Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

USP54 is a potential therapeutic target in castration-resistant prostate cancer.

BACKGROUND: USP54, a ubiquitin-specific protease in the deubiquitinase (DUB) family, facilitates the malignant progression of several types of cancer. However, the role of USP54 in prostate cancer (PCa), especially castration-resistant prostate cancer (CRPC), remains unknown. METHODS: We established the CRPC LNCaP-AI cell line from the hormone-sensitive prostate cancer (HSPC) LNCaP cell line. RNA-Seq was utilized to explore DUB expression levels in LNCaP and LNCaP-AI. USP54 was knocked down, and its effects on cell growth were evaluated in vitro and in vivo. Bioinformatics analyses were conducted to explore signaling pathways affected by USP54 in PCa. Quantitative polymerase chain reaction was used to confirm key signaling pathways involved. RESULTS: USP54 was the most strongly upregulated DUB in LNCaP-AI cells compared with LNCaP cells. USP54 levels were higher in PCa than in normal tissues. USP54 silencing suppressed the proliferation of PCa cell lines, both in vitro and in vivo. USP54 expression was positively correlated with the androgen receptor (AR) signaling level in PCa samples, and USP54 knockdown inhibited AR signaling in PCa cells. CONCLUSIONS: USP54 was upregulated during HSPC progression to CRPC. USP54 depletion suppressed CRPC cell proliferation both in vitro and in vivo. USP54 may facilitate PCa progression by regulating AR signaling.

ANP32B inhibition suppresses the growth of prostate cancer cells by regulating c-Myc signaling.

ANP32B is a histone chaperone that interacts with various transcription factors that regulate cancer cell proliferation, immigration, and apoptosis. c-Myc, a well-known oncogenic protein, is a principal player in the initiation and progression of prostate cancer (PC). The means by which ANP32B and c-Myc act remain unknown. We downloaded clinical data from the GEO, TCGA, and other databases to explore ANP32B expression and its effects on the survival of PC and normal tissues. ANP32B-knockdown cell lines were used to evaluate how ANP32B affected cell proliferation in vitro and in vivo. Gene set enrichment analysis and RNAseq were employed to define how ANP32B regulated PC pathways. Immunohistochemical measures were used to detect the expression levels of relevant proteins in xenografts and PC tissues. ANP32B expression increased in PC tissues; ANP32B knockdown inhibited cell growth but this was rescued by c-Myc signaling. ANP32B is thus a PC oncogene and may serve as a valuable therapeutic target when seeking to treat PC.

Total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates using side-chain Boc/tBu protecting groups.

A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/tBu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/tBu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.

The biological behavior of drug-resistantovarian cancer cells and changes in the CA125 and HE4 levels after CIK interventions.

OBJECTIVE: This study aimed to investigate the biological behavior of drug-resistant ovarian cancer cells and changes in the cancer antigen 125 (CA125) and human epididymal protein 4 (HE4) levels after the application of cytokine-induced killer (CIK) intervention. METHODS: Drug-resistant ovarian cancer cells (namely SKVCR) were treated with CIK at different concentrations to observe the changes in the cell survival and cell morphology and the CA125, HE4, cytokine transforming growth factor-α (TGF-α), and tumor necrosis factor-α (TNF-α) levels in the cell lines before and after intervention. RESULTS: With an increase in the CIK concentration, the survival rate of the SKVCR cell lines showed a decreasing trend. Under a constant CIK concentration, the survival rate of the SKVCR cell lines gradually decreased over time but become stable at 72 h. Before the CIK intervention, the SKVCR cells were full and rounded in shape, but after the CIK intervention, there was remarkable cell shrinkage and an increase in apoptotic cells. Compared with before the CIK intervention, the CA125 and HE4 levels were significantly decreased, but the TGF-α and TNF-α levels were increased (P<0.05). CONCLUSION: After the CIK intervention in the drug-resistant ovarian cancer cells, the cell survival rate decreases with an increase in the CIK concentration or an extension of the intervention time, and the cell morphology will be significantly improved, and the CA125, HE4, and other related cytokine levels will also change significantly, suggesting that CIK can kill drug-resistant ovarian cancer cells.

Berberine-Loaded Thiolated Pluronic F127 Polymeric Micelles for Improving Skin Permeation and Retention.

BACKGROUND: Challenges associated with local antibacterial and anti-inflammatory drugs include low penetration and retention of drugs at the expected action site. Additionally, improving these challenges allows for the prevention of side effects that are caused by drug absorption into the systemic circulation and helps to safely treat local skin diseases. METHODS: In the current study, we successfully prepared a thiolated pluronic F127 polymer micelles (BTFM), which binds to keratin through a disulphide bond, to produce skin retention. In addition, the small particle size of polymer micelles promotes the penetration of carriers into the skin. The current study was divided into two experiments: an in vitro experiment; an in vivo experiment that involved the penetration of the micelle-loaded drugs into the skin of rats, the skin irritation test and the anti-inflammatory activity of the drug-loaded micelles on dimethyl benzene-induced ear edema in mice. RESULTS: Results from our in vitro transdermal experiment revealed that the amount of drug absorbed through the skin was decreased after the drug was loaded in the BTFM. Further, results from the vivo study, which used fluorescence microscopy to identify the location of the BTFM after penetration, revealed that there was strong fluorescence in the epidermis layer, but there was no strong fluorescence in the deep skin layer. In addition, the BTFM had a very good safety profile with no potentially hazardous skin irritation and transdermal administration of BTFM could significantly suppress ear edema induced by dimethyl benzene. Therefore, these findings indicated that BTFM reduced the amount of drug that entered the systemic circulation. Our results also demonstrated that the BTFM had a certain affinity for keratin. CONCLUSION: Our experimental results suggest that the BTFM may be an effective drug carrier for local skin therapy with good safety profile.

Efficacy of bevacizumab combined with nedaplatin in the treatment of ovarian cancer and its effects on tumor markers and immunity of patients.

PURPOSE: To study the efficacy of bevacizumab combined with nedaplatin in the treatment of ovarian cancer and its effects on tumor markers and immunity of patients. METHODS: A total of 100 ovarian cancer patients treated in our hospital from January 2015 to December 2018 were enrolled and divided into experimental group (n=50) and control group (n=50) using a random number table. Patients in the control group were treated with carboplatin alone, while those in the experimental group were treated with bevacizumab combined with nedaplatin, based on the treatment in the control group. The efficacy, adverse reactions and quality of life (QoL) score of patients were observed. Moreover, the levels of serum human epididymis protein 4 (HE4), alpha fetoprotein (AFP) and macrophage migration inhibitory factor (MIF), tumor markers carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA) and CA19.9, immunity indexes cluster of differentiation 3+ (CD3+), CD4+, CD8+ and natural killer (NK) cells, and serum inflammatory factors interleukin-8 (IL-8), IL-6 and IL-10 were detected before and after therapy. RESULTS: In the experimental group, the efficacy was superior to that in control group (p<0.05), and the adverse reactions were significantly reduced (p<0.05), while the QoL score was significantly increased (p<0.05). Before treatment, there were no significant differences in the levels of HE4, AFP and MIF, tumor markers CA125, CEA and CA19.9, immunity indexes CD3+, CD4+, CD8+ and NK cells, and inflammatory factors IL-8, IL-6 and IL-10 between the two groups (p>0.05). After treatment, the levels of HE4, AFP and MIF, CA125, CEA and CA19.9 and inflammatory factors IL-8, IL-6 and IL-10 obviously declined in the experimental group compared with the control group (p<0.05), while the levels of immunity indexes CD3+, CD4+, CD8+ and NK cells were clearly increased (p<0.05). CONCLUSION: Bevacizumab combined with nedaplatin has good efficacy in the treatment of ovarian cancer, which can significantly improve the tumor markers, enhance the immunity and ameliorate the QoL of patients, with fewer adverse reactions, so it is worthy of popularization and application.

Lactate-fueled oxidative metabolism drives DNA methyltransferase 1-mediated transcriptional co-activator with PDZ binding domain protein activation.

Activity of transcriptional co-activator with PDZ binding domain (TAZ) protein is strongly implicated in the pathogenesis of human cancer and is influenced by tumor metabolism. High levels of lactate concentration in the tumor microenvironment as a result of metabolic reprogramming are inversely correlated with patient overall survival. Herein, we investigated the role of lactate in the regulation of the activity of TAZ and showed that glycolysis-derived lactate efficiently increased TAZ expression and activity in lung cancer cells. We showed that the reactive oxygen species (ROS) generated by lactate-fueled oxidative phosphorylation (OXPHOS) in mitochondria activated AKT and thereby inhibited glycogen synthase kinase 3 beta/beta-transducin repeat-containing proteins (GSK-3ß/ß-TrCP)-mediated ubiquitination and degradation of DNA methyltransferase 1 (DNMT1). Upregulation of DNMT1 by lactate caused hypermethylation of TAZ negative regulator of the LATS2 gene promoter, leading to TAZ activation. Moreover, TAZ binds to the promoter of DNMT1 and is necessary for DNMT1 transcription. Our study showed a molecular mechanism of DNMT1 in linking tumor metabolic reprogramming to the Hippo-TAZ pathway and functional significance of the DNMT1-TAZ feedback loop in the migratory and invasive potential of lung cancer cells.

Efficient Semi-Synthesis of Atypical Ubiquitin Chains and Ubiquitin-Based Probes Forged by Thioether Isopeptide Bonds.

The development of powerful and general methods to acquire ubiquitin (Ub) chains has prompted the deciphering of Ub-mediated processes. Herein, the cysteine-aminoethylation assisted chemical ubiquitination (CAACU) strategy is extended and improved to enable the efficient semi-synthesis of atypical Ub chain analogues and Ub-based probes. Combining the Cys aminoethylation and the auxiliary-mediated protein ligation, several linkage- and length-defined atypical Ub chains including di-Ubs, K27C-linked tri-Ub, K11/K48C-branched tri-Ub, and even the SUMOlated Ub are successfully prepared from recombinantly expressed starting materials at about a 9-20 mg L-1 expression level. In addition, the utility of this strategy is demonstrated with the synthesis of a novel non-hydrolyzable di-Ub PA probe, which may provide a new useful tool for the mechanistic studies of deubiquitinase (DUB) recognition.

Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer.

An efficient method for the assembly of polyUb chains using auxiliary-modified Ub isomers is reported. This strategy takes advantages of auxiliary-mediated native chemical ligation between the distal Ub C-terminal hydrazide and the auxiliary of proximal Ub. Using removable protecting groups, Lys48-linked and Lys6-linked tri-Ub and even a mixed-linkage Lys6, Lys48-linked triUb in multimilligram quantities was made. These results demonstrate that this strategy yields natural polyubiquitin chains of desired length and linkage by using Ub isomer.

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation.

Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.

Synthesis of l- and d-Ubiquitin by One-Pot Ligation and Metal-Free Desulfurization.

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one-pot native chemical ligation and metal-free desulfurization for chemical protein synthesis. This one-pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X-ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis coupled with a one-pot ligation-desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.

Accelerated Fmoc solid-phase synthesis of peptides with aggregation-disrupting backbones.

In this work, we describe an accelerated solid-phase synthetic protocol for ordinary or difficult peptides involving air-bath heating and amide protection. For the Hmsb-based backbone amide protection, an optimized acyl shift condition using 1,4-dioxane was discovered. The efficiency and robustness of the protocol was validated in the course of preparation of classical difficult peptides and ubiquitin protein segments.

Thiol-assisted one-pot synthesis of peptide/protein C-terminal thioacids from peptide/protein hydrazides at neutral conditions.

An efficient thiol-assisted one-pot synthesis of peptide/protein C-terminal thioacids was achieved by using peptide/protein hydrazides precursors at neutral pH and room temperature (about 20 °C). The transformation from hydrazides to thioacids was shown to be efficient for different C-terminal amino acids and was racemization-free. The in situ formed peptide-thioacids were further used for protein chemical synthesis and site-specific labelling successfully.