Identification of a regulatory segment of poly(ADP-ribose) glycohydrolase

Abstract

Coordinate regulation of PARP-1 and -2 and PARG is required for cellular responses to genotoxic stress. While PARP-1 and -2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16-residue N-terminal mitochondrial targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474, was required for PARG activity. This region of PARG was termed the "regulatory segment/MTS" (REG/MTS). The overall alpha-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation because the CD spectra of mutant Delta1-16 lacking the REG/MTS showed a higher alpha-helical content than hPARG59, indicating a secondary structure other than alpha-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in a complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. The presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARP-1 and -2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG because MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG.

FIGURE 1. Determination of the minimal catalytically active PARG

The fifteen PARG fragments synthesized and tested for enzymatic activity are shown in relation to the full-length hPARG111 protein. Each fragment is labeled according to its size in kilodaltons, and is indicated on the right as being either catalytically active (+) or inactive (−).

FIGURE 2. Preparation of PARG fragments and mutants

(A) Bacterial expression plasmid His₆-malE-TEV-hPARG used for the expression of the PARG fragments and mutants discussed in this study. (B) Coomassie blue staining of the Ni²⁺-purified hPARG59 fragment before (lane 1) and after (lane 2) removal of the His₆-MBP dual tag by TEV. The bands present following cleavage are the uncleaved fusion protein His₆-MBP-TEV-hPARG59 (103 kDa, band a), the cleaved hPARG59 protein (59 kDa, band b), the Pro-TEV protease (50 kDa, band c), the truncated TEV autoproteolysis product (47 kDa, band d) and the cleaved His₆-MBP tag (43 kDa, band e). (C) Enzyme kinetic parameters of bacterially expressed hPARG59 before and after cleavage of the His₆-MBP fusion tag. The K_m and V_max values and standard errors were determined using the GraphPad Prism® program, and are expressed as a mean value of a representative experiment carried out in triplicate.

FIGURE 3. Deletion and site-directed mutagenesis of the hPARG59 REG/MTS

The REG/MTS deletion and site-directed mutants are shown in relation to wild-type hPARG59. The exon structure is indicated on the top left. The REG/MTS residues present in each deletion mutant and those mutated by the site-directed mutagenesis are also noted. Basic residues of the REG/MTS are indicated with a plus sign (+) and those that are hydrophobic are underlined. To the right, the specific activity and percent activity of each fragment are listed, with the latter being expressed as a percentage of wild-type hPARG59. Data are shown as means of values from two separate experiments, each performed in triplicate.

FIGURE 4. Circular dichroism spectra

(A) The spectra of fragments hPARG42 (brown) and hPARG23 (green), and mutants Δ1-16 (purple) and L11D/L13D/L14D (red) are compared to that of wild type hPARG59 (black). (B) The molecular weight, mean residue ellipticity at 222 nm, [θ]₂₂₂, and helical fraction of hPARG42, hPARG23, Δ1-16 and L11D/L13D/L14D are compared to those of wild-type hPARG59.

FIGURE 5. Secondary structure predictions

Secondary structure predictions for the mostly α-helical domain and mixed α/β ADPRT catalytic domain of hPARP-1 (A) and hPARP-2 (B) are shown adjacent to their respective crystal structures (Protein Data Bank Identification numbers 2RCW and 3KJD for hPARP-1 and hPARP-2, respectively) and are compared to that of hPARG59 (C). The ADPRT catalytic domain of PARP-1 and PARP-2 and the ADPRT-like catalytic domain C of PARG are aligned to show structural similarity. The α-helices and β-sheets are indicated and β-sheet 5, containing the catalytic glutamate residues of each enzyme, is labeled by a red square. The predictions were carried out using the PHD and PROF_sec algorithms as described in Materials and Methods.

FIGURE 6. PARG REG/MTS is predicted to be in a loop conformation

(A) PROF_sec secondary structure predictions of the wild-type REG/MTS and L11D/L13D/L14D mutant. The three mutated residues are shown in red. Abbreviations: AA, amino acid; L, loop; E, β-sheet, Rel_sec, reliability index for PROF_sec prediction (0 = low to 9 = high); SUB_sec, a subset of the PROF_sec algorithm for all residues with an expected average accuracy >82 percent. (B) Predicted and observed changes in REG/MTS function induced by the L11D/L13D/L14D mutation. The export to mitochondria probabilities and cleavage site detections were obtained using the MitoProtII computational method (35), while the mitochondrial fraction values have been previously reported (12).

FIGURE 7. Deletion of the REG/MTS from full-length hPARG111

Deletion mutagenesis was used to characterize the role of the REG/MTS in the enzymatic activity of the full-length hPARG111 protein. The REG/MTS and its first and last amino acid residues are indicated. The specific activities and percent activities are shown as means of values from two separate experiments, each performed in triplicate.

FIGURE 8. Comparison of PARG amino acid sequences from different species

The amino acid sequence of the REG/MTS coded by exon 4 was aligned with five additional PARG sequences across a wide range of organisms. Residues shown in grey represent sequence identity in all six organisms, while those in red and green represent conservation of basic and hydrophobic residues, respectively. The accession numbers for PARG are as follows: Q86W56 for Homo sapiens, O02776 for Bacillus taurus, Q9QYM2 for Rattus norvegicus, O88622 for Mus musculus, Q4KLP9 for Xenopus laevis and B1H3J2 for Xenopus tropicalis.