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.

HBV promotes its replication by up-regulating RAD51C gene expression.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), pegylated-interferon-α(PEG-IFNα) and long-term nucleos(t)ide analogs (NUCs) are mainly drugs used to treat HBV infection, but the effectiveness is unsatisfactory in different populations, the exploration of novel therapeutic approaches is necessary. RAD51C is associated with DNA damage repair and plays an important role in the development and progression of tumors. Early cDNA microarray results showed that RAD51C expression was significantly increased in HBV-infected HCC cells, however, the relationship between HBV infection and abnormal expression of RAD51C has not been reported. Therefore, we conducted RT-PCR, western blot, Co-immunoprecipitation(Co-IP), and immunofluorescence(IF) to detect HBV-RAD51C interaction in RAD51C overexpression or interfering HCC cells. Our results showed that RAD51C and HBV X protein(HBX) produced a direct interaction in the nucleus, the HBV infection of HCC cells promoted RAD51C expression, and the increased expression of RAD51C promoted HBV replication. This indicated that RAD51C is closely related to the occurrence and development of HCC caused by HBV infection, and may bring a breakthrough in the the prevention and treatment study of HCC.

Macrophage migration inhibitory factor (MIF) acetylation protects neurons from ischemic injury.

Ischemia-induced neuronal death leads to serious lifelong neurological deficits in ischemic stroke patients. Histone deacetylase 6 (HDAC6) is a promising target for neuroprotection in many neurological disorders, including ischemic stroke. However, the mechanism by which HDAC6 inhibition protects neurons after ischemic stroke remains unclear. Here, we discovered that genetic ablation or pharmacological inhibition of HDAC6 reduced brain injury after ischemic stroke by increasing macrophage migration inhibitory factor (MIF) acetylation. Mass spectrum analysis and biochemical results revealed that HDAC6 inhibitor or aspirin treatment promoted MIF acetylation on the K78 residue. MIF K78 acetylation suppressed the interaction between MIF and AIF, which impaired MIF translocation to the nucleus in ischemic cortical neurons. Moreover, neuronal DNA fragmentation and neuronal death were impaired in the cortex after ischemia in MIF K78Q mutant mice. Our results indicate that the neuroprotective effect of HDAC6 inhibition and aspirin treatment results from MIF K78 acetylation; thus, MIF K78 acetylation may be a therapeutic target for ischemic stroke and other neurological diseases.

Development of a general bioluminescent activity assay for peptide ligases.

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labelling and cyclic peptide synthesis. To characterize various known peptide ligases or identify new ones, we propose a general bioluminescent activity assay via the genetic fusion of a recognition motif of peptide ligase(s) to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase(s) to the N-terminus of the low-affinity SmBiT complementation tag. After the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase(s), its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the bioluminescent activity assay using bacterial sortase A and plant-derived butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The present bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.

G3BP1 promotes human breast cancer cell proliferation through coordinating with GSK-3ß and stabilizing ß-catenin.

Ras-GTPase activating SH3 domain-binding protein 1 (G3BP1) is a multifunctional binding protein involved in the development of a variety of human cancers. However, the role of G3BP1 in breast cancer progression remains largely unknown. In this study, we report that G3BP1 is upregulated and correlated with poor prognosis in breast cancer. Overexpression of G3BP1 promotes breast cancer cell proliferation by stimulating ß-catenin signaling, which upregulates a number of proliferation-related genes. We further show that G3BP1 improves the stability of ß-catenin by inhibiting its ubiquitin-proteasome degradation rather than affecting the transcription of ß-catenin. Mechanistically, elevated G3BP1 interacts with and inactivates GSK-3ß to suppress ß-catenin phosphorylation and degradation. Disturbing the G3BP1-GSK-3ß interaction accelerates the degradation of ß-catenin, impairing the proliferative capacity of breast cancer cells. Our study demonstrates that the regulatory mechanism of the G3BP1/GSK-3ß/ß-catenin axis may be a potential therapeutic target for breast cancer.

Mitochondria autophagy: a potential target for cancer therapy.

Mitophagy is a selective form of macroautophagy in which dysfunctional and damaged mitochondria can be efficiently degraded, removed and recycled through autophagy. Selective removal of damaged or fragmented mitochondria is critical to the functional integrity of the entire mitochondrial network and cells. In past decades, numerous studies have shown that mitophagy is involved in various diseases; however, since the dual role of mitophagy in tumour development, mitophagy role in tumour is controversial, and further elucidation is needed. That is, although mitophagy has been demonstrated to contribute to carcinogenesis, cell migration, ferroptosis inhibition, cancer stemness maintenance, tumour immune escape, drug resistance, etc. during cancer progression, many research also shows that to promote cancer cell death, mitophagy can be induced physiologically or pharmacologically to maintain normal cellular metabolism and prevent cell stress responses and genome damage by diminishing mitochondrial damage, thus suppressing tumour development accompanying these changes. Signalling pathway-specific molecular mechanisms are currently of great biological significance in the identification of potential therapeutic targets. Here, we review recent progress of molecular pathways mediating mitophagy including both canonical pathways (Parkin/PINK1- and FUNDC1-mediated mitophagy) and noncanonical pathways (FKBP8-, Nrf2-, and DRP1-mediated mitophagy); and the regulation of these pathways, and abovementioned pro-cancer and pro-death roles of mitophagy. Finally, we summarise the role of mitophagy in cancer therapy. Mitophagy can potentially be acted as the target for cancer therapy by promotion or inhibition.

NOD1/RIP2 signalling enhances the microglia-driven inflammatory response and undergoes crosstalk with inflammatory cytokines to exacerbate brain damage following intracerebral haemorrhage in mice.

BACKGROUND: Secondary brain damage caused by the innate immune response and subsequent proinflammatory factor production is a major factor contributing to the high mortality of intracerebral haemorrhage (ICH). Nucleotide-binding oligomerization domain 1 (NOD1)/receptor-interacting protein 2 (RIP2) signalling has been reported to participate in the innate immune response and inflammatory response. Therefore, we investigated the role of NOD1/RIP2 signalling in mice with collagenase-induced ICH and in cultured primary microglia challenged with hemin. METHODS: Adult male C57BL/6 mice were subjected to collagenase for induction of ICH model in vivo. Cultured primary microglia and BV2 microglial cells (microglial cell line) challenged with hemin aimed to simulate the ICH model in vitro. We first defined the expression of NOD1 and RIP2 in vivo and in vitro using an ICH model by western blotting. The effect of NOD1/RIP2 signalling on ICH-induced brain injury volume, neurological deficits, brain oedema, and microglial activation were assessed following intraventricular injection of either ML130 (a NOD1 inhibitor) or GSK583 (a RIP2 inhibitor). In addition, levels of JNK/P38 MAPK, IκBα, and inflammatory factors, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, and inducible nitric oxide synthase (iNOS) expression, were analysed in ICH-challenged brain and hemin-exposed cultured primary microglia by western blotting. Finally, we investigated whether the inflammatory factors could undergo crosstalk with NOD1 and RIP2. RESULTS: The levels of NOD1 and its adaptor RIP2 were significantly elevated in the brains of mice in response to ICH and in cultured primary microglia, BV2 cells challenged with hemin. Administration of either a NOD1 or RIP2 inhibitor in mice with ICH prevented microglial activation and neuroinflammation, followed by alleviation of ICH-induced brain damage. Interestingly, the inflammatory factors interleukin (IL)-1ß and tumour necrosis factor-α (TNF-α), which were enhanced by NOD1/RIP2 signalling, were found to contribute to the NOD1 and RIP2 upregulation in our study. CONCLUSION: NOD1/RIP2 signalling played an important role in the regulation of the inflammatory response during ICH. In addition, a vicious feedback cycle was observed between NOD1/RIP2 and IL-1ß/TNF-α, which could to some extent result in sustained brain damage during ICH. Hence, our study highlights NOD1/RIP2 signalling as a potential therapeutic target to protect the brain against secondary brain damage during ICH.

Role of co- and post-translational modifications of SFKs in their kinase activation.

Src family kinases (SFKs) are non-receptor tyrosine kinases and are involved in various cellular functions (proliferation, differentiation, migration, survival and invasion) by regulating downstream pathways. Considerable evidence suggests that co- and post-translational modifications are highly related to the activation of SFKs and their downstream signals. How SFKs are activated and how their subsequent cascades were regulated has been reviewed in previous reports. However, the contribution of co- and post-translational modification to SFKs activation has not been fully elucidated. This review focuses on the effect of these modifications on SFKs activity according to structural and biochemical studies and uncovers the significance of co-and post-translational modifications in the regulation of SFKs activity.

Role and molecular mechanism of stem cells in colorectal cancer initiation.

In recent years, the rate of colorectal cancer has sharply increased, especially in China, where it ranks second for the number of cancer fatalities. Currently, the treatment of colorectal cancer patients involves the combination of resection surgery and treatment with postoperative anticancer drugs such as 5-FU and oxaliplatin. However, recurrence and metastasis after treatment are still the dominant reasons for the low survival rate. Colorectal cancer stem cells (CSCs) are regarded as the key contributors to tumour recurrence and metastasis due to their resistance to chemotherapy drugs and their extremely high tumourigenicity. Once CSCs overcome chemotherapy treatment, they continue to survive and reinitiate proliferation to form tumours, leading to recurrence. The dominant reason for CSC resistance is that most anticancer drugs are aimed at inhibiting proliferative pathways in cancer cells that differ from those in CSCs. Therefore, studies on the characteristics of CSCs and their intracellular molecular pathways are essential for the exploration of CSC-targeted drugs. In this report, we review recent advances in the research of CSCs and, in particular, review the important intracellular molecular pathways, such as HOXA5-catenin, STRAP-NOTCH and YAP/TAZ, related to the maintenance and differentiation of stem cells to generate a theoretical basis for the exploration of CSC-targeted drugs.

[Clinical characteristics and drug sensitivity in children with invasive pneumococcal disease: a multicenter study].

OBJECTIVE: To study the clinical characteristics, drug sensitivity of isolated strains, and risk factors of drug resistance in children with invasive pneumococcal disease (IPD). METHODS: The clinical characteristics and drug sensitivity of the isolated strains of 246 hospitalized children with IPD in nine grade A tertiary children's hospitals from January 2016 to June 2018 were analyzed. RESULTS: Of the 246 children with IPD, there were 122 males and 124 females. Their ages ranged from 1 day to 14 years, and among them, 68 (27.6%) patients were less than 1 year old, 54 (22.0%) patients were 1 to 2 years old, 97 (39.4%) patients were 2 to 5 years old, and 27 (11.0%) patients were 5 to 14 years old. Pneumonia with sepsis was the most common infection type (58.5%, 144/246), followed by bloodstream infection without focus (19.9%, 49/246) and meningitis (15.0%, 37/246). Forty-nine (19.9%) patients had underlying diseases, and 160 (65.0%) had various risk factors for drug resistance. The isolated Streptococcus pneumoniae strains were 100% sensitive to vancomycin, linezolid, moxifloxacin, and levofloxacin, 90% sensitive to ertapenem, ofloxacin, and ceftriaxone, but had a low sensitivity to erythromycin (4.2%), clindamycin (7.9%), and tetracycline (6.3%). CONCLUSIONS: IPD is more common in children under 5 years old, especially in those under 2 years old. Some children with IPD have underlying diseases, and most of the patients have various risk factors for drug resistance. Pneumonia with sepsis is the most common infection type. The isolated Streptococcus pneumoniae strains are highly sensitive to vancomycin, linezolid, moxifloxacin, levofloxacin, ertapenem, and ceftriaxone in children with IPD.

Antibiotic resistance profiles and multidrug resistance patterns of Streptococcus pneumoniae in pediatrics: A multicenter retrospective study in mainland China.

Emergent resistance to antibiotics among Streptococcus pneumoniae isolates is a severe problem worldwide. Antibiotic resistance profiles for S pneumoniae isolates identified from pediatric patients in mainland China remains to be established.The clinical features, antimicrobial resistance, and multidrug resistance patterns of S pneumoniae were retrospectively analyzed at 10 children's hospitals in mainland China in 2016.Among the collected 6132 S pneumoniae isolates, pneumococcal diseases mainly occurred in children younger than 5 years old (85.1%). The resistance rate of S pneumoniae to clindamycin, erythromycin, tetracycline, and trimethoprim/sulfamethoxazole was 95.8%, 95.2%, 93.6%, and 66.7%, respectively. The resistance rates of S pneumoniae to penicillin were 86.9% and 1.4% in non-meningitis and meningitis isolates, while the proportions of ceftriaxone resistance were 8.2% and 18.1%, respectively. Pneumococcal conjugate vaccine was administered to only 4.1% of patients. Penicillin and ceftriaxone resistance, underling diseases, antibiotic resistant risk factors, and poor prognosis appeared more frequently in invasive pneumococcal diseases. The incidence of multidrug resistance (MDR) was 46.1% in patients with invasive pneumococcal disease which was more than in patients with non-invasive pneumococcal disease (18.3%). Patients with invasive pneumococcal disease usually have several MDR coexistence.S pneumoniae isolates showed high resistance to common antibiotics in mainland China. Penicillin and ceftriaxone resistance rate of invasive streptococcal pneumonia patients were significantly higher than that of non-invasive S pneumoniae patients. Alarmingly, 46.1% of invasive clinical isolates were multidrug resistant, so it is important to continued monitor the resistance of S pneumoniae when protein conjugate vaccine (PCV13) is coming in mainland China.

The roles and mechanisms of G3BP1 in tumour promotion.

Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is a SH3 domain-binding protein that is overexpressed in a variety of tumour tissues and cancers, such as head and neck cancer, lung cancer, prostate cancer, colon cancer and breast cancer. G3BP1 promotes tumour cell proliferation and metastasis and inhibits apoptosis by regulating the Ras, TGF-ß/Smad, Src/FAK and p53 signalling pathways. At present, polypeptides targeting G3BP1 have shown anti-tumour activity and G3BP1 also involved in anti-cancer effects of some polyphenolic compounds (resveratrol and EGCG). Therefore G3BP1 may be a potential target for tumour treatment.

Identification of O-GlcNAcylation Modification in Diabetic Retinopathy and Crosstalk with Phosphorylation of STAT3 in Retina Vascular Endothelium Cells.

BACKGROUND/AIMS: Recently, we observed an increase in O-GlcNAc (O-linked-ß-N-acetylglucosamine) modification, and signal transducer and activator of transcription proteins 3 (STAT3) expression in primary retinal vascular endothelial cells (RVECs) under high glucose conditions and tissues altered by diabetic retinopathy (DR). In this study, we focused on the correlations between O-GlcNAcylation and STAT3 phosphorylation, and their potential effects with regards to DR. METHODS: Expression of O-GlcNAcylation and STAT3 were detected in DR-affected tissues and primary RVECs. The relationship between O-GlcNAcylation and STAT3 was further delineated by immunoprecipitation and Western blot analysis. Effects of O-GlcNAcylation on human RVEC apoptosis and involved protein expression were assayed with flow cytometry and Western blot. RESULTS: Global O-GlcNAcylation and pSTAT3 levels were significantly elevated in diabetic rat retina and primary RVECs under high glucose conditions. In vitro assays demonstrated that the Tyr705 site was sensitive to high glucose. While O-GlcNAcylation inhibited p727STAT3 expression, augmented O-GlcNAcylation could balance p705STAT3 expression within relatively high levels corresponding to vascular endothelial growth factor (VEGF) changes. Immunoprecipitation revealed that STAT3 was modified by O-GlcNAcylation and phosphorylation simultaneously. Next, we observed that overexpression of O-GlcNAcylation could relieve human RVEC apoptosis related to the JAK2-Tyr705STAT3-VEGF pathway. CONCLUSION: O-GlcNAcylation could relieve RVECs apoptosis through the STAT3 pathway in DR, and O-GlcNAcylation combined with STAT3 phosphorylation might open up new insights into the mechanisms of DR and other diabetic complications.

[Advances in researches on ß-carbonic anhydrases as anti-parasitic drug targets].

ß-carbonic anhydrases (ß-CAs) are ubiquitous metalloenzymes which active site contains a zinc ion (Zn²âº), and they could catalyze the hydration of carbon dioxide to bicarbonate and protons efficiently and are involved in many biological processes, such as respiration, pH and CO2 homeostasis, biosynthetic reactions, virulence regulation and so on, and may play a critical role in the life activity of many organisms which contain these enzymes. ß-CAs are widely distributed in fungi, bacteria, algae, plants and a small number of protozoan and metazoan except vertebrates. Therefore, as potential drug targets for designing and developing antibacterial and anti-parasitic drugs, ß-CAs promise a broad application prospect. This paper focuses on the distribution, physiological function and the progress of researches on ß-CAs in parasites and their vectors.

Peroxisome proliferator-activated receptor-γ agonist pioglitazone fails to attenuate renal fibrosis caused by unilateral ureteral obstruction in mice.

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction (UUO) was used to establish a different renal fibrosis model. PPAR? agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for a-SMA and PDGFR-ß, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-ß and MCP-1 had no significant changes, at the same time, CD4(+) T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.

[Diagnosis of an Imported Case of Plasmodium ovale Infection in Nanping City, Fujian].

An imported case previously misdiagnosed as vivax malaria was reviewed. The epidemiological data and blood sample were collected. The detection was conducted by microscopy, rapid diagnostic test (RDT) and nested PCR. The case was finally comfirmed as the first imported case of Plasmodium ovale infection in Nanping.