Structural and Biochemical Characterization of Thioredoxin-2 from Deinococcus radiodurans

Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H2O2 treated conditions. Both drtrx1 and drtrx2 genes were induced following H2O2 treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H2O2, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

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Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity

Biochemical Journal ◽

10.1042/bcj20180347 ◽

2018 ◽

Vol 475 (23) ◽

pp. 3827-3846 ◽

Cited By ~ 7

Author(s):

Alvin Gomez ◽

Christian Bindesbøll ◽

Somisetty V. Satheesh ◽

Giulia Grimaldi ◽

David Hutin ◽

...

Keyword(s):

Catalytic Activity ◽

Zinc Finger ◽

Electron Transfer Dissociation ◽

Biochemical Characterization ◽

Zinc Finger Domain ◽

Adp Ribosylation ◽

Aryl Hydrocarbon ◽

Adp Ribosyltransferase

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400–657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

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Faculty Opinions recommendation of BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1006519.81856 ◽

2002 ◽

Author(s):

Anjana Rao

Keyword(s):

Zinc Finger ◽

Germ Line ◽

Specific Protein ◽

Epigenetic Reprogramming ◽

Zinc Finger Domain ◽

Male Germ Line ◽

Insulator Protein

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Faculty Opinions recommendation of Biochemical characterization of the zinc-finger protein 217 transcriptional repressor complex: identification of a ZNF217 consensus recognition sequence.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1060750.512673 ◽

2007 ◽

Author(s):

Vitaly Citovsky

Keyword(s):

Zinc Finger ◽

Biochemical Characterization ◽

Zinc Finger Protein ◽

Transcriptional Repressor ◽

Recognition Sequence ◽

Finger Protein ◽

Repressor Complex

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Hsp70 Escort Protein: More Than a Regulator of Mitochondrial Hsp70

Current Proteomics ◽

10.2174/1570164615666180713104919 ◽

2018 ◽

Vol 16 (1) ◽

pp. 64-73 ◽

Cited By ~ 1

Author(s):

David O. Nyakundi ◽

Stephen J. Bentley ◽

Aileen Boshoff

Keyword(s):

Zinc Finger ◽

Zinc Finger Domain ◽

C Terminus ◽

Cellular Localisation ◽

The Matrix ◽

Mitochondrial Hsp70 ◽

Cellular Compartments ◽

Intramolecular Communication ◽

Self Aggregation ◽

J Protein

Hsp70 members occupy a central role in proteostasis and are found in different eukaryotic cellular compartments. The mitochondrial Hsp70/J-protein machinery performs multiple functions vital for the proper functioning of the mitochondria, including forming part of the import motor that transports proteins from the cytosol into the matrix and inner membrane, and subsequently folds these proteins in the mitochondria. However, unlike other Hsp70s, mitochondrial Hsp70 (mtHsp70) has the propensity to self-aggregate, accumulating as insoluble aggregates. The self-aggregation of mtHsp70 is caused by both interdomain and intramolecular communication within the ATPase and linker domains. Since mtHsp70 is unable to fold itself into an active conformation, it requires an Hsp70 escort protein (Hep) to both inhibit self-aggregation and promote the correct folding. Hep1 orthologues are present in the mitochondria of many eukaryotic cells but are absent in prokaryotes. Hep1 proteins are relatively small and contain a highly conserved zinc-finger domain with one tetracysteine motif that is essential for binding zinc ions and maintaining the function and solubility of the protein. The zinc-finger domain lies towards the C-terminus of Hep1 proteins, with very little conservation outside of this domain. Other than maintaining mtHsp70 in a functional state, Hep1 proteins play a variety of other roles in the cell and have been proposed to function as both chaperones and co-chaperones. The cellular localisation and some of the functions are often speculative and are not common to all Hep1 proteins analysed to date.

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