BACKGROUND: Juvenile amyotrophic lateral sclerosis (JALS) is an uncommon form of amyotrophic lateral sclerosis whose age at onset (AAO) is defined as prior to 25 years. FUS mutations are the most common cause of JALS. SPTLC1 was recently identified as a disease-causative gene for JALS, which has rarely been reported in Asian populations. Little is known regarding the difference in clinical features between JALS patients carrying FUS and SPTLC1 mutations. This study aimed to screen mutations in JALS patients and to compare the clinical features between JALS patients with FUS and SPTLC1 mutations. METHODS: Sixteen JALS patients were enrolled, including three newly recruited patients between July 2015 and August 2018 from the Second Affiliated Hospital, Zhejiang University School of Medicine. Mutations were screened by whole-exome sequencing. In addition, clinical features such as AAO, onset site and disease duration were extracted and compared between JALS patients carrying FUS and SPTLC1 mutations through a literature review. RESULTS: A novel and de novo SPTLC1 mutation (c.58G>A, p.A20T) was identified in a sporadic patient. Among 16 JALS patients, 7/16 carried FUS mutations and 5/16 carried respective SPTLC1 , SETX , NEFH , DCTN1 , and TARDBP mutations. Compared with FUS mutation patients, those with SPTLC1 mutations had an earlier AAO (7.9 ± 4.6 years vs. 18.1 ± 3.9 years, P  < 0.01), much longer disease duration (512.0 [416.7-607.3] months vs. 33.4 [21.6-45.1] months, P  < 0.01), and no onset of bulbar. CONCLUSION Our findings expand the genetic and phenotypic spectrum of JALS and help to better understand the genotype-phenotype correlation of JALS.
Humans ; Amyotrophic Lateral Sclerosis/genetics* ; DNA Helicases/genetics* ; Genetic Association Studies ; Multifunctional Enzymes/genetics* ; Mutation/genetics* ; RNA Helicases/genetics* ; RNA-Binding Protein FUS/genetics* ; Serine C-Palmitoyltransferase/genetics* ; Child, Preschool ; Child ; Adolescent ; Young Adult

As an excellent hosting matrices for enzyme immobilization, metal-organic framework (MOFs) provides superior physical and chemical protection for biocatalytic reactions. In recent years, the hierarchical porous metal-organic frameworks (HP-MOFs) have shown great potential in enzyme immobilization due to their flexible structural advantages. To date, a variety of HP-MOFs with intrinsic or defective porous have been developed for the immobilization of enzymes. The catalytic activity, stability and reusability of enzyme@HP-MOFs composites are significantly enhanced. This review systematically summarized the strategies for developing enzyme@HP-MOFs composites. In addition, the latest applications of enzyme@HP-MOFs composites in catalytic synthesis, biosensing and biomedicine were described. Moreover, the challenges and opportunities in this field were discussed and envisioned.
Metal-Organic Frameworks/chemistry* ; Porosity ; Enzymes, Immobilized/chemistry* ; Biocatalysis ; Catalysis

Enzyme-catalyzed CO₂ reduction to value-added commodities is important for alleviating the global environmental issues and energy crises due to high selectivity and mild conditions. Owing to high energy density, formic acid or methanol produced from CO₂ using formate dehydrogenase (FDH) or multi-enzyme cascades are promising target chemicals for CO₂ utilization. However, the low activity, poor stability and low reusability of key enzymes involved in such process hampered its large-scale application. Enzyme immobilization provides an effective solution to these problems and significant progress have been made in immobilization carriers. Moreover, integration of enzyme immobilization with other catalysis techniques have been explored extensively. This review summarized the recent advances in the immobilization of enzymes using membranes, inorganic materials, metal-organic frameworks, covalent organic frameworks and other carriers, and illustrated the characteristics and advantages of different immobilization materials and immobilization methods. The synergistic effects and applications of immobilized enzymes and electrocatalytic or photocatalytic coupling reaction systems for CO₂ reduction were further summarized. Finally, the current challenges of enzyme immobilization technology and coupling reaction systems were pointed out and their development prospects were presented.
Enzymes, Immobilized ; Carbon Dioxide ; Catalysis ; Formate Dehydrogenases ; Metal-Organic Frameworks

Objective: To explore the key deubiquitinating enzymes that maintain the stemness of liver cancer stem cells and provide new ideas for targeted liver cancer therapy. Methods: The high-throughput CRISPR screening technology was used to screen the deubiquitinating enzymes that maintain the stemness of liver cancer stem cells. RT-qPCR and Western blot were used to analyze gene expression levels. Stemness of liver cancer cells was detected by spheroid-formation and soft agar colony formation assays. Tumor growth in nude mice was detected by subcutaneous tumor-bearing experiments. Bioinformatics and clinical samples were examined for the clinical significance of target genes. Results: MINDY1 was highly expressed in liver cancer stem cells. The expression of stem markers, the self-renewal ability of cells, and the growth of transplanted tumors were significantly reduced and inhibited after knocking out MINDY1, and its mechanism of action may be related to the regulation of the Wnt signaling pathway. The expression level of MINDY1 was higher in liver cancer tissues than that in adjacent tumors, which was closely related to tumor progression, and its high expression was an independent risk factor for a poor prognosis of liver cancer. Conclusion: The deubiquitinating enzyme MINDY1 promotes stemness in liver cancer cells and is one of the independent predictors of poor prognosis in liver cancer.
Animals ; Mice ; Cell Line, Tumor ; Mice, Nude ; Liver Neoplasms/pathology* ; Prognosis ; Deubiquitinating Enzymes/metabolism* ; Neoplastic Stem Cells/pathology* ; Gene Expression Regulation, Neoplastic

Aims: This study was designed to examine the enzyme activity of selected virulent isolates of Ganoderma boninense against oil palm. In a separate in vitro assessment, the effect of macronutrients on the mycelial growth of four selected Ganoderma spp. was also tested. Methodology and results: The study involved a comparison of ligninolytic enzymes; lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) profiling of eight isolates of G. boninense, categorized into three levels of aggressiveness, with two control isolates (G. boninense PER71 and G. tornatum NPG1) using solid-state fermentation (SSF). The Principal Component Analysis (PCA) revealed that the isolates had a significant production of ligninolytic enzymes on day 80. The most aggressive isolate, ET61 had the highest Lac production. As for the macronutrient test, mycelial growth for all the Ganoderma spp. was highly affected by potassium (K). Conclusion, significance and impact of study The findings of this study elucidated the characteristics of G. boninense in relation to enzyme production for the degradation of oil palm lignin and the identification of essential nutrients involved in the survival and growth of Ganoderma spp. The study provides vital information on the pathogenic characteristics of G. boninense isolates involved in biomass degradation along with the role of nutrient on the growth of Ganoderma spp. that may influence basal stem rot (BSR) management in the field.
Enzymes ; Ganoderma ; Palm Oil

Aims: The present study was aimed to determine the diversity of the halophilic and halotolerant bacteria with hydrolase activities from salterns of the protected ecosystem Khnifiss lagoon (Morocco). Methodology and results: Bacteria were isolated from sediment samples collected at five hypersaline sites on three different growth media supplemented with various concentrations of NaCl. Thirty bacterial isolates were identified using the 16S rDNA sequencing method. Physiological characterization was examined on Columbia agar with different concentrations of NaCl and pH values and incubated at different temperatures. Enzymatic activities were tested in submerged cultures with appropriate substrates. A total of 246 halophilic bacterial isolates from the salines of the protected ecosystem of Khnifiss lagoon in the south of Morocco were able to produce different hydrolases (cellulases, amylases, DNases, lipases and proteases). Furthermore, the majority of the isolates were classified as moderately halophilic bacteria that grew optimally between 5% and 15% of NaCl, and they adapted to other extreme conditions than salt: some cases tolerated a pH range of 4.5 to 9.4, as well as a temperature range of 10 to 50 °C. The 16S rDNA sequencing and phylogenetic analysis results indicated that most of the bacteria isolated from the salt marshes studied belong to the genus Bacillus. Conclusion, significance and impact of study Phenotypic and phylogenetic characterization of halophilic and halotolerant bacteria isolated from the salines of the protected ecosystem of Khnifiss lagoon has shown great diversity. Screening of hydrolytic enzymes produced by these bacteria suggests that the salines of the Khnifiss lagoon are a rich source of technologically interesting bacteria.
Halophilic bacteria ; extracellular hydrolytic enzymes ; screening ; hypersaline environments ; Khnifiss lagoon

OBJECTIVES: To investigate the effects and mechanisms of deubiquitinating enzyme Josephin domain containing 2 (JOSD2) on susceptibility of non-small cell lung carcinoma (NSCLC) cells to anti-cancer drugs. METHODS: The transcriptome expression and clinical data of NSCLC were downloaded from the Gene Expression Omnibus. Principal component analysis and limma analysis were used to investigate the deubiquitinating enzymes up-regulated in NSCLC tissues. Kaplan-Meier analysis was used to investigate the relationship between the expression of deubiquitinating enzymes and overall survival of NSCLC patients. Gene ontology enrichment and gene set enrichment analysis (GSEA) were used to analyze the activation of signaling pathways in NSCLC patients with high expression of JOSD2. Gene set variation analysis and Pearson correlation were used to investigate the correlation between JOSD2 expression levels and DNA damage response (DDR) pathway. Western blotting was performed to examine the expression levels of JOSD2 and proteins associated with the DDR pathway. Immunofluorescence was used to detect the localization of JOSD2. Sulforhodamine B staining was used to examine the sensitivity of JOSD2-knock-down NSCLC cells to DNA damaging drugs. RESULTS: Compared with adjacent tissues, the expression level of JOSD2 was significantly up-regulated in NSCLC tissues (P<0.05), and was significantly correlated with the prognosis in NSCLC patients (P<0.05). Compared with the tissues with low expression of JOSD2, the DDR-related pathways were significantly upregulated in NSCLC tissues with high expression of JOSD2 (all P<0.05). In addition, the expression of JOSD2 was positively correlated with the activation of DDR-related pathways (all P<0.01). Compared with the control group, overexpression of JOSD2 significantly promoted the DDR in NSCLC cells. In addition, DNA damaging agents significantly increase the nuclear localization of JOSD2, whereas depletion of JOSD2 significantly enhanced the sensitivity of NSCLC cells to DNA damaging agents (all P<0.05). CONCLUSIONS Deubiquitinating enzyme JOSD2 may regulate the malignant progression of NSCLC by promoting DNA damage repair pathway, and depletion of JOSD2 significantly enhances the sensitivity of NSCLC cells to DNA damaging agents.
Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Antineoplastic Agents/pharmacology* ; Lung Neoplasms/genetics* ; DNA Damage ; DNA ; Deubiquitinating Enzymes/genetics*

The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.
Animals ; Cell Line ; Fibrosis ; Humans ; Kidney Diseases ; Mice ; Ubiquitin-Conjugating Enzymes/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Ubiquitination

Objective: To develop effective alternatives to natural enzymes, it is crucial to develop nanozymes that are economical, resource efficient, and environmentally conscious. Carbon nanomaterials that have enzyme-like activities have been extensively developed as substitutes for traditional enzymes. Methods: Carbide-derived carbons (CDCs) were directly synthesized via a one-step electrochemical method from a MAX precursor using an ammonium bifluoride electrolyte at ambient conditions. The CDCs were characterized by systematic techniques. Results: CDCs showed bienzyme-like activities similar to that of peroxidase and superoxide dismutase. We systematically studied the dependence of CDC enzyme-like activity on different electrolytes and electrolysis times to confirm activity dependence on CDC content. Additionally, the synthesis mechanism and CDC applicability were elaborated and demonstrated, respectively. Conclusion The demonstrated synthesis strategy eliminates tedious intercalation and delamination centrifugation steps and avoids using high concentrations of HF, high temperatures, and halogen gases. This study paves the way for designing two-dimensional material-based nanocatalysts for nanoenzyme and other applications.
Ammonium Compounds/chemical synthesis* ; Carbon/chemistry* ; Electrochemical Techniques ; Enzymes ; Fluorides/chemical synthesis* ; Humans ; Nanostructures ; Oxidation-Reduction

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.
Biocatalysis ; Enzymes, Immobilized/metabolism* ; Ferritins/metabolism* ; Glycoside Hydrolases/metabolism*