanisms in the regulation of tumor development in non-small cell lung cancers (NSCLC) remain largely unknown. Here we report that the levels of EIF4G1 expression are much higher in NSCLC cell lines and tumor tissues than those in the normal lung cells and adjacent normal tissues from the same patients. Using shRNA to knock down EIF4G1 expression stably, we found EIF4G1 required for NSCLC cell proliferation, anchorage-independent growth, migration and invasion. Furthermore, silencing of EIF4G1 induces NSCLC cell apoptosis and causes G0/G1 cell cycle arrest. To identify the partner protein network of EIF4G1 in NSCLC cells, we found that Ubiquitin-specific protease 10 (USP10) can directly interacts with EIF4G1, while acting as a negative regulator for EIF4G1-mediated functions. Together, our results indicate that EIF4G1 functions as an oncoprotein during NSCLC development, which may represent a novel and promising therapeutic target in lung cancer.

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.