Deoxyribozymes capable of catalyzing sequence-specific RNA cleavage have found broad applications in biotechnology, DNA computing and environmental sensing. Among these, deoxyribozyme 8-17 is the ...most common small DNA motif capable of catalyzing RNA cleavage. However, the extent to which other DNA molecules with similar catalytic motifs exist remains elusive. Here we report a novel RNA-cleaving deoxyribozyme called 10-12opt that functions with an equally small catalytic motif and an unusually short binding arm. This deoxyribozyme contains a 14-nucleotide catalytic core that preferentially catalyzes RNA cleavage at UN dinucleotide junctions (k
= 0.9 h
for UU cleavage). Surprisingly, the left binding arm contains only three nucleotides and forms two canonical base pairs with the RNA substrate. Mutational analysis reveals that a riboguanosine residue 3-nucleotide downstream of cleavage site must not form canonical base pairing for the optimal catalysis, and this nucleobase likely participates in catalysis with its carbonyl O6 atom. Furthermore, we demonstrate that deoxyribozyme 10-12opt can be utilized to cleave certain microRNA sequences which are not preferentially cleaved by 8-17. Together, these results suggest that this novel RNA-cleaving deoxyribozyme forms a distinct catalytic structure than 8-17 and that sequence space may contain additional examples of DNA molecules that can cleave RNA at site-specific locations.
(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the ...Marek's disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)
and UL36(480)
, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.
Herein, we investigated the effects of new cofactors and inhibitors on an oxidative cleavage of DNA catalysis, known as a pistol-like DNAzyme (PLDz), to discuss its catalytic mechanism. PLDz ...performed its catalytic activity in the presence of ascorbic acid (AA), in which Cu
promoted, whereas Fe
significantly inhibited the catalytic function. Since Fe
/AA-generated hydroxyl radicals are efficient on DNA damage, implying that oxidative cleavage of PLDz had no relation with hydroxyl radical. Subsequently, we used Fe
/H
O
and Cu
/H
O
to identify the role of hydroxyl radicals in PLDz catalysis. Data showed that PLDz lost its activity with Fe
/H
O
, but exhibited significant cleavage with Cu
/H
O
. Because Fe
/H
O
and Cu
/H
O
are popular reagents to generate hydroxyl radicals and the latter also produces superoxide anions, we excluded the possibility that hydroxyl radical participated in oxidative cleavage and confirmed that superoxide anion was involved in PLDz catalysis. Moreover, pyrogallol, riboflavin and hypoxanthine/xanthine oxidase with superoxide anion and hydrogen peroxide generation also induced self-cleavage of PLDz, where catalase inhibited but superoxide dismutase promoted the catalysis, suggesting that hydrogen peroxide played an essential role in PLDz catalysis. Therefore, we proposed a catalytic mechanism of PLDz in which superoxide anion and hydrogen peroxide mediated an oxidative cleavage process.
Ubiquitination and deubiquitination of cellular proteins are reciprocal reactions catalyzed by ubiquitination-related enzymes and deubiquitinase (DUB) which regulate almost all cellular processes. ...Marek's disease virus (MDV) encodes a viral DUB that plays an important role in the MDV pathogenicity. Chicken CD4
T-cell lymphoma induced by MDV is a key contributor to multiple visceral tumors and immunosuppression of chickens with Marek's disease (MD). However, alterations in the ubiquitylome of MDV-induced T lymphoma cells are still unclear. In this study, a specific antibody against K-ε-GG was used to isolate ubiquitinated peptides from CD4
T cells and MD T lymphoma cells. Mass spectrometry was used to compare and analyze alterations in the ubiquitylome. Our results showed that the ubiquitination of 717 and 778 proteins was significantly up- and downregulated, respectively, in T lymphoma cells. MDV up- and downregulated ubiquitination of a similar percentage of proteins. The ubiquitination of transferases, especially serine/threonine kinases, was the main regulatory target of MDV. Compared with CD4
T cells of the control group, MDV mainly altered the ubiquitylome associated with the signal transduction, immune system, cancer, and infectious disease pathways in T lymphoma cells. In these pathways, the ubiquitination of CDK1, IL-18, PRKCB, ETV6, and EST1 proteins was significantly up- or downregulated as shown by immunoblotting. The current study revealed that the MDV infection could exert a significant influence on the ubiquitylome of CD4
T cells.
Among many of the pathogens, virus is the main cause of diseases in livestock and poultry. A host infected with the virus triggers a series of innate and adaptive immunity. The realization of innate ...immune responses involves the participation of a series of protein molecules in host cells, including receptors, signal molecules and antiviral molecules. Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular or viral deubiquitinases (DUBs). DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. In this review, we briefly introduce the mechanisms of ubiquitination and deubiquitination, present antiviral innate immune response and its regulation by ubiquitin, and summarize the prevalence of DUBs encoded by viruses (Arteriviridae, Asfarviridae, Nairoviridae, Coronaviridae, Herpesviridae, and Picornaviridae) infecting domestic animals and poultry. It is found that these DUBs suppress the innate immune responses mainly by affecting the production of type I interferon (IFN), which causes immune evasion of the viruses and promotes their replication. These findings have important reference significance for understanding the virulence and immune evasion mechanisms of the relevant viruses, and thus for the development of more effective prevention and treatment measures.
Interleukin-2 (IL-2) is a pleiotropic cytokine regulating the immune and nervous systems. Mammalian and bird IL-2s have different protein sequences, but perform similar functions. In the current ...study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as much of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation reagents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulphide bond promotes homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the expansion of CD4
T cell in vivo or in vitro, and effectively bind to chIL-2 receptor. Overall, this study revealed that the recombinant chIL-2 purified from either
(
) or
(Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4
T cells.
An oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, ...cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.
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•An oxidative cleavage DNAzyme works with various cofactor X•Catalytic nucleotide conservation fluctuates with different cofactor X•The PL DNAzyme's minor secondary structure change affects electrophoretic mobility•Double-cofactor model of the variant T3G can be converted to sole-cofactor model
Chemistry; Biochemistry; Biocatalysis
Biosensors have been widely investigated and utilized in a variety of fields ranging from environmental monitoring to clinical diagnostics. Glucose biosensors have triggered great interest and have ...been widely exploited since glucose determination is essential for diabetes diagnosis. In here, we designed a novel dual-enzyme biosensor composed of glucose oxidase (GOx) and pistol-like DNAzyme (PLDz) to detect glucose levels in tears and saliva. First, GOx, as a molecular recognition element, catalyzes the oxidation of glucose forming H2O2; then PLDz recognizes the produced H2O2 as a secondary signal and performs a self-cleavage reaction promoted by Mn2+, Co2+ and Cu2+. Thus, detection of glucose could be realized by monitoring the cleavage rate of PLDz. The slope of the cleavage rate of PLDz versus glucose concentration curve was fitted with a Double Boltzmann equation, with a range of glucose from 100nM to 10mM and a detection limit of 5μM. We further applied the GOx–PLDz 1.0 biosensor for glucose detection in tears and saliva, glucose levels in which are 720±81μM and 405±56μM respectively. Therefore, the GOx–PLDz 1.0 biosensor is able to determine glucose levels in tears and saliva as a noninvasive glucose biosensor, which is important for diabetic patients with frequent/continuous glucose monitoring requirements. In addition, induction of DNAzyme provides a new approach in the development of glucose biosensors.
•Pistol-like DNAzyme (PLDz) as new ‘material’ is applied to construct a glucose biosensor.•PLDz and GOx are integrated in the cascade by H2O2 to develop a novel dual-enzyme glucose biosensor.•PLDz in the biosensor plays a dual role in both secondary signal recognition and signal output.•GOx–PLDz 1.0 is able to determine glucose levels in saliva and tears as a noninvasive biosensor.
I-R3 DNAzyme is a small, highly active catalytic DNA for DNA hydrolysis. In here, we designed two cis-structure DNAzymes (I-R3N and I-R3S) based on the different locates of the joint linker between ...I-R3 and its substrate. Data demonstrated that both DNAzymes were highly dependent on Zn2+, and worked at a narrow range around pH 7.0. They exhibited strong anti-interference with Mg2+ and Ca2+, but inhibited by Na+ and K+. Moreover, single and multiple-site mutations were generated within the catalytic core to carry out a comprehensive mutational study of I-R3 motif, in which most nucleotides were highly conserved and the nucleotides A5, T11 and T8 were identified as the mutational hotspots. Furthermore, an efficient variant A5G was obtained and its reaction condition was optimized. Finally, we constructed A5G to the 3’ end of a single-stranded DNA (ssDNA) and applied it for asymmetrical PCR amplification to produce a single and double-stranded DNA mixture, in which A5G within ssDNA can self-cleave to generate a shorter desired ssDNA by denaturing gel separation. This would provide a new non-chemical modification approach for preparation of the expected ssDNA for in vitro selection of DNAzymes.
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•Two cis-structure DNAzymes were designed by different locates of a joint linker.•The designed DNAzymes were highly dependent on Zn2+ but inhibited by Na+ and K+.•Conserved level of nucleotides within the catalytic core was mapped by mutations.•A free modification DNAzyme-based approach separates desired single-stranded DNA.
Abstract only
The spliceosome is responsible for the excision of introns from pre‐mRNA in eukaryotes and alterations of the spliceosome can cause serious consequences. Recently, studies have shown ...mutations in certain genes associated with the spliceosome are linked with certain types of Myelodysplastic Syndromes (MDS). Specifically, mutations in
SF3B1
, an U2snRNP associated splicing factor, have been found in approximately 20% of MDS cases. SF3B1 is thought to bind pre‐mRNA and to help link U2snRNP to pre‐mRNA. In our research, we investigated the corresponding mutations in the
S. cerevisiae
homolog,
HSH155
, which is well‐conserved between humans and yeast. We have introduced MDS‐related mutations into
S. cerevisiae HSH155
and showed the cells have no significant growth differences or temperature sensitive defects compared to the wild type. We expected splicing will be affected by the mutations, resulting in an increased ratio of pre‐mRNA to mRNA. To check this hypothesis, we carried out Reverse‐Transcription Polymerase Chain Reaction (RT‐PCR) but found no differences in splicing efficiency of tested pre‐mRNAs (
SUS1, DYN2, GLC7, YFR045w, PMI40, MATa1
) in
S. cerevisiae
. Our results suggest that changes in splicing due to SF3B1 mutations may not be apparent when studying endogenous yeast pre‐mRNAs. Simultaneously we are investigating the effects of mutations in a second spliceosomal protein associated with MDS cases, PRP21p. PRP21p is homologous to the human SF3A1 protein and is also associated with the U2 snRNP. For this project, we have produced the necessary yeast strains and have used growth assays to check for growth differences and temperature sensitivity as well as RT‐PCR experiments to test for splicing of endogenous genes. Additionally, we will use an ACT1‐CUP1 copper reporter gene that will help us determine splicing efficiency in cells. Through this assay, we will determine if splicing of wild type and mutated intron sequences are affected by mutations in
PRP21
.
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