The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

<p>Abstract</p> <p>Background</p> <p>The parvulin-type peptidyl prolyl <it>cis/trans </it>isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study.</p> <p>Results</p> <p>Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions.</p> <p>Conclusion</p> <p>Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae.</p

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-5

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>s. The resulting DNA sequences were aligned with respect to the Par17 and Par14 ATG codons; the respective sequence was obtained from Ensembl and aligned accordingly. Translated protein sequences in-frame with the respective ATG codons are given below the DNA sequences with stop codons written in red. Within the genomic sequences of and (obtained from Ensembl), the Par17 ATG codons are immediately preceded by an in-frame stop codon. The Q16/R18 to R16/S18 coupled SNPs that were described for the human sequence are highlighted in grey; comparison with the other primate sequences reveals the QR form to be the ancestral one. Phylogenetic clades used in scheme B are indicated by boxes here and the respective abbreviations from B. B. Phylogenetic scheme of primate relationships and Par17 sequence features. The Par17 elongation is preformed in sequence in all anthropoid genomic DNAs tested. Additional ATG codons at the putative starting position of Par17 exist in all tested species, but are out-of-frame relative to the Par14 coding sequence in New World and Old World monkeys. The gap of five nucleotides in New World monkeys shrinks to a gap of four nucleotides in Old World monkeys, still preventing a continuous ORF with Par14. The upstream ATG codons are in-frame with the Par14 sequence in the clade of apes, only. Within the gibbon sequence however, an in-frame stop codon prevents expression of an extended protein. This stop codon has been sequenced in three independent clones. Thus, only Hominidae (and ) possess an extended ORF that can lead to the expression of Par17. (The length of the branches in this scheme does not reflect any phylogenetic distances.) C. Exon 3 sequences were obtained as in A. Exon 3 sequences are highly conserved with only two amino acid substitutions between human and cow or human and mouse, respectively. Amino acid exchanges relative to the human sequence are in white on black. Phylogenetic groupings are indicated by boxes as in A

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-3

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>ne labeled Par17 and Par14 lysates for 15 min at 25°C; Porin and GFP lysates were used as controls. Mitochondria were separated from soluble protein by the addition 500 mM sucrose buffer, subsequent centrifugation and additional washing in a 250 mM sucrose containing SEM buffer to ensure efficient separation of mitochondria from lysate ingredients. Mitochondria were then suspended in buffer containing increasing salt concentrations (0, 80 and 600 mM NaCl) and re-isolated by centrifugation. The samples within each panel including the respective reference sample (L, 20% for Porin and GFP, 50% for Par14 and Par17) were separated on the same gel to allow direct comparison (M +, mitochondria added). The amount of associated radioactively labeled protein was analyzed by SDS-PAGE and autoradiography. Mitochondrial association of Par14 and Par17 as displayed as bars on the diagram represent data normalized to the highest value including error bars (standard deviation for n = 3). B. Par17, but not Par14, is imported into isolated human, rat and yeast mitochondria. Left. Par17-QR and Par14 S-methionine labeled lysates were incubated with different amounts of PK showing complete degradation of soluble parvulin proteins at a PK concentration of 50 μg/ml; at least 100 μg/ml PK were used for all following experiments. Right. Radiolabeled Par17-QR was incubated with mitochondria from human Jurkat cells, rat liver or from yeast either with or without subsequent proteinase K (PK) treatment (at least 100 μg/ml for 10 min). Mitochondria were then re-isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Par17 reached a PK protected compartment irrespective of the source of mitochondria indicating mitochondrial import. Par14, also present in this reaction, could be re-isolated together with mitochondria during centrifugation, but did not reach a protease-protected compartment. C. Time dependence of Par17 mitochondrial import. Mitochondria from yeast and human Jurkat cells were incubated with Par17-QR lysates for the times indicated followed by PK treatment. To assess relative import efficiencies, lysates of Su9-DHFR (amino acids 1–69 from subunit 9 of ATP synthetase fused to mouse DHFR [28]) were imported into the same mitochondrial preparations for equal time intervals at identical reaction conditions. Import reactions of Par17-QR and mature Su9-DHFR were analyzed by autoradiography and densitometric quantifications. Values were expressed as percent of added protein with correction for the different numbers of methionine residues in the precursor and processed proteins of Su9-DHFR. D. Par17 import depends on the mitochondrial membrane potential. Import reactions were performed as in B (right panel). Mitochondria were treated with the uncoupling reagent valinomycin to dissipate the mitochondrial membrane potential either before or after import reaction. Par17 was only imported into mitochondria with intact membrane potential (without valinomycin; or when the uncoupling agent was added after import). E. Par17 reaches the inner membrane of rat mitochondria. Porin and Par17-QR were imported into mitochondria isolated from rat liver. The outer mitochondrial membrane was removed by hypo-osmotic swelling as indicated by the disappearance of the porin signal. The Par17 band however only disappeared upon complete lysis of mitochondria by digitonin indicating that Par17 was imported at least to the inner mitochondrial membrane. F. Par17 is imported into the mitochondrial matrix. Import reactions with yeast mitochondria were performed with increasing amounts of digitonin leading to stepwise lysis. The amounts of Par17-QR, the inner membrane protein Tim23 and the matrix protein Mge1 were monitored in parallel samples using autoradiography for Par17 and Western blotting for Tim23 and Mge1. Tim23 is already degraded at a concentration of 0.1% digitonin, while Par17-QR and the matrix protein Mge1 resist PK. Degradation of these two proteins is only observed upon addition of 0.2% digitonin indicating that Par17 is transported to the mitochondrial matrix. Gels (upper part) and densitometric analysis (lower part) are shown

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-4

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>ncubation of dsDNA with protein lysates, bound protein was eluted with increasing KCl concentrations. 20% of lysate (Lys) and KCl fractions were analyzed by SDS-PAGE and autoradiography. Asterisk (*), unspecific band caused by free label. Prominent signals for both, Par17 and Par14, are seen at 100 and 200 mM KCl. Porin was used as negative control and was washed off at 0 and 50 mM KCl. B. Quantitative analysis. Par17-QR, -RS and Par14 fused to EGFP as well as EGFP without fusions were subjected to the DNA binding assay. Blots were quantitatively analyzed by densitometry. Data from each experiment were normalized to their sum. Standard deviations are given from duplicate experiments. Par17 and Par14 proteins were eluted from dsDNA cellulose at 100, 200 and to a lesser extent at 500 mM KCl pointing to similar DNA-binding of the two parvulin proteins at physiological salt concentrations. EGFP without fusions was completely eluted from DNA cellulose at 50 mM KCl

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae-0

<p><b>Copyright information:</b></p><p>Taken from "The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae"</p><p>http://www.biomedcentral.com/1741-7007/5/37</p><p>BMC Biology 2007;5():37-37.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2031878.</p><p></p>s. The resulting DNA sequences were aligned with respect to the Par17 and Par14 ATG codons; the respective sequence was obtained from Ensembl and aligned accordingly. Translated protein sequences in-frame with the respective ATG codons are given below the DNA sequences with stop codons written in red. Within the genomic sequences of and (obtained from Ensembl), the Par17 ATG codons are immediately preceded by an in-frame stop codon. The Q16/R18 to R16/S18 coupled SNPs that were described for the human sequence are highlighted in grey; comparison with the other primate sequences reveals the QR form to be the ancestral one. Phylogenetic clades used in scheme B are indicated by boxes here and the respective abbreviations from B. B. Phylogenetic scheme of primate relationships and Par17 sequence features. The Par17 elongation is preformed in sequence in all anthropoid genomic DNAs tested. Additional ATG codons at the putative starting position of Par17 exist in all tested species, but are out-of-frame relative to the Par14 coding sequence in New World and Old World monkeys. The gap of five nucleotides in New World monkeys shrinks to a gap of four nucleotides in Old World monkeys, still preventing a continuous ORF with Par14. The upstream ATG codons are in-frame with the Par14 sequence in the clade of apes, only. Within the gibbon sequence however, an in-frame stop codon prevents expression of an extended protein. This stop codon has been sequenced in three independent clones. Thus, only Hominidae (and ) possess an extended ORF that can lead to the expression of Par17. (The length of the branches in this scheme does not reflect any phylogenetic distances.) C. Exon 3 sequences were obtained as in A. Exon 3 sequences are highly conserved with only two amino acid substitutions between human and cow or human and mouse, respectively. Amino acid exchanges relative to the human sequence are in white on black. Phylogenetic groupings are indicated by boxes as in A

Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction.

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability

Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction

International audienceWhereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability