Stress-induced NEDDylation promotes cytosolic protein aggregation through HDAC6 in a p62-dependent manner.

Stress-coupled NEDDylation potentially regulates the aggregation of nuclear proteins, which could protect the nuclear ubiquitin-proteasome system from proteotoxic stress. However, it remains unclear how NEDDylation controls protein-aggregation responses to diverse stress conditions. Here, we identified HDAC6 as a direct NEDD8-binding partner that regulates the formation of aggresome-like bodies (ALBs) containing NEDDylated cytosolic protein aggregates during ubiquitin stress. HDAC6 colocalizes with stress-induced ALBs, and HDAC6 inhibition suppresses ALBs formation, but not stress-induced NEDDylation, suggesting that HDAC6 carries NEDDylated-proteins to generate ALBs. Then, we monitored the ALBs-associated proteostasis network and found that p62 directly controls ALBs formation as an acceptor of NEDDylated cytosolic aggregates. Interestingly, we also observed that ALBs are highly condensed in chloroquine-treated cells with impaired autophagic flux, indicating that ALBs rely on autophagy. Collectively, our data suggest that NEDD8, HDAC6, and p62 are involved in the management of proteotoxic stress by forming cytosolic ALBs coupled to the aggresome-autophagy flux.

A Novel Reciprocal Crosstalk between RNF168 and PARP1 to Regulate DNA Repair Processes.

Emerging evidence has suggested that cellular crosstalk between RNF168 and poly(ADP-ribose) polymerase 1 (PARP1) contributes to the precise control of the DNA damage response (DDR). However, the direct and reciprocal functional link between them remains unclear. In this report, we identified that RNF168 ubiquitinates PARP1 via direct interaction and accelerates PARP1 degradation in the presence of poly (ADP-ribose) (PAR) chains, metabolites of activated PARP1. Through mass spectrometric analysis, we revealed that RNF168 ubiquitinated multiple lysine residues on PARP1 via K48-linked ubiquitin chain formation. Consistent with this, micro-irradiation-induced PARP1 accumulation at damaged chromatin was significantly increased by knockdown of endogenous RNF168. In addition, it was confirmed that abnormal changes of HR and HNEJ due to knockdown of RNF168 were restored by overexpression of WT RNF168 but not by reintroduction of mutants lacking E3 ligase activity or PAR binding ability. The comet assay also revealed that both PAR-binding and ubiquitin-conjugation activities are indispensable for the RNF168-mediated DNA repair process. Taken together, our results suggest that RNF168 acts as a counterpart of PARP1 in DDR and regulates the HR/NHEJ repair processes through the ubiquitination of PARP1.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Isolation and characterization of a novel endo-beta-1, 4-glucanase from a metagenomic library of the black-goat rumen

ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Isolation and characterization of a novel endo-ß-1,4-glucanase from a metagenomic library of the black-goat rumen.

The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-ß-1,4-glucanase. The recombinant KG35 endo-ß-1,4-glucanase showed optimal activity within the range of 30-50°C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50°C at a pH of 5-7.

Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-ß-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.