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PubMed2024

Super Enhanced Purification of Denatured-Refolded Ubiquitinated Proteins by ThUBD Revealed Ubiquitinome Dysfunction in Liver Fibrosis.

Ubiquitination is crucial for maintaining protein homeostasis and plays a vital role in diverse biological processes. Ubiquitinome profiling and quantification are of great scientific significance. Artificial ubiquitin-binding domains (UBDs) have been widely employed to capture ubiquitinated proteins. The success of this enrichment relies on recognizing native spatial structures of ubiquitin and ubiquitin chains by UBDs under native conditions. However, the use of native lysis conditions presents significant challenges, including insufficient protein extraction, heightened activity of deubiquitinating enzymes and proteasomes in removing the ubiquitin signal, and purification of a substantial number of contaminant proteins, all of which undermine the robustness and reproducibility of ubiquitinomics. In this study, we introduced a novel approach that combines denatured-refolded ubiquitinated sample preparation (DRUSP) with a tandem hybrid UBD for ubiquitinomic analysis. The samples were effectively extracted using strongly denatured buffers and subsequently refolded using filters. DRUSP yielded a significantly stronger ubiquitin signal, nearly three times greater than that of the Control method. Then, eight types of ubiquitin chains were quickly and accurately restored; therefore, they were recognized and enriched by tandem hybrid UBD with high efficiency and no biases. Compared with the Control method, DRUSP showed extremely high efficiency in enriching ubiquitinated proteins, improving overall ubiquitin signal enrichment by approximately 10-fold. Moreover, when combined with ubiquitin chain-specific UBDs, DRUSP had also been proven to be a versatile approach. This new method significantly enhanced the stability and reproducibility of ubiquitinomics research. Finally, DRUSP was successfully applied to deep ubiquitinome profiling of early mouse liver fibrosis with increased accuracy, revealing novel insights for liver fibrosis research.

Animals↗

PubMed1999

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Agkistrodon↗

PubMed2001

Identification of key residues responsible for enzymatic and platelet-aggregation-inhibiting activities of acidic phospholipase A2S from Agkistrodon halys Pallas.

Site-directed mutagenesis was used to probe the structural and functional relationship of acidic phospholipase A2 from Agkistrodon halys Pallas. The mutants are AP-E6R (E6R), AP-D115K (D115K), AP-6R115K (E6R, D115K), AP-Y118M (Y118M), and AP-W119T (W119T). All mutants were inserted into a bacterial expression vector and effectively expressed in E. coli RR1. The purified recombinant enzymes were used to assay for enzymatic and inhibiting platelet aggregation activities. The enzymatic activities of AP-D115K, AP-Y118M and AP-W119T are close to that of denatured-refolded acidic phospholipase A2, while the enzymatic activities of AP-E6R, AP-6R1 15K are lower than that of denatured-refolded acidic phospholipase A2 (AP-WT). In these five mutants, AP-Y118M showed strongest inhibiting effect on platelet aggregation, which is the same as that of AP-WT, AP-W119T showed only modest activity and AP-E6R, AP-D115K, AP-6R115K showed little activity. To study the structural and functional relationships among these five mutants, molecular modeling of these five mutants was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activity of acidic phospholipase A2 are discussed.

Adult↗

PubMed1999

Expression and Biochemical Characterization of Acidic Phospholipase A(2)I from Agkistrodon acutus.

A cDNA encoding acidic phospholipase A(2)I(A.aAPLA(2)I)from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column Superose(TM)12. The enzymatic acti-vity and platelet aggregation inhibiting effect of the expressed A.aAPLA(2)I is close to those of denatured-refolded native acidic PLA(2) from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured-refolded basic phospholipase A(2) from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Journal Article↗

PubMed2002

[The hemolytic site of the basic phospholipase A(2) from Agkistrodon halys pallas].

The gene of the basic phospholipase A(2) from Agkistrodon halys Pallas (BPLA(2) )was mutated site-directedly by polymerase chain reaction (PCR) and the residue Arg(34) of the encloding protein was substituted by Glu and Gln respectively. The mutant gene has been cloned into the expression vector pBLMVL2 and has been expressed in E.coli RR1 effectively. The protein was produced as insoluble inclusion bodies. After partial purification, the inclusion bodies were denatured and renatured into active form, and the renatured recombinant protein was purified by gel-filtration. The expression product has the same enzymatic activity as the denatured-refolded BPLA(2) and its hemolytic activity dropped distinctly, which suggest that the basic residue Arg(34) of BPLA(2) is a crucial amino acid residue during the process of hemolysis.

Agkistrodon↗